<%BANNER%>

Targeting Tumor Metastasis-Src Inhibition

Permanent Link: http://ufdc.ufl.edu/UFE0041250/00001

Material Information

Title: Targeting Tumor Metastasis-Src Inhibition
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Dong, Meiyu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: azd0530, invasion, metastases, src
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Src is a non-receptor tyrosine kinase that is frequently over-expressed in malignances and increased Src activation/phosphorylation has been associated with a poor patient prognosis. Src signaling pathways are known to affect critical cellular processes of metastases. This project examined the impact of Src inhibition on tumor metastases by using a specific Src tyrosine kinase inhibitor (AZD0530). In the first part of this project the effect of AZD0530 on tumor cell functions related to metastases were evaluated. AZD0530 was found to inhibit tumor cell proliferation, migration, invasion, adhesion, and to induce tumor cells to accumulate in the G1 phase of cell cycle in several mouse and human cancer models. These results suggested that AZD0530 treatment could inhibit tumor cell functions related to metastasis. The second part of this project was to study the effect of AZD0530 on metastatic process in vivo. A single dose treatment immediately after tumor cell injection via tail vein effectively blocked formation of lung nodules in mice. AZD0530 was also found to inhibit tumor cell induced angiogenesis. However, AZD0530 had no major effect on endothelial cell growth, tube formation and migration. The level of secreted VEGF from KHT cells was found to decrease after AZD0530 treatment. This suggested that the effect on angiogenesis may be caused by inhibiting the secretion of angiogenic factors by tumor cells. The third part was to establish the KHT mouse sarcoma and 4A4 breast tumor models to non-invasively assess tumor growth as well as metastases using the Xenogen imaging system. In both the KHT-Luc and 4A4-Luc spontaneous metastases models, bioluminescent signals from the primary tumors were detected and the intensity was found to increase with tumor growth. In the KHT-Luc experimental metastasis model, bioluminescent signals were observed from lung metastases, and the signal intensity increased with time. These tumor models may allow us to study the effect of Src inhibition on metastases non-invasively and efficiently in the future.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meiyu Dong.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Siemann, Dietmar W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041250:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041250/00001

Material Information

Title: Targeting Tumor Metastasis-Src Inhibition
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Dong, Meiyu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: azd0530, invasion, metastases, src
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Src is a non-receptor tyrosine kinase that is frequently over-expressed in malignances and increased Src activation/phosphorylation has been associated with a poor patient prognosis. Src signaling pathways are known to affect critical cellular processes of metastases. This project examined the impact of Src inhibition on tumor metastases by using a specific Src tyrosine kinase inhibitor (AZD0530). In the first part of this project the effect of AZD0530 on tumor cell functions related to metastases were evaluated. AZD0530 was found to inhibit tumor cell proliferation, migration, invasion, adhesion, and to induce tumor cells to accumulate in the G1 phase of cell cycle in several mouse and human cancer models. These results suggested that AZD0530 treatment could inhibit tumor cell functions related to metastasis. The second part of this project was to study the effect of AZD0530 on metastatic process in vivo. A single dose treatment immediately after tumor cell injection via tail vein effectively blocked formation of lung nodules in mice. AZD0530 was also found to inhibit tumor cell induced angiogenesis. However, AZD0530 had no major effect on endothelial cell growth, tube formation and migration. The level of secreted VEGF from KHT cells was found to decrease after AZD0530 treatment. This suggested that the effect on angiogenesis may be caused by inhibiting the secretion of angiogenic factors by tumor cells. The third part was to establish the KHT mouse sarcoma and 4A4 breast tumor models to non-invasively assess tumor growth as well as metastases using the Xenogen imaging system. In both the KHT-Luc and 4A4-Luc spontaneous metastases models, bioluminescent signals from the primary tumors were detected and the intensity was found to increase with tumor growth. In the KHT-Luc experimental metastasis model, bioluminescent signals were observed from lung metastases, and the signal intensity increased with time. These tumor models may allow us to study the effect of Src inhibition on metastases non-invasively and efficiently in the future.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meiyu Dong.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Siemann, Dietmar W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041250:00001


This item has the following downloads:


Full Text

PAGE 1

1 TARGETING TUMOR METASTASIS SRC INHIBITION By MEIYU DONG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIV ERSITY OF FLORIDA 2009

PAGE 2

2 2009 Meiyu Dong

PAGE 3

3 To my husband, parents and son for their love, support and encouragement

PAGE 4

4 ACKNOWLEDGMENTS I would like to first thank to my advisor Dr. Dietmar W. Siemann for h is encourage ment, instruction, and patience in guiding my research. His insight in this research provide d a key path through the whole research process. I would also like to thank to my committee members : Dr. Edwin M. Meyer Dr. Wolfgang Streit, Dr. Thoma s Rowe, and Dr. Brian K. Law for their help and support in my re search. Their help really played an important role in the completion of this research. I would also like to thank Dr. Lung Ji Chang for his kind help. I would also like to thank the past and present members of Dr. Siemanns laboratory including Dr. Lori Rice, Dr. Yao D ai, Sharon Lepler, Chris Pampo, Dr. Wenyin shi, Dr. William D. Brazelle, Dr. Beth A. Salmon, Dr. Christina M. Norris, Dr Howard Salmon and Dr Ananya Guha and Nikolett Molnar for their help, suggestions, and technical support. I really enjoy ed working with all my co -workers. I would also like to thank Nicole Teoh Parker for her kindly help with editing and revising this dissertation. I also greatly appreciate the faculty, sta ff and student groups in the College of Medicine IDP program for their help. Finally, I would like to thank my husband Zhihong Hu for his unconditional love and support. I would also like to thank m y son, Robert Hu. His innocent smile always entertains me when I am tired and strengthen me to finish my research.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 12 CHAPTER 1 INTRODUCTION ....................................................................................................................... 14 Cancer and Metastases ................................................................................................................ 14 Targeting Tumor Metastases ...................................................................................................... 15 Src Implication in Metastasis ..................................................................................................... 18 Src Family .................................................................................................................................... 18 Src ................................................................................................................................................. 19 Src Functions ............................................................................................................................... 19 T he R ole of Src in the M etastatic P rocess ................................................................................. 20 Proliferation and Survival ................................................................................................... 20 Cell Migration and Invasion ............................................................................................... 20 Adhesion............................................................................................................................... 21 Angiogenesis ........................................................................................................................ 21 Kinase Inhibitors ......................................................................................................................... 22 Src Inhibitors ............................................................................................................................... 22 SH2/SH3 Inhibitors (AP22408, UCS15A) ........................................................................ 22 Src -destabilizing Agents (G eldanamycin, Herbimycin A, Benzoquinone Ansamycins) ..................................................................................................................... 23 ATP -competitive Src Kinase Inhibitors (AZD0530, M475271, SKI 606) ...................... 23 AZD0 530 ..................................................................................................................................... 23 Project Significance .................................................................................................................... 23 2 THE EFFECT OF SRC INHIBITION ON TUMOR CELL FUNCTIONS ............................ 31 Introduction ................................................................................................................................. 31 Materials and Methods ................................................................................................................ 32 Tissue Culture ...................................................................................................................... 32 Drug Preparation .................................................................................................................. 33 Western Immunoblotting ..................................................................................................... 33 Cell Growth .......................................................................................................................... 34 Clonogenic Cell Survival Assay ......................................................................................... 34 Cell Cycle Analysis ............................................................................................................. 35

PAGE 6

6 Cell Migration ...................................................................................................................... 35 Scratch assay ................................................................................................................. 35 Modified Boyden -chamber cell migration assay ....................................................... 35 Cell Invasion Assay ............................................................................................................. 35 ELISA MMP9 ..................................................................................................................... 36 Gelatin Zymography ............................................................................................................ 36 Cell Detachment Assay ....................................................................................................... 36 Immunofluorescence Microscopy ...................................................................................... 37 Statistical Analysis ............................................................................................................... 37 Results .......................................................................................................................................... 37 Src Signaling ........................................................................................................................ 37 Cell Growth .......................................................................................................................... 38 Cell Migration ...................................................................................................................... 38 Cell Invasion ........................................................................................................................ 38 MMP 2 and MMP 9 Secretion ........................................................................................... 39 MMP 2 and Total MMP 9 Activities ................................................................................. 39 Cell Detachment .................................................................................................................. 39 Discussion .................................................................................................................................... 39 3 THE EFFECT OF SRC INHIBITION ON IN VIVO TUMOR METASTSES ...................... 54 Introduction ................................................................................................................................. 54 Materials and Methods ................................................................................................................ 55 Drug Preparation .................................................................................................................. 55 Animals ................................................................................................................................ 56 Experimental Metastases (1) ............................................................................................... 56 E xperimental Metastases (2) ............................................................................................... 56 Experimental Metastases (3) ............................................................................................... 57 Spontaneous Metastases ...................................................................................................... 57 Maintenance of Cell Lines and Tissue Culture In Vitro .................................................... 57 SDS -PAGE Sample Preparation and Immunoblot Analysis ............................................. 57 HMVEC -L Morphology ...................................................................................................... 58 Cellular Growth Assay ........................................................................................................ 58 Tube Formation Assay (1) .................................................................................................. 58 Tube Formation Assay (2) .................................................................................................. 58 Tube Formation Assay (3) .................................................................................................. 59 Migration As s ay (1) ............................................................................................................. 59 Migration Assay (2) ............................................................................................................. 59 Cell cell Adhesion Assay: ................................................................................................... 59 ELISA ................................................................................................................................... 60 Intra -dermal Assay (1) ......................................................................................................... 60 Intra -dermal Assay (2) ......................................................................................................... 60 Results ................................................................ .......................................................................... 60 Effect of AZD0530 Treatment on Lung Colony Formation in an Experimental Metastases Model ............................................................................................................. 60 Effect of AZD0530 Treatment on Lung Colony Formation in a Spontaneous M etastases Model ............................................................................................................. 61

PAGE 7

7 Effect of AZD0530 on Src Phosporylation in HMVEC -L ................................................ 62 Effect of AZD0530 on HMVEC -L Morphology ............................................................... 62 Effect of AZD0530 on HMVEC -L Growth ....................................................................... 62 Effect of AZD0530 on HMVECL Tube Formation .......................................................... 62 Effect of AZD0530 on HMVECL Migration .................................................................... 63 pSrc Recovery after AZD0530 Withdrawal ....................................................................... 63 Effect of AZD0530 on KHT HMVEC L Adh esion .......................................................... 63 Effect of AZD0530 on VEGF Secretion ............................................................................ 63 Effect of AZD0530 on Angiogenesis ................................................................................. 64 Discussion .................................................................................................................................... 64 4 ESTABLISHING TUMOR MODELS IN WHICH THE DEVELOPMENT OF METASTASES CAN BE NON INVASIVELY ASSESSED ................................................. 85 Introduction ................................................................................................................................. 85 Materials and Methods ................................................................................................................ 87 Cell P reparation ................................................................................................................... 87 Animals ................................................................................................................................ 87 Imaging ................................................................................................................................. 87 KHT Fibrosarcoma Experimental Metastasis Model ........................................................ 88 KHT Fibrosarcoma Spontaneous Metastasis Mode l ......................................................... 88 4A4 Breast Tumor Spontaneous Metastasis Model .......................................................... 88 Histology .............................................................................................................................. 88 Results .......................................................................................................................................... 88 Established KHT Luc and 4A4 Luc Cell Lines ................................................................ 88 KHT Fibrosa rcoma Experimental Metastasis Model ........................................................ 89 KHT Fibrosarcoma Spontaneous Metastasis Model ......................................................... 89 4A4 Breast Tumor Spontaneous Metastasis Model .......................................................... 89 Discussion .................................................................................................................................... 90 5 SUMMARY ............................................................................................................................... 107 LIST OF REFERENCES ................................................................................................................. 111 BIOGRAPHICAL SKETCH ........................................................................................................... 120

PAGE 8

8 LIST OF TABLES Table page 1 1 List of FDA approved small molecul e kinase inhibitors ..................................................... 30

PAGE 9

9 LIST OF FIGURES Figure page 1 1 The tumor metastatic process ................................................................................................ 25 1 2 Src tyrosine kinase in colorectal cancer.. .............................................................................. 26 1 3 The structure of Src family kinases ....................................................................................... 26 1 4 Mechanisms involved in acti vation of Src tyrosine kinase. ................................................ 27 1 5 Potential signal tran sduction pathways involving Src ......................................................... 28 1 6 Structure of AZD0530 ........................................................................................................... 29 2 1 Effects of AZD0530 on pSrc and Src expression in tumor cells after 24 hr treatment. .... 43 2 2 Effects of 24 hr exposure of KHT cells to AZD0530 on FAK, pFAK, Stat3, pStat3, AKT, pAKT, Erk, pErk and Met. .......................................................................................... 44 2 3 Cell growth in the presence of AZD0530 of a variety of tumor cell lines ......................... 45 2 4 KHT cell colonogenic survival after 24 hr exposure to AZD0530 ..................................... 46 2 5 Effect of AZD0530 on tumor cell cycle distribution assessed by flow cytometry 24 hr after expos ure to 0 5 M AZD0530. ................................................................................ 47 2 6 Effects of AZD0530 on tumor cell migration assayed by scratch assay after 24 hr drug treatment. ........................................................................................................................ 48 2 7 Effects of AZD0530 on tumor cell migration assayed by modified Boyden-chamber cell migration assay after 24 hr drug treatment. ................................................................... 49 2 8 Effects of AZD0530 on tumor cell invasion assayed by transwell invasion assay after 24 hr drug treatment.. ............................................................................................................. 50 2 9 Effects of AZD0530 on secreted MMP 2 and MMP 9 measured by ELISA in KHT cells after 24 hr treatment. ..................................................................................................... 51 2 10 Effects of AZD0530 on MMPs activity after 24 hr treatment. .......................................... 52 2 11 KHT cell detachment determined 24 hr after AZD0530 treatment. ................................... 52 2 12 Distribution of pSrc (Y423), Src, pFAK (Y861) and FAK after 24 hr exposure to 5 M AZD0530 analyzed by immunofluorescent confocal microscopy. .............................. 53 3 1 Effects of 24 hr pre treatment of AZD0530 on the ability of KHT cells to form lung colonies when injected into mice via the tail veins.. ............................................................ 67

PAGE 10

10 3 2 Effects of AZD0530 on the ability of KH T cells to form lung colonies when injected into mice via the tail veins. .................................................................................................... 68 3 3 Effects of AZD0530 on the ability of KHT cells to form lung colonies when injected into mice via the tail veins with three different treatment schedules. ................................. 69 3 4 Effects of AZD0530 on the ability of KHT cells to form lung colonies when injected into the leg of mice intra -muscularly.. .................................................................................. 70 3 5 Effects of AZD0530 on pSrc (Y416) and Src in HMVEC L cells. .................................... 71 3 6 Effects of AZD0530 on HMVEC -L cell morphology after 24 hr treatment. ..................... 72 3 7 Effects of AZD0530 on HMVEC -L cell growth over a 7 day period in the presence of AZD0530. ........................................................................................................................... 73 3 8 Effects of AZD0530 on tube f ormation by HMVEC -L cells pre -treated with drug for 24 hr. ....................................................................................................................................... 74 3 9 Effects of AZD0530 on tube formation by HMVEC -L cells pre -treated with drug for 48 hr. ....................................................................................................................................... 75 3 10 Effects of AZD0530 on tube formation by HMVEC -L cells in the presence of AZD0530. ............................................................................................................................... 76 3 11 Effects of AZD0530 on complete tubes formed by HMVEC L cell. ................................. 77 3 12 Effects of AZD0530 on HMVEC -L cell migration after the cells were pre treated with drug for 48 hr. Data shown are representative of two experiments. .......................... 78 3 13 HMVEC -L cell migration in the presence of 1 10 M AZD0530 for 24 hr. ..................... 79 3 14 pSrc (Y416) recovery in KHT cells at various times after AZD0530 removal following a 24 hr drug treatment. .......................................................................................... 80 3 15 Effects of AZD0530 on KHT cell adhesion to HMVEC L monolayer. ............................. 81 3 16 Effects of AZD0530 on secret ed VEGF in the medium of KHT cells after 24 hr treatment. ................................................................................................................................ 82 3 17 Effects of 24 hr pre treatment of AZD0530 on the ability of KHT cells to induce angiogenesis. ........................................................................................................................... 83 3 18 Effects of AZD0530 on the ability of KHT cells to induce angiogenesis. ......................... 84 4 1 Xenogen imaging system. ...................................................................................................... 92 4 2 Bioluminescent imaging of KHT Luc and 4A4Luc cells after lentivirus infection ........ 93

PAGE 11

11 4 3 Bioluminescent imaging of mouse #1 after tail vein injection of KHT Luc cells. ............ 94 4 4 Bioluminescent imaging of mouse #2 after tail vein injection of KHT Luc cells. ............ 95 4 5 Bioluminescent imaging of mouse #3 after tail vein injection of KHT Luc cells. ............ 96 4 6 Bioluminescent imaging of mouse #4 after tail vein injection of KHT Luc cells.. ........... 97 4 7 Bioluminescent imaging of mice after KHT-Luc cells are injected intramuscularly. ....... 98 4 8 Bioluminescent imaging immediately after 4A4 Luc cell injection into the fat pads of mice. ........................................................................................................................................ 99 4 9 Bioluminescent imaging of mouse #1 after 4A4 Luc cell injection into the fad pad. ..... 100 4 10 Bioluminescent imaging of mouse #2 a fter 4A4 Luc cell injection into the fad pad. ..... 101 4 11 Bioluminescent imaging of mouse #3 after 4A4 Luc cell injection in the fad pad.. ....... 102 4 12 Bioluminescent imaging of mouse #4 after 4A4 Luc cell injection in the fad pad.. ....... 103 4 13 Bioluminescent imaging of mouse #5 after 4A4 Luc cell injection in the fad pad.. ....... 104 4 14 Bioluminescent imaging of mouse #1 with 4A4-Luc breast tumor. ................................. 105 4 15 Bioluminescent imaging of mouse #2 with 4A4-Luc brea st tumor.. ................................ 106

PAGE 12

12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TARGETING TUMOR METASTASIS SRC INHIBITION By Meiyu Dong December 2009 Chair: Dietmar W. Siemann Department: Pharmacology and Therapeutics Src is a non -receptor tyrosine kinase that is frequently over expressed in malignances and i ncreased Src activation/phosphorylation h as been associated with a poor patient prognosis. Src signaling pathways are known to affect critical cellular processes of metastases. This project examine d the impact of Src inhibition on tumor metastases by using a specific Src tyro sine kinase inhibit or (AZD0530) In t he first part of this project the effect of AZD0530 on tumor cell functions related to metastases were evaluated AZD0530 was found to inhibit tumor cell proliferation, migration, invasion, adhesion, and to induce tumor cell s to accumul ate in the G1 phase of cell c ycle in several mouse and human cancer models. T h ese results suggest ed that AZD0530 treatment c ould inhibit tumor cell functions related to metastas i s. The second part of this project was to study the effect of AZD0530 on met astatic process in vivo. A single dose treatment immediately after tumor cell injection via tail vein effectively blocked formation of lung nodules in mice. AZD0530 was also found to inhibit tumor cell induced angiogenesis. However, AZD0530 ha d no major effect on endothelial cell growth, tube formation and migration. The level of secreted VEGF from KHT cells w as found to decrease after AZD0530 treatment. T his suggest ed that the effect on angiogenesis may be caused by inhibiting the secretion of a n giogenic factors by tumor cells. The third part was to establish the KHT mouse sarcoma and 4A4 breast tumor models to non -invasively assess tumor growth as well as

PAGE 13

13 metastases using the Xenogen imaging system. In both the KHT L uc and 4A4 Luc spontaneous metastas es model s, bioluminescent signal s from the primary tumors were detected and the intensity was found to increase with tumor growth. In the KHT Luc experimental metastasis model, bioluminescent signals were observed from lung metastases and the signal inte nsity increased with time. These tumor models may allow us to study the effect of Src inhibition on metastases non invasively and efficiently in the future.

PAGE 14

14 CHAPTER 1 INTRODUCTION Cancer and Metastases Cancer is a serious health problem in the world today. One in three people will develop cancer in their lifetime. One in four deaths in the United States is caused by cancer (The Cancer Cure Foundation, 2009) In 2009 about 1.5 million new cancer cases and 562,340 cancer deaths are expected in the Unit ed States (Jemal et al. 2009) Convention al treatment s for cancer include chemotherapy, radiation ther apy and surgery. Although t hese treatments can be helpful in some types and early stages of cancer, in many cases they will fail. The most common reason for these failures is that these treatme nts do not prevent tumor cells from spreading to distant s ites by the process of metastasi s. About 90% of cancer deaths are attributed to distant metastases (Wittekind and Neid, 2005) Approximately one third of cancer patients have detectable metastases at the time of diagnosis. Another t hird have micro metastases which are not clinically detectable (Cancer Medic ine, 2009) These undetected tumor cell deposits severely limit the success of local therapy even if the pri mary tumor has been controlled. Clearly, new treatment strategies against metastases are needed to improve cancer patient survival. Metastasis is the spread of cancer cells from the original tumor site through the blood and/or lymph vessels to distant sites to form secondary cancers. The metastatic p rocess is a very complicated, multi -step event (Figure 1 1) As the primary tumor grows it secret es angiogenic factors to stimulate new vasculature formation. This new vasculature provides an avenue for cancer cells to escape from the original tumor. Some of the cancer cells invade the blood vessels and travel to distant sites. In order to escape t he blood vessel, they attach and extrav a sate by destroying the vessel walls. After invasi on at the new site, they use local facto rs in the

PAGE 15

15 environment and/or factors released from themselves to proliferate and grow to form new colonies (Wittekind and Neid, 2005) Not all cancer cells make it through this entire process. Out of the many thousands of cancer cells th at reach the blood circulation, only one will likely survive to form a secondary tumor (Cancer Research UK, 2009) Some cancer cells can be killed by lymphocytes in the immune system; others by the shear force in fast flowing blood. Due to the complexity of this process, a reasonable strategy to target tumor metastasis would be to discover which steps are rate limiting in the cancer metastatic cascade and interrupt them. Targeting Tumor Metastases With the rapidly expanding knowledge base in the area of metastasis as well as the recent advances in drug discovery, several anti -metastasis strategies are now being explored. These strategies usually aim to target a specific step of metastasis or a specific molecule involved in the metastatic cascade. If su ccessful, such strategies may have profound effects on cancer therapy. Components of the blood clotting pathway may contribute to metastasis by trapping cells in capillaries or by facilitating adherence of cells to capillary walls. This concept was originally confirmed in experimental animal models of metastatic cancer (Francis et al. 1992; Schneider et al. 1996) This suggests the possibility that substantial benefit may be realized from this treatment approach in patients with malignancy. P latelets may serve as aids to cancer cells by adhering to them and helping them to survive in the blood stream and stick to the walls of the blood vessels. Once the cancer cells have adhered to the bl ood vessel walls, they a re able to pass through the walls and form metastatic lesions in a new location in the body. However, some of the clinical trials did not show dramatic benefit For example, Dahl reported no improvement when porcine plasmin was use d (Dahl, 1966) The availability of newer and potentially more

PAGE 16

16 effective therapeutic agents holds promise for even greater gains (Francis et al. 1992; Schneider et al. 1996; Zacharski et al. 1984; Zacharski et al. 1992) For a tumor cell to metastasize, it must pass through surrounding stroma l elements as it migrates towards a blood vessel. One important class of molecules that facilitate s tumor invasion is the matrix metalloproteinases (MMP s ) (Dove, 2002) These enzymes are secreted by tumor cells and destroy the extracellular m atrix (ECM) and basement membrane to facilitate invasion. MMPs have been thought to be promising targets for cancer therapy on the basis of their extensive upregulation in malignant tissues and their unique ability to degrade all components of the extrac ellular matrix. Preclinical studies testing the efficacy of MMP suppression in tumor models were so compelling that synthetic metalloproteinase inhibitors (MPIs) were rapidly developed for human clinical trials However cli nical trials with MPIs such as marimastat and batimastat yielded disappointing results, highlighting the need for better insight into the role of these enzymes in tumor metastasis. It is now recognized that MMP activity is tightly regulated at several levels, suggesting new therapeuti c strategies for cancer metastasis (Maekawa et al. 2 000) In order for microme tastases to grow to a size that is clinica lly detectable, tumor cells induce new blood vessels by the process of angiogenesis (Ausprunk and Folkman, 1977) This angiogenesis can serve as the turning point for the metastatic lesions in to a fast growing phase that will be lethal (Mahabeleshwar and Byzova, 2007) As mentioned earlier angiogenesis in the primary tumor also provides channels for the tumor cells to escape into th e blood stream and t ravel to distant sites. For these reason s, many anti angiogenic agents are currently under investigation Several are currently in clinical trials with promising results (Underiner et al.

PAGE 17

17 2004) However, the emphasis in these studies has been on evaluating drug efficacy in primary tumor s not in distant lesions (Isayeva et al. 2004) The adhesive interaction between tumor cells and host cells or the extracellular matrix (ECM ) plays a crucial ro le in metastasis formation. The tumor cells need to adhere to the endothelial cell s and ECM for both intravasati on and extravasta ti on steps. Several studies showed the significance of adhesion in the metastatic process. For example, expression of E cadh erin, the most abundant adhesion molecule in adherent junctions of epithelia is downregulated in most epithelial cancers. Several research groups have shown that reconstitution of a functional E -cadherin adhesion complex suppresses the invasive phenotype of many different tumor cell types (Frixen et al. 1991; Hsu et al. 2000; Luo et al. 1999; Vleminckx et al. 1991) In addition, impaired cell -matrix adhesion is related to an increase in tumor cell migration and invasion (Schlaepfer et al. 2004) I ntegrins are important molecules involved in the interaction of tumor cells with extracellular matrix. Integrin inhibitors can reduce lung metastasis and also decrease the si ze of tumor nodules (Elliott et al. 1994; Newton, 1995) Other a nti adhesion agents are also under investi gation as anti -metastatic agents (Gomes et al. 2004) M otility is involved in several steps in metastatic cascade, including tumor cell dissociation, intra vasation, and extrava sation from the blood vessels G -protein -couple d IP3 is involved in stimulat ion of cell migration (Kohn and Liotta, 1990) L651582 an agent which can inhibit I P3 was found to interfere with cancer cell motility and prolong the survival time of nude mice bearing ovari an tumors (Kohn and Liotta, 1990) Successful metastatic cells have undergone changes in their proliferative, surv ival, migratory and invasive abilities. During tumor progression and metastasis an active crosstalk occurs between tumor cells and their microenvironment (Krstein Mseide, 2005) A number of

PAGE 18

18 si gnal tr ansdu ction kinases have been identified as being important in tumor metastatic processes Consequently, novel agents that target specific pathways, such as the Src kinase pathway, have shown promise in interrupt ing the tumor metastatic cascade and are under active investigation (Koppikar et al. 2008; Lee and Gautschi, 2006) Src Implication in Metastasis Over -expression of Src occurs in many solid tumors including colon, breast, bladder and pancreas (Cartwright et al. 1994; Egan et al. 1999; Lutz et al. 1998) In addition, elevated Src expression in tumors may be related to poor prognosis (Aligayer et al. 2002) (Figure 1 2). A number of r ecent s tudies have shown that Src tyrosine kinase may play an important role in tumor progression and metastasis. For example, i ncrease of Src activity was observed in metastatic lesions compared to the primary tumors (Termuhlen et al. 1993) It has also been shown that increase a kinase activity in tumors is correlated to Duke tumor stages, size and metastases (Aligayer et al. 2002) suggest ing that Src activation may contribute to the progression of cancer cells toward a metastatic phenotype by promoting survival, proliferation, migration and invasion (Cartwright et al. 1994; Cartwright et al. 1990) Src kinase activity is also apparently related to the process of angiogenesis through the regulation of VEGF expression (Schenone et al. 2007) as well as VEGF -mediated vascular permeability (Park et al. 2007) Based on these observations, Src tyrosine kinase appears to be a reasonable tar get to treat metastatic disease Src Family Src is a counterpart of the transforming gene of the Rous sarcoma virus (vSrc) in normal cells (Martin, 2001) It is an important m ember of the Src family. The Src family is composed of non receptor protein tyrosine kinases which play critical roles in a variety of cellula r signal transduction pathways, regulating diverse processes such as cell division, motility, adhesion,

PAGE 19

19 angiogenesis, and survival (Martin, 2001) Constitutively activated v ariants of Src family kinases, including the viral oncoproteins vSrc, are capable of inducing malignant transformation in a variety of cell types (Kadono et al. 1998; Tavoloni et al. 1994) There are nine member s in this family: Src, Yes, Fyn, Lyn, Lck, Hck, Blk, Fgr, and Yrk (Martin, 2001; Toyoshima et al. 1987) Src, Fyn, and Yes are expressed in most tissues. All Src fa mily kinases are comprised of a poorly conserved amino termi nal membrane localization sequence also known as t he SH4 (Src homology 4) domain an SH3 domain, an SH2 domain, a tyrosine kinase domain, and a regulatory sequence (Frame, 2002) (Figure 1 3 ). Members o f the family share similar structures and may have redundant functions in cells. Src The best studied member of the Src family is Src which is a 60 kD cytoplasmic protein tyrosine kinase Src tyrosine kinase is normally held in an inactive state by the interaction between phosphorylated tyrosine 527 (p Y527) in the C terminal tail and the SH2 domain (Thomas and Brugge, 1997) T he interaction s among the SH3 SH 2 and the catalytic domain s also contribute to stabilizatio n of the structure Activation of the kinase occurs by either a utophosphorylation at Y416 or dephosphorylation at Y527 by C -terminal Src Kinase (CSK) (Thomas and Brugge, 1997) (Figure 1 4 ). Src Functions Src is important in embryonic and developmental stages of life. However in the adult, the function of Src is still not clear. Src is usually expressed at low levels in most cell types and remains in an inactive conformation in the absence of appropriate extracellular stimuli. It has been found to be associated with cellular membranes. Studies show that it is involved in signal transduction events regulating cell growth and proliferation via activated growth factor receptors. Src also plays a role in regulatin g cytoskeletal structure and motility (Thomas and Brugge, 1997) Src knock -out mice develop normall y without severe abnormalities except for a non -

PAGE 20

20 lethal defect in osteoclast function (Soriano et al. 1991) Therefore, the development of Src inhibitors as anti -cancer agents is attract ive because they are likely to have limited side effects in normal tissue. The R ole of Src in the M etastatic P rocess In cancer cells Src is found to be involved in several cellular functions associated with the important steps of the metastatic cascade. These include cell survival, proliferation, migration, invasion and in duction of angiogenesis (Figure 1 5 ) (Saad, 2009) Proliferation and Survival Proliferation is an important step in metast asis. When the tumor cells reach a secondary site only a small portion grow s and proliferate s to form harmful metastatic lesions. Src has been shown to contribute to cell growth both in vitro and in vivo (Frame, 20 02; Moasser et al. 1999) Src is required for inducing cell proliferation by several growth factor receptors such as EGF and PDGF. Src stimulates DNA synthesis when these growth receptors are activated (Broome an d Hunter, 1996; Garcia et al. 1991; Zheng et al. 2005) Src also has been found to regulate the G2/M phase transition of the cell cycle (Basu and Cline, 1995; Roche et al. 1995; Taylor and Shalloway, 1996) and to rescue cells from apoptosis (Khwaja et al. 1997) For example, in anoikis resistant lung carcinoma cells Src was observed to be up regulated, suggesting that Src may contribute to cancer cell survival during dissemination and metastasis (Wei et al. 2004) Cell Migration and Invasion In addition to cell growth and survival, Src has also been found to be involved in cell migration and invasion. A cell mus t be able to continuously remodel focal complexes into focal adhesions, and vice versa, to migrate. Src as well as focal adhesion kinase ( FAK ), are important regulators of focal adhesion turnover. Src generally causes a reduction of focal adhesions and d ecreased cell adhesion (Parsons and Parsons, 1997; Timpson et al. 2001) In addition Src -/

PAGE 21

21 fibroblasts from knockout mice showed decreased motility compared to wild type fibroblasts. This characteristic was show n to be reversible by re -introducing Src (Kaplan et al. 1995; McLean et al. 2000; Schwartzberg et al. 1997) Tumor cell invasion involves the proteolytic degradation of ECM components by proteases secreted by th e cells. Src is also important in this process. For example, evidence shows that Src promotes invasion by forming Src -FAK complexes. These complexes enhance the expression of MMPs, uPAR and calpain that help degrade ECM proteins and provide proteolytic cleavage (Hauck et al. 2002; Hiscox et al. 2006; Pongchairerk et al. 2005) Adhesion Cancer cells exhibit dysfunctional adhesion systems that aid their ability to metastasize. Cell cell adhesion normally acts t o hold cells together and is indispensable for the proper organization of tissue s and organs in the human body. Src was found to deregulate the expression of E -cadherin ( a major adhesion molecule ) by activating the MEK/ERK and MLCK/myosin pathway. Deregu l ation of E -cadherin by Src over -expression can cause normal epithelial cells to change to a malignant phenotype and gain migratory and invasive abilities (Avizienyte et al. 2005; Felsenfeld et al. 1999; McLean et al. 2000) Cell -matrix adhesion s organize ce lls into tissues and coordinate their cellular functions. The matrix also provides a route for cell migration. It has been suggested that Src has a function in the contro l of focal adhesion turnover that affe ct s cell attach ment and promote s cell motility in the surrounding ECM by interacting with the integrin family (Playford and Schaller, 2004) Angiogenesis T umors can not grow beyond the size of a few millimeters without inducing their own blood supply via angiogenesis (Folkman, 1990) Src exp ression has been associated with the angiogenic process. For example, Src modulates the expression of proangiogenic factors such

PAGE 22

22 as VEGF (Eliceiri et al. 1999; Ellis et al. 1998; Folkman, 1990; Marx et al. 2001) a k ey contr ibutor to tumor cell induced vessel formation. In addition, Src can directly regulate the migration of endothelial and smooth muscle cells and promote endothelial tube formation in vitro (Kilarski et a l. 2003; Kumar et al. 2003) Kinase Inhibitors Protein kinases are enzymes that can transduce their signals and regulate cell functions by phosp h orylating their downstream substrates. There are three major categories of protein kinase inhibitors. Firs t are the serine/threonine kinase inhibitors, second are the tyrosine kinase inhibitors and third are the histidine kinase inhibitors which have recently been recognized (Johnson and Lewis, 2001) There are now many kinase inhibitors that have received FDA approval. For example, ZD6474 is VEGFR2 and EGFR inhibitor which is currently in phase III clinical trial s for a variety of cancers including non -small cell lung carcinoma (NSCLC) (Morabito et al. 2006) (Table 1 1) There are several strategies to synthesize kinase inhibitors including analogue synthesis, st ructure -informed design and fragment -based assembly strategies They can be used either alone or in combination (Zhang et al. 2009) Src Inhibitors SH2/SH3 Inhibitors ( AP22408, UCS15A) The SH2 and SH3 are protein recognition domains. The SH2 domain recognizes specific sequences containing phosphotyrosine and the SH3 domain binds to specific proline rich sequences (Thomas and Brugge, 1997) These domains guide Src to i ts substrates; thus, drugs designed to sterically block SH2or SH3 -mediated interactions inhibit specific subsets of Src protein protein interactions.

PAGE 23

23 Sr c -destabilizing Agents ( Geldanamycin, Herbimycin A, Benzoquinone A nsamycins) Th e destabilizing inhibi tors that can interfere with the association between Src and its associated molecular chaperone heat shock protein 90 (Hsp90) can increase the degradation of Src protein (Whitesell et al. 1994) ATP -comp etitive Sr c Kinase Inhibitors ( AZD0530, M475271, SKI 606) ATP -competitive inhibitors bind to the ATP binding pocket, then block ATP binding and inhibit phosphotransferase activity, thus inhibiting catalytic function. AZD0530 AZD0530, N (5 -chloro 1, 3 -benzodioxol 4 y l) 7 [2 (4 -methylpiperazin 1 yl)ethoxy] 5 (tetrahydro 2H -pyran 4 yloxy)quinazolin4 amine (Figure 1 6) is a small molecule, highly selective, orally available Src kinase inhibitor t hat interferes with auto phosphorylation at Y 4 1 6 (Hennequin et al. 2006) This agent has been shown to suppress the mobility and invasiveness of thyroid (Schweppe et al. 2009) lung (Purnell et al. 2009) and prostate cancer cells (Chang et al. 2008) in preclinical studies. It is currently undergoing phase II evaluation in a number of clinical settings including h ead and n eck s quamous cell carcinoma (Koppikar et al. 2008) and lung cancer (Lee and Gautschi, 2006) Project Significance Tumor metastasis remains the principal cause of treatment failure and indicator of poor prognosis in patients with cancer. Successful treatment of metasta ses is vital to improving survival. Src tyrosine kinase has been shown to play an important role in the metastatic cascade of tumor cells affecting survival, proliferation, migration, invasion, and induction of angiogenesis. Experiments proposed in this project are directed at examining the impact of a small molecule Src inhibitor on Src associated cellular characteristics and functions in vitro and in vivo The central goal of this project is to determine whether molecular modulation of Src

PAGE 24

24 signaling has the potential to improve therapy outcome by reducing the metastatic spread of tumor cells.

PAGE 25

25 Clearance X NO f Passing endothelial defense: Tumor cell apoptosis and clearance by endothelium derived NO Basement membrane b Invasion d Intravasation e Adhesion to blood vessel wall in distant organ g Extravasation h Migration Primary tumor a Blood vessel i Micrometastasis c A ngiogenesis j Metastasis Clearance X NO f Passing endothelial defense: Tumor cell apoptosis and clearance by endothelium derived NO Basement membrane b Invasion d Intravasation e Adhesion to blood vessel wall in distant organ g Extravasation h Migration Primary tumor a Blood vessel i Micrometastasis c A ngiogenesis j Metastasis Figure 1 1. The tumor metastatic process. It illustrates the sequential steps of the metastatic cascade including angiogenesis, migration, i n vasion, extra va s ation and proliferation. Reprinted with permission (Wang et al. 2005)

PAGE 26

26 Figure 1 2. Src tyrosine kinase in colorectal cancer. It illustrates Src protein leve l and activity correlates with the tumor progression. Reprinted with permission (Yeatman, 2004) Figure 1 3 The structure of Src f amily kin ases Modified with permission (Yea tman, 2004) SH4 SH3 SH2 Kinase domain Y Y

PAGE 27

27 Figure 1-4. Mechanisms involved in activation of Src tyrosine kina se. Src tyrosine kinase can be activated either by phos phorylation at Y416 (in chic ken cells), Y423 (in mouse cells) or Y419 (in human cells) or dephos phorylation at Y527. Reprinted with permission (Yeatman, 2004)

PAGE 28

28 Figure 1 5 Potential signal transduction pathways involving Src Src may mediate growth factor receptors to regulate the processes involved in growth survival, migration, invasio n and angiogenesis Reprinted with permis sion (Saad, 2009)

PAGE 29

29 Fi gure 1 6. Structure of AZD0530. http://www.selleckchem.com/ProductDetail.asp?ProdId=S1006.

PAGE 30

30 Table 1 1. List of FDA approved small molecule kinase inhibitors (Fabian et al. 2005) Name Code Trade Company Target Indication imatinib STI 571 Gleevec Novartis ABL, c KIT, PDGFR CML, GIST gefitinib ZD1839 Iressa AstraZeneca EGFR NSCLC erlotinib OSI 774 Tarceva Genentech/ OSI/Roche EGFR NSCLC nilotinib AM N107 Tasigna Novartis ABL CML (imatinib resistant) dasatinib BMS35482 5 Sprycel BMS Src, ABL, c KIT, PDGFR, Eph CML, melanoma sunitinib SU11248 Sutent Pfizer c KIT, PDGFR, VEGFR RCC, GIST (imatinib resistant) sorafenib BAY 54 9085 Nexavar Bayer PDGFR, c KIT, BRAF, VEGFR2 RCC, HCC lapatinib GW572016 Tykerb GSK EGFR, HER2 Breast cancer (HER2+) saracatinib AZD0530 AstraZeneca ABL, Src PrCa, BrCa, NSCLC cediranib AZD2171 Recentin AstraZeneca VEGFR NSCLC, kidney cancer, CRC vandetanib ZD6474 Zactima As traZenaca VEGFR, EGFR Multiple cancers (NSCLC) temsirolimus CCI 779 Torisel Wyeth mTOR RCC everolimus RAD 001 Afinitor Novartis mTOR RCC (after failure of Sutent/Nexavar)

PAGE 31

31 CHAPTER 2 THE EFFECT OF SRC IN HIBITION ON TUMOR CE LL FUNCTIONS I ntroduction Recent studies show that Src signaling may play an im portant role in cancer progression. For example, in s o me solid tumors including colon, breast, bladder and pancreas (Cartwright et al. 1994; Egan et al. 1999; Lutz et al. 1998) Src was found to be over -express ed compared to corresponding normal tissue s E levated Src expression or Src activity in tumors w as found to be related to poor prognosis (Aligayer et al. 2002) At the cellular level, s tudies have demonstrated that Src kinase activity may correl ate with t he malignant potential of cells In particular Src has been shown to contribute to ce ll growth (Frame, 2002; Moasser et al. 1999) and to be involved in regulating cell motility and invasiveness which are important cell functions related to the metastatic process (Kaplan et al. 1995; McLean et al. 2000; Schwartzberg et al. 1997) Several approaches to Src inhibition have been considered (Chapter 1). One of these Src kinase inhibitions was investigated in this study. AZD0530 belongs to a class of anilinoquinazoline drug s As noted above, i t is a small molecule, which has high affinity and specificity for the tyrosine kinase domain of Src (Hennequin et al. 2006) This drug can inhibit Src kinase activity by interfering with Src phosphorylation at tyrosine 4 19human/423-mouse (Hennequin et al. 2006) The IC50 for Src kinase inhibition is about 0.0027 M AZD0530 selectivity for Src kinase is~1000 -fold greater than for VEGFR 2 and ~100-fold greater than for c -Kit and EGFR (Hennequi n et al. 2006) In humans, t he half life of AZD0530 is about 36 hr (Neil 2008). In other species t he half life of this drug is 5 to 6 hrs in rat and 7 19 hrs in dog the volume of distribution is 10 L/Kg in rat and11.6 2.5 in dog and the c learance i s about 1 (L/h)/Kg in rat and 0.7 0.1 in dog (Hennequin et al. 2006) The drug is currently on phase I/II clinical trials i ncluding for h ead and n eck s quamous cell carcinoma (Koppikar et al. 2008) and

PAGE 32

32 lung cancer (Lee and Gautschi, 2006) It is orally available clinically in a form requiring only one dose per day. T he side effect s are mild and include potential rash and muscle pain (Neil, 2008) Several human and mouse tumor model s including the KHT (a mouse fibrosarcoma) SCCVII (a mouse squ amous cell carcinoma), 4A4 (a human breast tumor) and 2C5 (a human breast tumor) were used in this project. All of them are known to metastasize to distant organs T he KHT mouse sarcoma is a highly aggressive tumor which arose spontaneously in a C3H/Km m ouse in 1967 (Kallman et al. 1967) Donald et al showed 100% of the mice died within about 30 days from metastases following subcutaneous tumor implantation. W hen the primary tumor wa s controlled by irradi ation the mice died of extensive metastatic disease(Baker et al. 1981) A ttempts to control KHT lung metastases by irradiating the lungs re sulted in mice d ying fr om ovarian and renal metastases co n firmed to have disseminated from the lung nodules (Siemann and Mulcahy, 1984) This model thus may mimic highly malignant and metastatic diseases presented clinically, in which controlling the development of tumors at distant sites is necessary to impr ove treatment outcomes. In this chapter, the potential roles of Src involved pathways were examined with respect to cell functions associated with metastatic cascades, using AZD0530 induced inhibition. These cellular functions included cell proliferation migration, invasion and adhesion, which were evaluated in the four tumor cell types described above. Materials and Methods Tissue Culture Murine KHT sarcoma cells (Kallman et al. 1967) and SCCVII squamou s cell carcinoma cells were grown in alpha minimal essential medi u m ( -MEM) supplemented with 10% fetal b ovine serum (FBS) and 2 mmol/L L -glutamine. Human breast tumor cell lines 4A4 and 2C5

PAGE 33

33 were grown in Dulbecco's Modified Eagle (DMEM) med iu m supplemented with 10% FBS and 2 mmol/L L -glutamine. Drug Preparation AZD0530 (AstraZeneca, Macclesfield, U.K. ) was dissolved in DMSO at a concentration of 10 mM and subsequently diluted in PBS immediately before use in c oncentrations ranging from 0.1 to 10 M In all cases the final DMSO concentration was less than 0.5%. Western Immunoblotting Src and pSrc, wer e assayed by western immunoblotting in KHT, and 4A4 cells Focal a dhesion k inase ( FAK ), pFAK, Stat 3, pStst3, AKT, pAKT, ERK, pERK and Met were also as sayed by w estern immunoblotting in KHT cells. Tumor cells were grown in 10 0 mm dishes. A fter treatment w ith 0.5 10 M AZD0530 for 24 hr the cells were washed with cold PB S twice, harvested in RIPA buffer (50 mM HEPES, pH 7.4; 150 mM NaCl; 1% Triton X 10; 0.1% SDS; 0.5% sodium deoxycholate; 1 M sodium orthovanade; 5 M EDTA; 5 M sodium fluoride) containing a 1:20 dil ution of mammalian protease inhibitor cocktail containing 4 -(2 aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E 64, bestatin, leupeptin, and aprotinin. ( Sigma -Aldrich St. Louis, MO ), then collected and centrifuged at 14000 rpm (4C for 10 min). Total protein conc entration was measured by Micro BCA Protein Assay (Pierce Biotechnology Rockford, IL ). The cell lysates were subjected to SDS -polyacrylamide gel electrophoresis, and the proteins were transferred to PVDF membranes ( Amersham Biosciences Piscataway, NJ). The membranes were blocked in 5% BSA buffer for 1 h r at room temperature, and then the blots were incubated at 4C overnight with the Src, pSrc [ Y423-mouse / Y41 6 chicken/Y419-human ], FAK, p FAK [Y861] (Biosource International, Inc. Cama rillo, CA ), p FAK [Y397] (CHEMICON International, Inc. Billerica, MA ), Stat 3 (Cell Signaling

PAGE 34

34 Technology, Inc. Danvers, MA ), pSt a t3 (Cell Signaling Technology, Inc. Danvers, MA) AKT (Cell Signaling Technology, Inc. Danvers, MA), pAKT (Cell Signaling Technol ogy, Inc. Danvers, MA ), ERK (Cell Signaling Technology, Inc. Danvers, MA ) pERK (Cell Signaling Technology, Inc. Danvers, MA ) and Met (Cell Signaling Technology, Inc. Danvers, MA ), followed by incubation for 1 hr at room temperature with secondary antibody (species -specific horseradish peroxidase conjugated). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences). Cell Growth KHT cells (5x103) were seeded into triplicate 6 -well plates and exposed to 0 10 M AZD0530 24 hr after cell seeding Plates were trypsinized and cells counted daily for a period of 6 days. Two separate experiments were carried out. SCCVII cells (5x103) were seeded into triplicate 6 -well plates and exposed to 0 10 M AZD0530. Plate s were trypsinized and cells counted daily for a period of 6 days. 4A4 cells (2x104) were seeded into triplicate 6 -well plates and exposed to 0 -10 M AZD0530. Plates were trypsinized and cells counted every other day for a period of 8 days. 2C5 cells (2 x104) were seeded triplicate into 6 -well plates and exposed to 0 -10 M AZD0530. Plates were trypsinized and cells counted counted every other day for a period of 8 days Clonogenic Cell Survival A ssay KHT cells were treated with 0 10 M AZD 0530 for 24 hr Then cells were collect ed and plated at various densities (5 dishes per density) in 60 mm dishes. Two weeks later, the dishes were stained with crystal violet and the colonies that contain ed more that 50 cells were counted at 5x magnification under a l ight microscope. The ratio of surviving colonies to the number of

PAGE 35

35 cells plated at that density was calculated as the plating efficiency (PE). The PE for each treatment group divided by the PE for control cells was then calculated to give the surviving fr action of cells for each treatment Cell Cycle Analysis KHT SCCVII, 4A4 and 2C5 cells were treated with 0 5 M AZD0530 for 24 hr or 48 hr T he cells were then harvested, fixed with ethanol, treated with RNase stained with propidium iodide, and analyzed by flow cytometry. The proportion of cells in the variou s phases of the cell cycle was det ermined using Modfit software. Cell Migration Scratch a ssay A scrape of uniform width (2 mm) was made in a monolayer of confluent KHT or SCCVII cells prior to tre atment with 0 10 M AZD0530. Tumor cells were exposed to drug for 24 hr, then were fixed and stained with crystal violet. Cells entering the denuded area were counted at 10X magnification in four randomly selected fields in each well. Modified Boyden-chamber c ell migration ass ay KHT cells (1x105), SCCVII cells (7x 104), 4A4 cells (3 x105) and 2C5 cells (3 x105) were seeded in modified Boyden chambers (BD Biosciences San Jose, CA ) and treated with 0 5 M AZD0530. After 24 hr cell s were fixed and stained with crystal violet Cell s on top of the 8 M -por e membrane were removed using cotton swabs. Cells migrating through the membrane were counted at 20X magnification using 12 randomly selected fields on each filter. Cell Invasion Assay KHT cells ( 5x105), SCCVII cells (3x105), 4A4 ( 5x105), and 2C5 cells (5x105) suspended in 0.1% FBS medium were seeded in Matrigel Invasion Chambers (BD Biosciences San Jose, CA ) and treated with 0 5 M AZD0530. The lo wer chamber contained medium with 10% FBS.

PAGE 36

36 After 24 or 72 hr the cells and M atrigel in the upper chamber were removed using cotton swabs. Cells that invad ed through the membrane were fixed and stained with crystal violet and counted at 20X magnification in 12 randomly selected fields on each filter ELISA MMP -9 KHT cells (1.6x106) were seeded in 60 mm dishes and then treated with 0 10 M AZD0530. After 24 hr, supernatants were collected, and the concentrations of total matrix metalloproteinase 9 ( MMP 9 ) and total matrix metalloproteinase 2 (MMP 2 ) in the media were determined by ELISA according to the manufacturers protocol (R&D Syste ms Minneapolis, MN ). Gelatin Z ymography KHT cells (1.6x106) were seeded in 60 mm dishes and then treated with 0 10 M AZD0530. After 24 hr, supernatants were collected and the proteins were separated on a 10% SDS -PAGE gel containing 1 mg/ml gelatin. Aft er electrophoresis, the gel was washed for 15 min in a rinse buffer containing 2.5% Triton X 100 and then incubated overnight in the same buffer at room temperature. After washing with deionized water, the gel was incubated for an additional 24 hr at 37C in an incubation buffer containing calcium and zinc. The gel was s tained with Coomassie brilliant blue R 250 and visualized using a transilluminator and scanned. Cell Deta chment Assay KHT cells were seeded into 6 -well plates (3x105 /well) and incubated a t 37C under 5% CO2 in humidified air for 24 hr prior to exposure to AZD0530 (05 M). Twenty -four hr later the media were collected and the number of detached cells was counted using a hemocytometer

PAGE 37

37 Immunofluorescence Microscopy KHT cells were cul tured on chamber -slides, and treated with 5 M AZD0530 for 24 hr. The cells were fixed in 3.7% formaldehyde and permeabilized in 0.5% Triton X 100 in TBS. Cells were labeled with primary antibodie s raised against Src, pSrc [Y423 ] (Cell Signaling Technology, I nc. Danvers, MA), FAK and pFAK [Y861] (Biosource International) and then stained with the appropriate secondary antibody conjugates to Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Inc.). Images of the stained cells were captured using a Leica scanning confocal microscope. Statistical A nalysis The statistical significance of differences of in vitro data was determined by Students t test (two tailed) ; in vivo data were analyzed by Wilcoxon rank sum test. Results Src Signaling T he effect s of A ZD0530 on protein expression and phosphorylation of Src in KHT and 4A4 cells are shown in F ig ure 2 1 AZD0530 did not affect the leve l of Src protein expression, but did reduce the autophosporylation of Src at tyrosine 4 23-mouse/419 -human in a dose depend ent manner in each cell line AZD0530 treatment also affected FAK signaling. When KHT cells were exposed to 10 M AZD0530 for 24 hr phospho rylation of FAK at t yr osine 861, a Src dependent phosphorylation site (Brunton et al. 2005) was reduced (Fig ure 2 2 ), but as expected, no change in phospho rylation was seen at t yr osine 397, a Src independent site Met, a tyrosine kinase receptor has also been shown to be critically involved in cancer progression and metastases (Gentile et al. 2008) To evaluate whether Src inhibition affected Met expression, Met protein levels were measured in AZD0530 treated KHT cells. The results (Fig ure 2 2 ) indicated reduction of Met protein 24 hr after exposure to 10 M AZD0530. pStat3, pAKT, and

PAGE 38

38 pErk were also examined as potential Src downstream effectors were also examined. However, no change was detected after a 24 hr AZD0530 treatment. Cell G r owth KHT sarcoma cell growth over a six day period in the presence of AZD0530 was slowed in a dose dependent manner (Fig ure 2 3 ). Cell cyc le analysis (Fig ure 2 5 ) indicated that compared to control cells the percentage of KHT cells treated with 2.5 or 5 M AZD0530 increased in the G1 phase from ~51 to ~83%, and the percent age of G2 and S phase decreased from ~13 to ~7% and ~35 to ~11% respectively after a 24 hr exposure In 4A4 and 2C5 cells neither growth nor cell cycle changed dramatically in response to AZD0530 treatment (Fig ure 2 3) Cell Migration T o investigate wheth er AZD0530 treatment could inhibit tumor cell motility, both a scratch and a mo dified B oyden-chamber cell migration assa y were used In the scratch assay KHT cell migration into the denuded area (Fig ure 2 6 ) was reduced by ~2 5 % by a 0.5 M dose of AZD0530 and ~ 6 0% by a 2.5 M dose of drug. I n the mo dified Boyden-chamber (Fig ure 2 7 ) concentrations greater than 0.1 M AZD0530 significantly impaired the ability of K H T sarcoma cell to migrate through a porous membrane. Sim ilar result s were observed in SCCVII, 4A4 and 2C5 cells. Cell Invasion AZD0530 treatment significantly reduced the ability of KHT cell to invade through a matrigel layer (Fig ure 2 8 ). Th e results indic ate increasing inhibition of KHT cell invasion with increasing doses of AZD0530 ranging from 0.5 to 5 M. Similar results were observed in SCCVII, 4A4 and 2C5 cells.

PAGE 39

39 MMP -2 and MMP -9 Secretion Total MMP 2 and MMP 9 protein level including proactive and active form s w ere not significantly change d in KHT cells after 24 hr exposure to AZD0530 (Fig ure 2 9 ). MMP -2 and T otal MMP -9 Activities G elatin zymography analysis showed that the activity of MMP 9 was decreased in a dose dependent manner after 24 hr AZD0530 treatment in KHT cells However, MMP 2 activity was not change d (Fig ure 2 10). Cell Detachment KH T cells treated with AZD0530 developed a rounded shape a nd increasingly de tached from tissue culture plates in a dose dependent manner (Fig ure 2 11). The number of detached cells was increased by ~1.8 fold a t 2.5 M and ~2. 6 -fol d at 5 M. I mmunoflurescent staining of Src, pSrc ( Y423), FAK and pFAK ( Y 861) indicated a reduction in Src and FAK phosphorylation and a relocalization of pSrc and p FAK from the focal adhesions at the edge of cells to the cytoplasm followin g AZD0530 treatment (Fig ure 2 12). Discussion Accumulating evidence suggests that Src tyrosine kinase s may play critical roles in regulating cancer progression. Increase s of Src activity or o ver -expression o f Src have been detected in a variety of human tumors (Cartwright et al. 1994; Egan et al. 1999; Lutz et al. 1998) and associated with late stage disease (Wiener et al. 2003) Consequently, several Src inhibitors including AZD0530, BMS 354825, and SKI 606 are currently under investigation as novel therapeutic agents for the treatment of various type s of cancer (Rucci e t al. 2008) In the present study, the activity of one such i n hibitor AZD0530 was assessed in four tumor cell lines including two rodent tumor cell line s (KHT sarcoma and SCCVII lung carcinoma ) and two human tumor cell lines (4A4 and 2C5 breast tumor c ell lines).

PAGE 40

40 Westernblotting results showed that pSrc was inhibited by AZD0530 at 1 M in KHT cells and 0.5 M in 4A4 cells. It showed that different cell lines had different sensitivity to this drug. This was consistant with previous reports on thyroid (Schweppe et al. 2009) breast (Hiscox et al. 2006) and prostate cancer cells (Chang et al. 2008) Initial ex periments demonstrated that inhibition of cell proliferation by AZD0530 likely was due to an influence on the cell cyc le [accu mulation of cells in G1/S phase ], as the doses of drug used did not result in tumor cell cytotoxicity as measured by clonogenic cell survival in KHT cells These findings are similar to those reported by Schweppe et al in thyroid (C6 43, TPC1, BCPAP, and SW1736 cells) and prostate cancer cells (DU145 and PC 3) (Schweppe et al. 2009) However, in 4A4 and 2C5 cells, no effect on cell proliferation by AZD0530 w as observed and consistently, no ce ll cycle effect w as detected either. Sim ilar findings have also been reported in K1 tyroid cancer cells (Schweppe et al. 2009) These data also indicate that different cell types may hav e variable responses to thi s drug. T he prese nt investigation clearly demonstrates that both tumor cell migration and invasion were significantly impaired by AZD0530 treatment in a dose dependent manner in all four tumor cell lines used in this study These findings are consistent with previous reports on AZD0530 on cell motility and invasion (Purnell et al. 2009; Schweppe et al. 2009) Import antly in KHT cells cel l migration and invasion are affected at doses which had litt le effect on cell proliferation Met (Gentile et al. 2008) a tyrosine kinase receptor, also is recognized as a crucial molecule involved in cancer progression and metastases It has b een shown to regulate Src activity in a variety of cancer cells (Herynk et al. 2007; Mueller et al. 2008) Interestingly, Emaduddin (Emaduddin et al. 2008) showed that w h en Sr c activity wa s inhibited, M et phosphorylation was reduced in four colon cancer cell lines, indicating a reversed signal flow from Src to Met. In our

PAGE 41

41 study, western blot analysis shows that AZD0530 exposure decreases Met protein levels indicat ing tha t Src is involved in regulation of Met expression T he present findings also suggest that this AZD0530 action may contribute to its effect on the migration and invasion of tumor cells. Finally, tumor cell invasion may involve the proteolytic degradation o f ECM ( extracellular matrix ) components by metalloproteinases and MMP 9 and MMP 2 which are known to be regulated by Src (Wu et al. 2008) In the present studies MMP 9 and MMP 2 secretion levels were unchanged af ter AZD0530 treatment in KHT cells However, the activity of MMP 9 was decreased after AZD0530 treatment suggest ing that impairing MMP 9 activity by AZD0530 may contribute to the observed reduction in KHT cell invasion capacity The ability of cells to migrat e and inva de is dependent on their integrin-mediated interaction with the ECM which in turn is regulated by focal adhesion molecules. FAK is a non receptor tyrosine kinase and serves as a major component of focal adhesions. FAK can regulate the dyn amics of focal adhesion by interacting with Src (Mitra and Schlaepfer, 2006) Upon integrin engage ment, FAK is phosphorylated at tyrosine 397 and this allows FAK to recruit Src. Src subsequently phosphorylat es FAK at several sites including tyrosine 861. The present results show that AZD0530 treatment not only decrease s activated Src, but FAK phosphorylation at tyrosine 861 as well In addition, the immunofluorescent staining studies show that AZD0530 treat ment changes in the cell morphology and causes a relocalization of both p FAK and pSrc from the edge of tumor cells to the cytoplasm These observations along with the results of the detachment studies strongly suggest that the focal adhesions of tumor ce lls are significantly disrupted by AZD0530induced Src inhibition In summary t he results show ed that AZD0530, a specific Src inhibitor, has significant effects on key cellular processes associated with the metastatic cascade. The y further suggest

PAGE 42

42 that AZD0530 treatment may have an anti -metastatic effect on in vivo studies as well, which was studied in the next chapter.

PAGE 43

43 Figure 2 1. Effect s of AZD0530 on pSrc and Src expression in tumor cells after 24 hr treatment A) KHT ce lls B) 4A4 cells. The results shown are a representative experiment of three pSrc Actin Src c 0.5 1 5 10 M pSrc Actin Src c 0.5 1 5 10 M A B c 0.5 2.5 5 10 M pSrc Src Actin

PAGE 44

44 Figure 2 2. Effects of 24 hr exposure of KHT cells to AZD0530 on FAK, pFAK, Stat3, pStat3, AKT pAKT Erk, pErk and Met The results s hown are a representative experiment of two. pSrc (Y416) Src pStat3 (Y705) Stat3 pFAK (Y861) FAK act in pFAK (Y397) 0 0 10 10 M pAkt (S473) Akt pErk (T202/Y204) Erk 0 0 10 10 M 0 0 10 10 M

PAGE 45

45 Figure 2 3. C ell growth in the presence of AZD0530 of a variety of tumor cell lines The results shown are a representative experiment of two. day -2 0 2 4 6 8 10 12 14average cell number 1e+3 1e+4 1e+5 1e+6 1e+7 DMSO 0.5 uM 5 uM 10 uM 20 uM 2C5 day -2 0 2 4 6 8 10average cell number 1e+4 1e+5 1e+6 1e+7 DMSO 0.5 M 5 M 10 M 20 M day -2 0 2 4 6 8average cell number 1e+3 1e+4 1e+5 1e+6 1e+7 DMSO 0.5 uM 1.0 uM 2.5 uM 5 uM 10 uM KHT 4A4 Treatment Treatment Treatment

PAGE 46

46 Figure 2 4. KHT cell colonogenic survival after 24 hr exposure to AZD0530. Results represent two independent experiments.

PAGE 47

47 A B Control 0.5 mM 2.5 M 5.0 MDip G1: 40.95 % Dip G2: 16.10 % Dip S: 42.95 % Dip G1: 41.94 % Dip G2: 15.19 % Dip S: 42.86 % Dip G1: 41.03 % Dip G2: 15.94 % Dip S: 43.03 % Dip G1: 40.00 % Dip G2: 16.40 % Dip S: 43.60 % Fig ure 2 5 Effect of AZD0530 on tumor cell cycle distr ibution assessed by flow cytometry 24 hr after exposure to 0 5 M AZD0530. A) in KHT cells B) in 4A4 cells. The results shown are a representative experiment of three Control 0.5 M Dip G1: 51.56% Dip G2: 13.27% Dip S: 35.17% Dip G1: 62.10% Dip G2: 9.96% Dip S: 27.94% 2.5 M Dip G1: 81.86% Dip G2: 6.95% Dip S: 11.19% Dip G1: 83.07% Dip G2: 6.34% Dip S: 10.58% 5.0 M

PAGE 48

48 A B KHT Migration Assay vs AZD0530Drug Concentration 0.001 0.01 0.1 1 10 100Mean cells/field 0 100 200 300 400 0 0.5 2.5 5* ** ** **( uM ) SCCVII Migration Assay vs AZD0530Drug Concentration 0.001 0.01 0.1 1 10 100Mean cells/field 0 50 100 150 200 250 300 350 0 0.5 2.5 5* ** ** ( uM ) Figure 2 6. Effect s of AZD0530 on tumor cell mig ration assayed by scratch assay after 24 hr drug treatment A) Representative pictures of scratch assay s B) Tum or cell migration was inhibited by AZD0530 in a dose dependent manner (mean cells per field SE for three experiments) p <0. 05, **p <0.0 1 vs control Control 0.5 uM 2.5 uM

PAGE 49

49 KHT Migration Assay AZD0530 ( uM ) .001 .01 .1 1 10 100% migrated cell 0 20 40 60 80 100 120 Exp. 1 Exp. 2 Exp. 3 0 0.5 5 2.5 SCCVII Migration Assay AZD0530 ( uM ) -1 01234567% migrated cell 0.0 .2 .4 .6 .8 1.0 1.2 Exp.1 Exp.2 Exp.3 0.5 2.5 4A4 Migration Assay AZD0530 ( uM ) .001 .01 .1 1 10 100% migrated cell 0 20 40 60 80 100 120 Exp. 1 Exp. 2 Exp. 3 0 0.5 5 2C5 Migration Assay AZD0530 ( M ) .001 .01 .1 1 10 100% Migrated Cell 0 20 40 60 80 100 120 Exp.1 Exp.2 Exp.3 Exp.4 0.5 2.5 5 0 0 Figure 27. Effect s of AZD0530 on tumor cell migration assayed by modified B oyden-chamber cell migration assay after 24 hr drug treatment Each bar represents one independent experiment.

PAGE 50

50 KHT Invasion Assay AZD0530 ( uM ) .001 .01 .1 1 10 100% invaded cell 0 20 40 60 80 100 120 Exp. 1 Exp. 2 Exp. 3 Col 5 0 0.5 5 2.5 SCCVII Invasion Assay AZD0530 ( uM ) -1 01234567% invaded cell 0.0 .2 .4 .6 .8 1.0 1.2 Exp.1 Exp.2 Exp.3 2.5 0.5 4A4 Invasion Assay AZD0530 ( M ) .001 .01 .1 1 10 100% invaded cell 0 20 40 60 80 100 120 Exp. 1 Exp. 2 Exp. 3 0 0.5 5 2.5 2C5 Invasion Assay AZD0530 ( M ) .001 .01 .1 1 10 100% invaded cell 0 20 40 60 80 100 120 Exp. 1 Exp. 2 Exp. 3 0 0.5 5 2.5 Figure 2 8. Effect s of AZD0530 on tumor cell invasion assayed by transwell inva sion assay after 24 hr drug treatment. Each bar represents one independent experiment.

PAGE 51

51 A B AZD0530 ( M) -2 0 2 4 6 8 10 12Secreted MMP-9 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Secreted MMP -9 Normalized to Control AZD0530 ( M) -2 0 2 4 6 8 10 12Secreted MMP-9 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Secreted MMP -9 Normalized to Control Figure 2 9. Effect s of AZD0530 on secreted MMP 2 and MMP 9 measured by ELISA in KHT cells after 24 hr treatment. A) MMP 2. B) MMP 9. Each bar represents one independent experiment.

PAGE 52

52 Figure 210. Effect s of AZD0530 on MMPs activity after 24 hr treatment. The results shown are a representative experiment of three Figur e 2-11. KHT cell detachment determined 24 hr after AZD0530 treatment. Data shown are the mean number of cells that detached per well from 6well plates ( SE for 3 experiments). **p<0.01 vs control

PAGE 53

53 Fiugre 2 12. Distribution of pSrc (Y4 23), Src, pFAK (Y861) and FAK a f ter 24 hr exposure to 5 M AZD0530 analyzed by immunofluorescent confocal microscopy. Red: Alexa Fluor 594 staining Src and FAK Green: Alexa Fluor 488 staining pSrc and pFAK Blue: DAPI staining nuclei

PAGE 54

54 CHAPTER 3 T HE EFFECT OF SRC INHIBITION ON IN VIV O TUMOR METASTSES Introduction As discussed in C hapter 1, the metastatic process is complicated and involves multi steps. When tumor cells begin to metastasize, they escape from the primary tumor, enter the blood vessels, survive in t he circulation, and arrest at distant sites. Then they leave the blood vessels by first adhering to the endothelial cells and then invading between the se cells as well as smooth muscle cells, breaking down the ext ra cellular matrix At the distant sites, they enter the parenchyma and resp ond to the microenvironment to proliferate and form colonies (Widel and Widel, 2006) The key steps in this process include cell migration, invasion, adhesion and proliferation. The in vitro studies in Chapter 2 showed that the Src inhibitor AZD0530 can impair some key steps in this process To understan d how these interruption s of cell functions affect tumor metastases in vivo two kinds of experimental model s were used The fi rst model was the experimental metastas e s model. The experimental metastases model bypas s es the initial steps of the metastatic cascade, i.e. tumor cell s escaping from the primary tumor intrava sat ing into the vasculature It instead begins with introdu ct ion of tumor c ells into the circulation by tail vein injection, where they can arre st at a second site and extrav a sate from the vasculature In order to survive at the new site and start prolifera ting they need to be compatible with the local microenviro nment (Takahashi, 1999) One advantage of this model is that it is relative ly simple compared to a spontaneous metastases model since it contains fewe r steps. Also, the tumor cell number injected into the circulation is known in each mouse so there is less variation th a n when tumor cells shed from a growing primary tumor. The second experimental model is the spontaneous metastases model. It may be a rgued that this is a more physiologically relevant

PAGE 55

55 metastatic model since i t contains all steps in the metastases process (Ito et al. 2002) These models allow the investigation of metastasis from different perspe ctives. Bot h experimental and spontaneous metastas i s experiments were carried out in the KHT sarcoma mouse model. This model is well established and characterized i n our lab. For the experiment al metastas e s study KHT cells were injected via the tail v ein a nd the mice were monitered for 18 days. Then the mice were euthanized and the lungs were removed for examination. In the spontaneous metastases experiment s the KHT cells were injected intra muscularly to allow primary tumor growth Then the mice w ere euthanized 18 days later and the lung s were removed for examination. The effect s of AZD0530 (in vivo half life 16 hr) on tumor cell growth and ability to form secondary tumor deposit s were evaluated by examining the number and size of the lung metasta tic nodules in these two models In the experimental metastasis model, f our tr eatment schedules including one four and 18 day treatments were designed. The different schedule s helped determined that extravasation and establishment of lung nodules were a ffected by AZD0530. To fu rther investigate th ese mechanism s the effect of AZD0 530 on interactions between tumor and endothelial cell s were evaluated by a cell cell adhesion assay In addition, in vitro studies on endothelial cell functions related to an giogenesis, and secretion of proa n giogenic factors such as VEGF by the cancer cells were examined. The effect of AZD0530 on tumor cell induced angiogenesis also was exami ned in vivo Materials and Methods Drug P reparation AZD0530 difumarate was dissolv ed in a mixture of 0.5% hydroxypropyl methyl cellulose and 0.1% polysorbate (Tween 80) at a dose at 10mg/kg or 25 mg/kg body weight for mice. The

PAGE 56

56 drug was store d at room temperature in the dark and prepare d as a fresh suspension every 14 days. Animals Al l research was governed by the principles of the Guide for the Care and Use of Laboratory Animals (USPHS), and approved by the University of Florida Institutional Animal Care and Use Committee (Gainesville, FL). S ix -to -eight -week old female C3H mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were maintained in a specific pathogen-free environment (University of Florida Health Science Center, Gainesville, FL), with food and water provided ad libitum. Experimental M etastases (1) Untreated K HT cells or cells treated with 1 or 5 M AZD0530 for 24 hr were injected (1x 105) into C3H mice via the tail vein ( 10 mice /group). Three weeks later, the mice were euthanized and the lun gs were removed and fi xed in Bouins solution for 24 hr. The number of lung nodules was count ed and the s izes were determined using an eyepiece with a vernier scale under a light microscope. Experimental M etastases (2) KHT cells were injected (2x 103) into C3H mice via the tail vein ( 10 mice /group) One group was kept as control. A second group received 10 mg/kg AZD0530 daily by gava ge. T he third group received 25 mg/kg AZD0530 daily by gava ge. Eighteen days after tumor cell injection the mice were euthanized and the lun gs were removed and fi xed in Bouins solution for 24 h r. The number of lung nodules was count ed and the sizes were determined using an eyepiece with a vernier scale under a light microscope.

PAGE 57

57 Experimental M etastases (3 ) KHT cells were injected (2x 103) into C3H mice via the tail vein ( 10 mice /group) Three treatment groups were evaluated All three received a 10 mg/kg dose of AZD0530 immediately after tumor cell injection. The first group received no additional drug treatment. The second and third groups were given 3 or 17 additional 10 mg/kg doses of AZD0530 administered on a daily ba sis. Eighteen days after tumor cell injection the mice were euthanized and the lun gs were removed and fi xed in Bouins solution for 24 hr. The number of lung nodules was count ed and the sizes were determined using an eyepiece with a vernier scale under a light microscope. Spontaneous M etastases KHT cells (1x 105) were injected into the leg of C3H mice intramuscularly at day 0 for three groups ( 10 mice /group ). Starting on day 3 mice were given daily doses of either 10 or 25 mg/kg AZD0530 on a daily basi s. At day 7 the mice received one dose 35 Gy irradiation in an attempt to control the primary tumor. At day 18, the mice were euthanized and the lun gs were removed and fi xed in Bouins solution for 24 hr. The number of lung nodules was count ed and the s izes were determined using an eyepiece with a vernier scale under a light microscope. Maintenance of Cell Line s and Tissue C ulture I n V itro Human microvascular endothelial cells of lung (HMVEC -L) were grown in EBM 2 -MV media supplemented with 5% FBS SDSPAGE Sample Preparation and Immunoblot A nalysis (1) HMVEC L cells were treated with various concentrations of AZD0530 for 24 hr. Then the cell lysates were collected and subjected to SDS -polyacrylamide gel electrophoresis, and analyzed by Western i mmunobl ot fo r pSrc and Src as described in C hapter 2.

PAGE 58

58 (2) HMVEC L cells were treated with 10 M AZD0530 for 1 4 or 24 hr respectively. Then the cell lysates were collected and subjected to SDS -polyacrylamide gel electrophoresis, analyzed by i mmunoblot fo r pSrc and Src as described in C hapter 2. (3) KHT cells were treated with AZD0530 for 24 hr. Then the drug was removed by changing with fresh media, and after 24 hr, 48 hr and 72 hr the cell lysates were collected and subjected to SDS polyacrylamide gel electrophoresis, analyzed by Western i mmunoblot for pSrc and Src as descr ibed in Chapter 2. HMVEC -L M orphology HMVEC -L cells (2x 104) were seeded in triplicate into 6 -well plates and exposed to varying concentrations of AZD0530 as indicated After 24 hr, the cells were observed under light microscope. Cellular Growth Assay HMVEC -L cells (2x 104) we r e seeded in triplicate into 6 -well plates and exposed to varying concentrations of AZD0530 as indicated Cells were trypsinized and quantified with a hemocytometer daily over a period of 7 days. Tube Formation Assay (1) HMVEC -L cells were pre-treat ed with 0 10 M AZD0530 for either 24 hr or 48 hr. Then the pre treated cells (6x104) were plated in triplicate on Matrigel pre coated 24 well plates (BD Biosciences, San Jose, CA). The cells were incubated for 24 hours at 37 C. Then tube formation was observed and photographed. Tube Formation Assay (2) HMVEC -L cells (6x104) were plated in triplicate on Matrigel pre -coated 24 -well plates (BD Biosciences, San Jose, CA) in the presence of 0 10 M AZD0530. Tube formation was observed and photographed at 24 hr, 48 hr a nd 72 hr later.

PAGE 59

59 Tube Formation Assay (3 ) HMVEC -L cells (6x104) were plated on Matrigel pre -coated 24 -well plates (BD Biosciences, San Jose, CA). The cells were incubated for 6 hours at 37 C until they form ed complete tubes. Then 010 M AZD0530 was added into the wells in triplicate. Tube formation was observed and photographed at 24 hr, 48 hr and 72 hr later. Migration A s s ay (1) HMVEC -L cells (5x102) were seeded in modified Boyden chambers (BD Biosciences San Jose, CA) and treated with 0 10 M of AZ D0530. After incubation for 24 hours at 37 C c ell s on top of the 8 m -por e membrane were removed using cotton swabs. Migrated c ell s on the other side of the membrane were fixed, stained with crystal violet and counted. Migration A ssay (2) AZD0530 (0 1 0 M) p re treated HMVEC -L cells (5x102), were seeded in modified Boyden-chambers (BD Biosciences San Jose, CA ) and treated with various concentrations of AZD0530. After incubation for 48 hours at 37 C stationary c ell s on top of the 8 m -por e membrane w ere removed using cotton swabs. Migrated c ell s on the other side of the membrane were fixed and stained with crystal violet. Cells that migrated through the membrane were counted at 5X magnification Cell-cell Adhesion A ssay: AZD0530 pre treated KHT cell s (106 /well) were seeded on a confluent HMVEC L cell monolayer in 6 -well plate. After 2 hr incubation at 37C under 5% CO2 in humidified air the plates were washed with PBS and the KHT cells which had not attached to the HMVEC -L monolayer were collected and counted using a hemocytometer.

PAGE 60

60 ELISA KHT cells (1.6x106) were seeded in 60 mm dishes and then treated with 0 10 M AZD0530. After 24 hr, supernatants were collected, and the concentrations of VEGF in the media were determined by ELISA according to t he manufacturers protocol (R&D Systems. Minneapolis, MN ). Intra dermal A ssay (1) Untreated KHT cells or cells treated with 1 or 5 M AZD0530 for 24 hr were injected (1x 105) intra -dermal ly in 4 ventral locations of the nude mice (3 mice /group). On day 4 the skin containing the 4 nodules was r e moved and vessels counted under a dissection microscope Intra dermal A ssay (2) KHT cells were injected (1x 105) intra dermally in 4 ventral locations of the nude mice (3 mice /group) for 3 groups The first trea tment group received 10 mg/kg AZD0530 daily by gavage T he second treatment group received 25 mg/kg AZD0530 daily by gavage On day 4 the skin containing the 4 nodules was r e moved and vessels counted under a dissection microscope. Results Effect of AZD0 530 Treatment on Lung Colony F ormation in a n Experimental Metastases M odel To determine if AZD0530 can affect the in vivo metastatic activity of KHT cells, tumor cells were pre treated with the agent for 24 hr and then injected via the tail vein. Three we eks later, the resultant number of lung nodules was counted. T he data showed a significant reduction in the number of lung colonies formed by AZD0530 pretreated tumor cells compared to control (Fig 3 1 b ). However, no significant change in size w as detect ed (Fig 3 1 c ).

PAGE 61

61 To de termine if AZD0530 treatment ha d similar effect s on lung colony formation when the mice were treated with the drug, another experiment was carried out. The mice were injected with un treated KHT cells via tail vein, and then treated with AZD0530 daily. Three weeks later the number of lung nodules was counted. The number of lung nodules was significantly reduced in the treated groups compared to control. However, no signif icant difference was detected between the 10 mg/kg group and 2 5 mg/kg groups (Fig 3 2). To further study the anti -metastatic effect of AZD0530, a third type of experimental metastases experiment was designed. Three different treatment schedules were evaluated. The mice in the first drug treatment group received on ly one dose of AZD0530 immediately after tumor cell injection. The mice in the second drug treatment group received 3 more daily treatment s after the first given dose. And the mice in the third drug treatment group were treated with drug daily until the end of the experiment. The data showed that t he median of lung col onies in the control group was 13, in group 2 (one dose tre atment group) was 6.5 in group 3 (four day treatment group) was 4.5 and in group 4 (daily treatment group) was 5.5 There was a significant difference in the number of lung colonies in all treated groups compared to control. However, there was no s ignificant difference between drug -tre ated groups (Fig 3 3) Effect of AZD0530 Treatment on Lung Colony F ormation in a Spontaneous Met astases M odel To study the anti -metastatic effect of AZD0530, a sponta neous metastases experiment was also carried out. KHT tumor cells were injected intramuscularly in the mice to form primary tumors. AZD0530 treatment was started three days after tumor cell injection. No significant difference was detected between the AZD0530 treated animals and the untreated control mice (Fig 3 4).

PAGE 62

62 Effect of AZD0530 on Src P hosporylation in HMVEC -L The effect of AZD0530 on Src phosphorylation in HMVEC L was examined by Western immunoblot ( Fig 3 5 ). The pSrc was decreased after 24 hr exposure to 1 10 M AZD0530 and this decrease wa s dose dependent Src expression level was not c hanged after drug treatment (Fig 3 5a ). Fig 3 5b show s that pSrc decrease could be detecte d as early as 15 min after drug treatment when exposed to 10 M AZD0530. Effect of AZD0530 on HMVEC -L M orphology The potential effect s of AZD0530 on HMVEC L morphology w ere examined under the light microscope after 24 hr exposure to AZD0530. No dramatic c hange in cell morphology was observed after this interval of 1 10 M AZD0530 treatment ( Fig 3 6 ). Effect of AZD0530 on HMVEC -L G rowth HMVEC -L cell growth over an eight day period in the presence of AZD0530 was evaluated. There was no significant differenc e in the initial cell growth rate in the treatment groups compared to control However, there was a reduction in the number of cells in the drugtreat ed groups in the plateau phase (Fig. 3 7 ). Effect of AZD0530 on HMVECL Tube F ormation The effect s of AZD0 530 on pre treated HMVEC L cell s w ere examined by tube formation assays. There was no significant difference in the capacity of KHT cells to form tubes after 24 hr or 48 hr AZD0530 pretreatment compared to control ( Fig 3 8, Fig 3 9 Fig 3 10). Similarly, when the HMVEC L cell s were seeded on the Matrigel in the presence of the drug, t here was no significant difference in the capacity of KHT cells to form tubes after 24 hr or 48 hr AZD0530 pretreatment compared to control Further, when complete tubes wer e formed, AZD0530 treatment did not appear to affect them up to 72 hr treatment ( Fig 3 11).

PAGE 63

63 Effect of AZD0530 on HMVECL M igration To investigate whether AZD0530 treatment could inhibit HMVEC -L motility, two different treatment schedules were carried out. The first pre -treated KHT cells with 1 10 M AZD0530 for 48 hr; in this schedule there was no significant difference in cell migration compared to control (Fig 3 12). The second paradigm was to compare the ability of cells to migrate in the presence of th e drug. No significant difference in the migration ability was detected when KHT cells were in the presence of 1 10 M AZD0530 (Fig 3 13). pSrc Recovery after AZD0530 W ithdrawal To investigate how long it would take for pSrc to recover to normal levels a fter AZD0530 was removed from media, a Western blot study was carried out. Fig 3 14 shows that pSrc increased when the drug was removed, and this increase was in a time dependent manner. However, the pSrc level was still ~50% less than that found in the c ontrol group even after the drug was removed from media for 72 hr. Effect of AZD0530 on KHT -HMVEC L A dhesion To study if AZD0530 could affect the abili ty of KHT cells to adhere to an endothelial cell monolayer, a cell -cell adhesion assay was carried out. After KHT cells (106) were pre treated with drug for 24 hr, the ability of KHT cells to adhere to HMVEC -L was decreased. This decrease was in a dose dependent manner (Fig 3 15). Effect of AZD0530 on VEGF S ecretion To study the effects of AZD0530 on VEGF secretion in KHT cells, ELISA was carried out. Data showed that the secreted VEGF in KHT cell supernatant was decreased compared to controls when trea t ed with 0.5 or 1 M AZD0530 for 24 hr (Fig 3 16).

PAGE 64

64 Effect of AZD0530 on A ngiogenesis To study the effect of AZD0530 on angiogenesis, an intra -dermal assay was conducted in two ways. First, AZD0530 -pretreated KHT cells were injected intra dermally into the skin of the mice, and thr ee days later the number of vessels that grew to the tu mor cell inoculation was determined The result showed that t he number of vessels in the treated groups was significantly decreased compared to control (Fig 3 17). Sec ond, KHT cells were inj ected intra dermally into the skin of the mice, and then AZD0530 was given orally each day. In this model, a result was obtained that was similar to the first one. There were fewer vessels growing into the tumor cell inoculation in the treated groups comp ared to the control group (Fig 3 18). Discussion Src expression/activity has been shown to be involved in many types of cancer (Cartwright et al. 1994; Egan et al. 1999; Lutz et al. 1998) It is an important kin ase related to cancer cell survival, proliferation, migration, invasion and adhesion. Chapter 1 showed that inhibit ion of Src activity by AZD0530 impaired the cell functions involved in metastatic process To examine whether the in vitro effects of AZD05 30 treatment on tumor cell function would affect the ability of KHT sarcoma cells to establish secondary tumor deposits in the lungs of mice, tumor cells pretreated with non -cytotoxic doses of AZD0530 and untreated control tumor cells were injected via the tail vein. The results demonstrated that when similar numbers of tumor cells were injected, fewer AZD0530pretreated KHT cells were able to form tumor nodules (Fig 3 1 ). Mechanistically, AZD0530 exposure impair ed KHT cell proliferation migration, invas ion and adhesion, the early steps in the metastatic cascade required by the tumor cells to establish new tumor growth When the mice were treated with AZD0530 orally and daily after injecting KHT cells via tail vein, a similar re sult was achieved as above ; i.e. there were fewer lung nodules

PAGE 65

65 in the treated groups compared to control (Fig 3 2). These results showed that orally administering AZD0530 could achieve similar effects as pre -treating the tumor cells. These findings suggested that the effect of AZD0 530 on inhibiting lung colony formation may be at early steps of the establishment of secondary tumor deposits To further investigate our hypothesis, another in vivo experiment was designed. Three different treatment schedules were evaluated. The mice in the first drug -treatment group received only one dose of AZD0530 immediately after tumor cell injection. Based on lab previous experiences, if the KHT tumor cells could not get out of the blood stream in the first 24 to 48 hrs, they would never form me tas tases. T he mice in the second drug treatment group receive d four daily treatment s T he mice in the third drug treatment group were treated with drug daily until the end of the experiment. The data showed that the number of lung nodules was decreased in all the drug treatment groups. However, no significant difference was detected with in the se groups (Figure 3 3) further support the notion that the effect of AZD0530 on inhibiting lung colony formation may be at early steps of the establish ment of seco ndary tumor deposits. In addition, our lab data showed that when starting treatment 3 days after tumor cell injection via tail vein, there was little anti -metastatic effect which was also consistent with this hypothesis The finding that the size distri butions of colonies arising from control and treated groups were similar (Fig 3 1 C) also supports the notion that the reduction in the number of lung colonies formed in the drug treatment groups was predominantly a consequence AZD0530 exposure impeding the ability of tumor cells to establish secondary tumor deposits. To further understand which factors reduce lung colony formation, additional mechanistic studies were carried out. A KHT HVEC -L adhesion assay showed that after KHT cells were exposed to AZD 0530, the ability of these cells to adhere to the HMVEC L monolayer was

PAGE 66

66 decreased (Figure 3 15) This cell fu nction is very important for cells in the metastatic process to adhere to the endothelial cell mo nolayer before they can extrav a sate from the bloo d vessels. A lso, in the extrav a sating step, cell s need to secret enzymes to degrade ECM to leave the blood vessels for subsequent cell migration and invasion (Iiizumi et al. 2007) Our data in Chapter 1 also demonstrated that tumor cell migration and invasion ability were inhibited by AZD0530. No dramatic effect of AZD0530 on HMVEC L cel l growth, migration and tube formation was observed in our studies In intra dermal assays there were fewer vessels growing into KHT tumor cell inoculation either after injecting pre treated KHT tumor cells or by treating mice after KHT cell injections in to the mouse skin. These findings suggested that AZD0530 could inhibit angiogenesis predominantly by interrupting the KHT tumor cells ability to induce new vasculature formation. VEGF secretion level by KHT cells was observed to be decreased after AZD0530 treatment further support ing this hypothesis. In summary, in vivo studies showed that AZD0530 can interrupt t he ability of KHT tumor cells from form ing metastatic lung nodules. One potential mechanism is that AZD0530 may impair key early steps required by tumor cells to secondary deposits by affecting tumor cell ad hesion, migration, and invasion so that by inhibiting the tumor cells from adhering to the endothelial layer of the vessels and preventing them from extravasating into a secondary site While it is possible that a nti angiogenic effects of AZD0530 may also contribute to the anti metastatic effect, this appears not to be a major factor in the KHT tumor model. Since our data implied that AZD0530 affected KHT tumor metastases at a very early stage, t his provides a new approach for using this agent in clinical settings that would involve administrating AZD0530 at an early stage to be more helpful for the patients who have high risks of metastatic diseases.

PAGE 67

67 Figure 3 1. E ffect s of 24 hr pre treatment of AZD0530 on the ability of KHT cells to form lung colonies when injected into mice via the tail veins. A) Representative pictures of the lung nodules. B ) Data shown are the median number of lung nodules (line s) and the 25th a nd 75th percentiles ( box boundaries) respectively Whiskers above and below the box indicate the 90th and 10th percentiles The results shown are representative of two experiment s p <0.05 vs control **p <0.0 1 vs control C ) Size distributions of the lung nodules obtained in (A ). Data shown are representative of two experiment s C A B Number of Lung Nodules 0 5 10 15 20 25 30 Control 1 M 5 M* ** Size (mm3) 0.2-0.4 0.4-0.6 0.6-0.8 0.8-1.0 1.0-1.2 1.2-1.4 1.4-1.6 1.6-1.8Number of Lung Nodules 0 5 10 15 20 25 Control 1 M 5 M C ontrol 1 M 5 M C

PAGE 68

68 Figure 3 2 E ffect s of AZD0530 on the ability of KHT cells to form lung colonies when injected into mice via the tail veins. Data shown are the median number of lung nodules (line s) and the 25th and 75th percentiles ( box boundaries) respectively Whiskers above and below the box indicate the 90th and 10th percent iles **p <0.0 1 vs control Data shown are representative of two experiment s 0 1 2 3 4Number of Lung Nodules 0 5 10 15 20 25 30 control 10 mg/kg 25 mg/kg** **

PAGE 69

69 F igure 3 3 E ffect s of AZD0530 on the ability of KHT cells to form lung colonies when injected into mice via the tail veins with three different treatment schedules. D ata shown are the median number of lung nodules (lines) and the 25th and 75th percentiles (box boundaries) respectively. Whiskers above and below the box indicate the 90th and 10th percentiles Group 1 is the co ntrol group. The mice in group 2 were tre ated with one dose AZD0530 immediately after tumor cell injection. The mice in group 3 were treated with 4 doses of AZD0530 daily. The mice in group 4 were treated with 18 doses AZD0530. **p <0.0 1 vs control Dat a shown are representative of two experiment s Group 1 = control Group 2 = one dose Group 3 = four doses Group 4 = 21 doses Group 0 1 2 3 4 5Number of Lung Nodules 0 5 10 15 20 25 30 ** ** **

PAGE 70

70 Figure 3 4. E ffect s of AZD0530 on the ability of KHT cells to form lung colonies when injected into the leg of mice intra -muscularly. Data shown are the median number of lung nodules (line s) and the 25th and 75th percentiles ( box boundaries) respectively Data shown are representative of two experiment s Number of Lung Nodules 0 10 20 30 40 50 Control 10 mg/kg 25 mg/kg

PAGE 71

71 Figure 3 5. Effects of AZD0530 on pSrc (Y416) and Src in HMVEC -L cells A) pSrc and Src were examined after K HT cells were expos ed to drug at the indicated doses for 24 hr. B) pSrc and Src were examined after KHT cells were treated with 10 M drug for the indicated time. Data shown are representative of two experiment s

PAGE 72

72 Figure 3 6. Effect s of AZD0530 on HMVEC -L cell morphology after 24 hr treatment. Data shown are representative of three experiment s control 5 M 1 M 10 M

PAGE 73

73 Figure 3 7. Effect s of AZD0530 on HMVEC -L cell growth over a 7 -day period in the presence of A ZD0530. Data shown are representative of two experiment s day -2 0 2 4 6 8Number of Cells 1e+4 1e+5 1e+6 DMSO 0.5 M 1 M 5 M 10 M Treatment

PAGE 74

74 Figure 3 8. Effect s of AZD0530 on tube formation by HMVEC -L cells pre -treated with drug for 24 hr. Data shown are representative of three experiment s control 1 M 5 M 10 M

PAGE 75

7 5 Figure 3 9. Effect s of AZD0530 on tube formation by HMVEC -L cells pre -treated with drug for 48 hr. Data shown are representative of two experiment s Data shown are representative of two experiment s control 1 M 5 M 10 M

PAGE 76

76 A Control 1 M 5 M 10 M Control 1 M 5 M 10 M B Control 1 M 10 M 5 M C Control 1 M 10 M 5 M Figure 3 10. Effect s of AZD0530 on tube formation by HMVEC L cells in the presence of AZD0530. A) P hoto was taken after 24 hr drug treatment. B) P hoto was taken after 48 hr drug treatment. C) Photo was taken after 72 hr drug treatment. Data shown are representative of one experiment

PAGE 77

77 A control 1 M 10 M 5 M B 1 M control 10 M 5 M C control 1 M 10 M 5 M Figure 3 11. Effect s of AZD0530 on complete tubes formed by HMVEC -L cell. A) Picture was taken after 24 hr drug treatment. B) P hoto was taken after 48 hr drug treatment. C) Photo was taken after 72 hr drug treatment. Data shown are representative of one experiment

PAGE 78

78 Figure 3 12. Effect s of AZD0530 on HMVEC L cell migration after the cells were pre -treated with drug for 48 hr. Data shown are representative of two experiment s AZD0530 ( M) -2 0 2 4 6 8 10 12 14Cell Number 0 20 40 60 80 100 1 5 A B C 1 M 1 M 10 M

PAGE 79

79 Figure 3 13. HMVEC L cell migration in the presence of 1 10 M AZD0530 for 24 hr Data shown are representative of one experiment AZD0530 ( M) -2 0 2 4 6 8 10 12 14Cell Number 0 20 40 60 80 100 1 5 C 1 M 5 M 10 M A B

PAGE 80

80 Figure 3 14. pSrc (Y416) recovery in KHT cells at various times after AZD0530 remov al following a 24 hr drug treatment. Data shown are representative of two experiment s

PAGE 81

81 A B C AZD0530 ( M ) 0.01 0.1 1 10 0 2.5 5 0.5** ***Number of Cells Not Adhered (x104)0 2 4 6 8 10 12 14 16 18 Figure 3 15. Effect s of AZD0530 on KHT cell adhesion to HM VEC -L monolayer. A) HMVEC -L monolayer B) KHT cell adhering HMVEC L after 2 hr incubation C) Fewer p retreated KHT cells adhere to HMVEC -L monolayer ( Mean SE for 3 experiments). *p<0.05 vs control **p <0.01 vs control Cont rol 5 M

PAGE 82

82 AZD0530 ( M ) 0.1 1 10VEGF Expression (% of control) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Exp.1 Exp.2 Exp.3 0 0.5 0 Figure 3 16. Effect s of AZD0530 on s ecreted VEGF in the medium of KHT cells after 24 hr treatment. Data shown are three independent experiment s

PAGE 83

83 A B C AZD0530 ( M) Number of Vessles 0 10 20 30 40 Control 1 M 5 M ** ** Figure 3 17. E ffect s of 24 hr pre treatment of AZD0530 on the ability of KHT cells to induce angiogenesis. A) Representative pictures showed that the vessel grew into tumor inoculation. B) Representative pictures taken at a higher magnification. C) Data shown are the m edian number of vessels (lines) and the 25th and 75th percentiles (box boundaries) respectively. Whiskers above and below the box indicate the 90th and 10th percentiles **P<0.0 1 vs control Data shown are repr esentative of two experiment s control 5 M

PAGE 84

84 Figure 3 1 8 E ffect s of AZD0530 on the ability of KHT cells to induce angiogenesis. Data shown are the median number of vessels (lines) and the 25th and 75th percentiles (box boundaries) respec tively. Whiskers above and below the box indicate the 90th and 10th percentiles **P<0.0 1 vs control Data shown are representative of two experiment s Number of Vessles 0 10 20 30 40 50 Control 10 mg/kg 25 mg/kg ** **

PAGE 85

85 CHAPTER 4 ESTABLISHING TUMOR MODELS IN WHICH THE DEVELO PMENT OF METASTASES CAN BE NON INVASIVELY ASSESSED Introduction Metastas i s is a complicated process. It includes multiple steps as discussed in C hapter 1 I t is still a poorly understood process. T o study this process w hole -body imaging met hods offer a v ery useful approach for the visualiz ation of biological process es non -invasively. Techniques available to perform such imaging include microcomputed tomography (CT), micro positron emission tomography (PET) and magnetic resonance imaging (MRI) dynamics of metastatic cancer. R ecently, optical imaging of bioluminescence (BLI) and optical imaging of fluorescence also have been applied in this field as well (Lyons, 2005) One of the most popular optical imaging systems is the Xenogen imaging system, a highly sensitive procedure developed by Xenogen of Caliper Life Sciences It can detect photon emissions generated either by fluorescent or luminescent. For fluorescent imaging, g reen fluorescent op rotein (GFP) and red fluorescent p rotein (RFP) are commonly used as labels (Matsumoto et al. 2009) However there are some disadvantages with the fluorescent imaging method including ba ckground autofluorescence in animals and high signal absorption by tissues As for luminescent imaging, it depends on enzyme substrate reaction s to generate photons that can be detected by a CCD camera. The most commonly used enzyme is luciferase and its substrate is D -l uciferin By capturing the photons from the luciferase transfected cells, it allows researchers to quantify the signals generated from living animals (Henriquez et al. 2007) This Xenogen system has been used in a variety of fields including oncology research, infectious disease, inflammation, metabolic diseases, neurology, gene therapy, stem cell biol o gy, cardiovascular disease immunology and transplantation biology, toxicology, and drug metabolism studies (Caliper LifeSciences 2009) When used in oncology, it allows users to

PAGE 86

86 non invasively image tumor growth and met astase s formation longitudinally. For example, Matsumoto et al (Matsumoto et al. 2009) showed that the Xenogen system allow ed them to visualiz e the metastases and facilitated a faster and more accurate counting of disseminated foci in the pleura (as compared to the previous standard of measuring body weight changes) in a lung ca ncer model. Darlene et al showed that b ioluminescent human breast cancer cell lines permit ted rapid and sensitive in vivo detection of primary mammary tumors and multiple metastases in immune deficient mice (Jenkins et al. 2005) In infectious disease s Cook et al (Cook and Griffin, 2003) showed that the Xenogen system m onitor ed the locat ion and extent of virus replication in live mice instead of using a large numbers of animals to obtain similar data In drug metabolism studies Palframan showed that this imaging system is effective for detect ing the distribution of the interested therap eutic agent s in tissues in mice with collagen -ind uced arthritis (Palframan et al. 2009) The Xenogen imaging system is composed of three major parts (F ig ure 4 1 ). (1) A 1 inch ultra -sensitive CCD camera cooled wi th a closed cycle mechanical refrigeration unit to 105C; (2) a light -tight imaging chamber; (3) a computer for operating the Xenogen and imaging analysis. Since the CCD camera is cryogenically cooled to 105C, the electronic background is minimized and the sensitivity maximized The l ight tight imaging chamber allows this machine to accommoda te lighting environments in standard labs. The powerful operating system makes it easy to analyze the data. It can adjust the field of view for 10 25 cm so that it can image up to 5 mice at one time. This chapter describes studies undertaken to establish the use of the Xenogen imaging system in two tumor models, the KHT fibrosarcoma and the 4A4 breast cancer tumor model.

PAGE 87

87 Materials and Methods Cell P reparation KHT and 4A4 cells (1 x 104) were infected with a lentivirus containing luciferase gene. Three weeks following the expansion of the cells, the cells were collected and luminescent signals were examined using Xenogen imaging system. Animals All research was go verned by the principles of the Guide for the Care and Use of Laboratory Animals (USPHS), and approved by the University of Florida Institutional Animal Care and Use Committee (Gainesville, FL). S ix -to -eight -week old female athymic nude mice were obtained from Jackso n Laboratories (Bar Harbor, ME) and were maintained in a specific pathogen-free environment (University of Florida Health Science Center, Gainesville, FL), with food and water provided ad libitum. I maging Bioluminescent imaging was performed us ing Xenogen imaging system. For in vitro imaging, 4A4 and KHT cells were serially diluted from 1x105 to 100 c ells in media into 96 -well plates. Ten minutes before imaging, D -luciferin ( Caliper Life Sciences Inc .) diluted in Dulbecco's Phosphate Buffered Saline (DPBS) at 150 g/ml was added to each well The imaging exposure time wa s 1 min For in vivo imaging, the mice were injected intrapeitoneally with 200 ul of 1 50 mg/ ml D luciferin in DPBS 15 min before imaging. Then the mice were anesthetized in the animal chamber of X enogen imaging system using 1 .5 2.5 % isoflurane Then the anesthetized m ice were imaged in the imaging chamber. T he exposure time was from 1s to 5 min decided by the tumor model. The images were analyzed by Xenogen imaging software. For ex vivo imaging, 200 ul of 1 50 mg/ ml D luciferin in DPBS were injected into the mice intrapeitoneally before the mice were euthanized Then the tissue were removed and put into

PAGE 88

88 PBS in the 6 -well plates. The imaging exposure time wa s 3 min Then the tissue was fixed in 10% buffered formalin and examined by histology. KHT F ibrosarcoma Experimental Metastasis M odel Female nude mice were injected with 5x 103 KHT Luc cells via tail v ein on day 0. Then the mice were imaged by the Xenogen imaging system on day 0. After the 1st imaging the mice were imag ed every 7 days for up to 6 weeks. KHT F ibrosarcoma S pontaneous M etasta sis M ode l Female nude mice were injected with 1x 105 KHT Luc cells intramuscularly on day 0. Then the mice were imaged by the Xenogen imaging system on day 0. After the 1st imaging the mice were imag ed every 3 days for up to 12 days 4A4 Breast Tumor S pontaneous M etastasis M odel Female nude mice were with 106 4A4 cells into the fat pad. The mice were imaged by the Xenogen imaging system on day 0. After t he 1st imaging the mice were imag ed by imaging every 7 days. The size of the primary tumors was measured by caliper. When the primary tumor grew to 1.5 cm in diameter, the primary tumors were surgically removed. The mice were imaged both before and after surgery. T he removed tumors were also imaged ex vivo for 3 min. Histo l ogy Selective tissue s were fixed in 10% buffered formalin after ex vivo imaging. Tissue sections and HE staining were prepared by Histology Tech Services (Gainesville, FL). Results Established KHT Luc and 4A4 -Luc C ell Lines After infect ion with lenti virus expressing the luciferase gene, the KHT and 4A4 cells stably expressed this enzyme The bioluminescent signals could be detected by the Xenogen

PAGE 89

89 imaging system. The intensity of the signals was related to the cell number as shown in Fig ure 4 2 KHT F ibrosarcoma E xperimental M etastasis M odel Before injecting KHT Luc cell s the cells were checked using the Xenogen imaging system and found to express bi oluminescent signals at high inte nsity. After KHT Luc cell injection via the tail vein no signals were initially detected. By day 21 signals from lung metastases could be detected from 2 out of 5 mice (F igures 4 3 through 4 6) The intensity of the signals increased along with the gro wth of lung metastases. By day 42, signals from the lung metastases were detected in all mice. Lungs removed from the mice also displayed luciferase signals. The lung metastases were confi rmed by histology (Figure s 4 3 through 4 6 ). KHT F ibrosarcoma S po ntaneous M etastasis M odel After the intra -muscular injection of KHT Lu c cell s, signals were detected in all 4 mice (Figure 4 7) The signals increased with the growth of the primary tumor. Lung metastases were not detected by the time the mice had to be euthanized due to the aggressive growth of the primary tumor (day 12) However, micro -metastases were detected ex vivo after the lungs were removed from the mice (Figure 4 7) 4A4 Breast Tumor S pontaneous M etastasis M odel After 4A4 -Luc cell injection, th e signals from the injected ce ll were detected in all 5 mice (Figure 4 8) The signals increased with the growth of the primary tumor (Figure 4 9 through 4 13). In most cases the intensity of bioluminescent signals reflected changes in tumor measurement by calipers. The tumor growth rates were clearly different in different animals. In mice #3, #4 and #5, it grew slower than in mouse #1 or #2. Those tumors which grew larger than 1.5 cm in diameter (mice #1 and #2) were surgically removed. The signals from the removed tumor s were examined by imaging (Fig ure 4 14 B and 4 15 B) One of the mice

PAGE 90

90 subjected to surgery showed signals after surgery indicating tumor residue, which had invaded the body wall and could not be removed completely (Figure 4 15 A). T he tumor of the other mouse did not invade the body wall and was removed completely Imaging the mouse showed no evidence of tumor residue. Both mice are still being evaluated and to date no metastases have be en detected. Discussion Bioluminescent imagin g provides a way to non -invasively detect the primary tumor and metastasis. It has been successfully used in several tumor models including prostate cancer (Jenkins et al. 2003; Scatena et al. 2004) breast cancer (Jenkins et al. 2005) and lung cancer (Matsumoto et al. 2009) In our study, two metastatic models were investigated including one mouse KHT fibrosarcoma tumor model and one human 4A4 breast cancer tumor. Initial i n vitro imaging studies showed that the bioluminescent signal intensity obtained from the KHT Luc and 4A4 Luc cell lines correlated with the number of the cells containing luciferase genes. Subsequent studies aimed to examine whether the Xenogen system could be used to study t hese two tumor model s noninvasively for both the growth of primary tumor and the metastases. Traditionally, to study the metastases and determine when and where metastases occur requires large number s of animals that are euthanized at different time points Using non -invasive imaging can greatly reduce the number of animals and the effort required to perform such studies. In addition, it allow s us to study the tumor growth or tumor response in the same animal at different time points. In t he KHT experimental metastasis model we detected bioluminescent signals from lung metastases in all four mice injected Importantly, the signal intensity increased with the growth of the lung nodules.

PAGE 91

91 The bioluminescent signal s from lung metastases were not detected before the mice had to be euthanized likely due to t he aggressiveness of the primary tumor i n KHT spontaneous metastases model However, micro -metastases were observed ex vivo after the lungs were taken out of the mice. This indicate d that the Xenogen imaging system is very sensitive. T o study spontaneou s metastases, the 4A4 human breast tumor model was used. This tumor grows slowly and is known to form lung metastases from orthotopically implant ed primary tumor s (Urquidi et al. 2002) In this model, the metasta ses can be imaged over time because the primary tumor can be surgically removed before it overwhelms the mouse For this reason, the 4A4 model more closely resembles a clinical situation where the removal of the primary tumor from the patients still leave s them at risk of dying from secondary tumors. Our study showed that the bio luminescent signals increased with the size of the primary tumor over time, indicating that the Xenogen system can semi -quantify the growth rate These mice are still being foll owed for lung metastases after surgery. So far, the lung metas tases have not been detected In summary, these studies have show n the potential of the Xenogen s ystem to image primary and metastatic tumors. In the future, it may provide a tool to evaluate the effect of anti tumor drugs, including Src tyrosine kinase inhibitors

PAGE 92

92 Figure 4 1 Xenogen imaging system.

PAGE 93

93 KHT Luc 4A4 -Luc Figure 4 2 Bioluminescent imaging of KHT Luc and 4A4 Luc cells after lentivirus infection Cells 10 5 Cells 10 4 Cells 10 3 Cells 10 2 Cells

PAGE 94

94 Figure 4 3. Bioluminescent imaging of mouse #1 after tail vein injection of KHT Luc cells. A) Bioluminescent signals of lung metastases in the live mouse. B) Quantified bioluminescent signal from A. C) Bioluminescent imaging of the lung ex vivo D) HE staining of the lung for metastases.

PAGE 95

95 Figure 4 4. Bioluminescent imaging of mouse #2 after tail vein injection of KHT Luc cells. A) Bioluminescent signals of lung metastases in the live mo use. B) Quantified bioluminescent signal from A. C) Bioluminescent imaging of the lung ex vivo D) HE staining of the lung for metastases.

PAGE 96

96 Figure 4 5. Bioluminesce nt imaging of mouse #3 after tail vein injection of KHT Luc cells. A) Biolumin escent signals of lung metastases in the live mouse. B) Bioluminescent imaging of the lung ex vivo C) HE staining of the lung for metastases.

PAGE 97

97 Figure 4 6. Biol uminescent imaging of mouse #4 after tail vein injection of KHT Luc cells. A) Biolu minescent signals of lung metastases in the live mouse. B) Bioluminescent imaging of the lung ex vivo C) HE staining of the lung for metastases.

PAGE 98

98 Figure 4 7. Bioluminescent imaging of mice after KHT Luc cells are injected intramuscularly. A) Bioluminescent signal s from KHT Luc primary tumor s B) Bioluminescent signal s from micro -metastases in ex vivo lungs.

PAGE 99

99 Fiugure 4 8. Bioluminescent imaging immediately after 4A4 -Luc cell injection into the fat pads of mice.

PAGE 100

100 Figure 4 9. Bioluminescent imaging of mouse #1 after 4A 4 Luc cell injection in to the fad pad. A) Bioluminescent signals from primary tumor in the live mouse a t indicated time point. B) Quantified bioluminescent signal from A. C) Tumor size measured by caliper s

PAGE 101

101 Figure 4 10. Bioluminescent imaging of mouse #2 after 4A 4 -Luc cell injection in to the fad pad. A) Bioluminescent signals from primary tumor in the live mouse at indicated time point. B) Quantified bioluminescent signal from A. C) Tumor size meas ured by caliper s

PAGE 102

102 Figure 4 11. Bioluminescent imaging of mouse #3 after 4A 4 -Luc cell injection in the fad pad. A) Bioluminescent signals from primary t umor in the live mouse at indicated time point. B) Quantified bioluminescent signal from A. C) Tumor size measured by caliper s

PAGE 103

103 Figure 4 12. Biolum inescent imaging of mouse # 4 after 4A 4 -Luc cell injection in the fad pad. A) Bioluminescent signals from pr imary tumor in the live mouse at indicated time point. B) Quantified bioluminescen t sign al from A. C) Tumor size measured by caliper s

PAGE 104

104 Figure 4 13. Biolum inescent imaging of mouse # 5 after 4A 4 -Luc cell injection in the fad pad. A) Bioluminescent signals from pr imary tumor in the live mouse at indicated time point. B) Quantified biolum inescent signal from A. C) Tumor size measured by caliper s

PAGE 105

105 Figure 4 14. Bioluminescent imaging of mouse #1 with 4A4 Luc breast tumor A) Bioluminescent imaging before and after surgery. B) Bioluminescent imaging of the tumor ex vivo

PAGE 106

106 Figure 4 15. Bioluminescent imaging of mouse #2 with 4A4 Luc breast tumor. A) Bioluminescent imaging before and after surgery. B) Bioluminescent imaging of the tumor ex vivo

PAGE 107

107 CHAPTER 5 S UMMARY Src tyrosine kinase was the first oncogenic protein kinase to be discovered (Bishop et al. 1978) Studies have shown that Src tyrosine kinase plays an important role in cancer progress (Alper and Bowden, 2005) Evidence shows that Src tyrosine kinase is involved in multiple cellular functions related to the metastatic processes In addition, the protein expression level and activation of Src tyrosine kinase are related to the malignant grades of cancer (Yeatman, 2004) Also, Src tyrosine kinase is expressed at a low level in most normal tissues (Alper and Bowden, 2005) Therefore, Src tyrosine kinase may be a promising therapeutic target in inhibiting metasta ses with less toxicity. AZD0530 is a small molecule kinase inhibitor which can specifically inhibit Src activation by competitively blocking its ATP pocket (Hennequin et al. 2006) The goal of this dissertation was to examine the potential anti -metastatic effects of this agent in rodent and human tumor models. Chapter two mainly focused on the effect of Src inhibition by AZD0530 on tumor cells. It showed that AZD0530 can inhibit cell migration, invasion and adhesion in all tested tumor cell lines. However, its anti proliferation effect was found to be somewhat cell line dependen t In KHT fibrosarcoma cells, AZD0530 treatment resulted in tumor cell accumulation in the G1/S cell cycle phase leading to a slowing of the growth of the tumor cells. In contrast, no such effects were observed in 4A4 and 2C5 human breast cancer cells. Ch apter three focused on the effect of Src inhibition by AZD0530 on mouse tumor models. In vivo studies showed that the ability of the KHT tumor cell s to f orm metastatic lung nodules was de creased after the tumor cell s had been treated with AZD0530 in a exp erimental metastasis model. In this model, when mice were treated with three different dose schedules ; a single dose

PAGE 108

108 after tumor cell injection, four daily treatment s and daily treatment until the end of the experiment, similar anti -tumor effects were ob s erv ed. In the control group, the mean number of lung nodules was 13. I t decreased to ~5 in all drug -treatment groups. This i mplied that the effect of AZD0530 may be primarily on the early stages of the metastatic process These results suggested that ad ministration of this drug to patients with no evidence of metastases but at high risk of developing metastases may be most helpful The metastatic process is complicated and involves multi ple steps. It includes tumor cell s escap ing from the primary tumor enter ing the blood vessels, surviv ing in the circulation, arresting at distant sites extrava sating the blood vessel, surviving in the parenchyma and proliferating at one or more distant organs (Carter, 1982) Our data indicated that AZD050 play s an important role in impeding the ability of tumor cells to initially establish secondary tumor depos its. For example in the extrava sating step, the tumor cells need to adher e to the endothelial cells invad e through the endothelial cells a nd smooth muscle cells, break down the extra -cellular matrix and finally migrat e from the blood vessels (Wittekind and Neid, 2005) O ur data showed that AZD0530 interrupt ed the ability of tumor cells t o adhere to the endothelial cells. Further, AZD0530 inhibit s t umor cell migration, invasion and the activity of type IV collagenase MMP9. T he in vitro adhesion assa y showed that when treated with AZD0530, the KHT cells were detached from the plat es suggesting that cell adhesions w ere disrupted by the drug. Together the results imply that Src inhibition by agents such as AZD0530 have potential to significantly impair the spread of cancer cells. Future studies of agents targeting metastases such as AZD0530 would benefit from models allowing the noninvasive assessment of the metastatic cascade. For this reason KHT Luc and 4A4 -Luc metastatic cell s were established (Chapter 4). These cell s allowed us to non-invasively

PAGE 109

109 observe the growth of the prim ary tumor as well as the metastatic spreading of tumor cells using the Xenogen imaging system. One adva ntage of these models is that they can precisely provide in formation about when and where metastases occur while using very few mice ( Chapter 4 ). I n th e traditional method s, to obtain such informa tion, a much larger number of mice would be used. Another advantage is that t hese models offer a more sensitive way to examine metastatic deposit s than traditional ways. For example, i n the KHT -Luc spontaneous metastasis model the ex vivo imaging of the lungs showed m icro -metastases which are normally difficult to be detect The third advantage is that these models may allow us quantifying the signals from the primary or metastatic tumor. For example, i n the 4A4 spontaneous metastasis model, the signals correlated nicely with the size of the primary tumor. Also, in the KHT experimental metastasis m odel, the signals increased as the lung metastatic nodules grew. One disadvantage of the 4A4 tumor model is that this tumor grows so slowly that it takes a very long time to get second ary metastatic tumor growth A model that may have some desirable features for studying metastases may be the 4T1 cell line, which was derived from a spontaneously arising BALB/c mamma ry tumor When introduced orthotopically, the 4T1 line grows rapidly at the primary site and forms metastases in lungs, liver, bone and brain over a period of 3 6 weeks. When introduced via the tail vein or arterially, metastases are apparent in these same organs after 1 2 weeks (Aslakson and Miller, 1992; Pulaski and Ostrand-Rosenberg, 2001) T his model is being established in our laboratory now. Based on its apparent characteristics, this model may be extremely useful to pre -clinically evaluating anti -cancer strategies in future. In summary, this dissertation has show n that Src play s an important role in cancer metastases. Specifically our studies showed that i nhibiting Src signaling by AZD0530 treatment can im pair the metastatic process of tumor cells. In the future, we will continue our study on

PAGE 110

110 non invasively evaluating the effect of Src in hibition on metastases using Xenogen imaging system. In addition, we will investigate the effect on metastases when com bining this strategy with other interventions.

PAGE 111

111 LIST OF REFERENCES Aligayer H, Boyd DD, Heiss MM, Abdalla EK, Curley SA, Gallick GE (2002). Activation of Src kinase in primary colorectal carcinoma: an indicator of poor clinical prognos is. Cancer 94: 34451. Alper O, Bowden ET (2005). Novel insights into c Src. Curr Pharm Des 11: 111930. Aslakson CJ, Miller FR (1992). Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mo use mammary tumor. Cancer Res 52: 1399405. Ausprunk DH, Folkman J (1977). Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14: 5365. Avizienyte E, Brunton VG, Fincham VJ Frame MC (2005). The SRC induced mesenchymal state in late -stage colon cancer cells. Cells Tissues Organs 179: 73 80. Baker D, Elkon D, Lim ML, Constable W, Wanebo H (1981). Dose local x-irradiation of a tumor increase the incidence of metastases? Cancer 48: 23948. Basu A, Cline JS (1995). Oncogenic transformation alters cisplatin-induced apoptosis in rat embryo fibroblasts. Int J Cancer 63: 597603. Bishop JM, Baker B, Fujita D, McCombe P, Sheiness D, Smith K et al (1978). Genesis of a virus transformin g gene. Natl Cancer Inst Monogr : 21923. Bjorge JD, Jakymiw A, Fujita DJ (2000). Selected glimpses into the activation and function of Src kinase. Oncogene 19: 562035. Broome MA, Hunter T (1996). Requirement for c Src catalytic activity and the SH3 domain in platelet -derived growth factor BB and epidermal growth factor mitogenic signaling. J Biol Chem 271: 16798806. Brunton VG, Avizienyte E, Fincham VJ, Serrels B, Metcalf CA, 3rd, Sawyer TK et al (2005). Identification of Src -specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. Cancer Res 65: 133542. Caliper LifeSciences. IVIS Imaging Systems Identify Pathways, Monitor Diseases http://www.caliperls.com/products/opticalimaging/ (accessed May 2009). Cancer Research UK. How a cancer spreads http://www.cancerh elp.org.uk/help/default.asp?page=101 (accessed October 2009). Cancer Medicine, 2009 Cancer Medicine. Invasion and Metastases http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi ?book=cmed6&part=A2597 (assessed October, 2009)

PAGE 112

112 Carter RL (1982). Some aspects of the metastatic process. J Clin Pathol 35: 10419. Cartwright CA, Coad CA, Egbert BM (1994). Elevated c -Src tyrosine kinase activity in premalignant epithelia of ulcerative c olitis. J Clin Invest 93: 50915. Cartwright CA, Meisler AI, Eckhart W (1990). Activation of the pp60c -src protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci U S A 87: 55862. Chang YM, Bai L, Liu S, Yang JC, Kung HJ, Evans CP ( 2008). Src family kinase oncogenic potential and pathways in prostate cancer as revealed by AZD0530. Oncogene 27: 636575. Cook SH, Griffin DE (2003). Luciferase imaging of a neurotropic viral infection in intact animals. J Virol 77: 53338. Dahl S (1966). Treatment of cancer patients with infusions of plasmin. Oncology 20: 358. Dove A (2002). MMP inhibitors: glimmers of hope amidst clinical failures. Nat Med 8: 95. Egan C, Pang A, Durda D, Cheng HC, Wang JH, Fujita DJ (1999). Activation of Src in human br east tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530. Oncogene 18: 122737. Eliceiri BP, Paul R, Schwartzberg PL, Hood JD, Leng J, Cheresh DA (1999). Selective requirement for Src kinases during VEGF induced angiogenesis and vascular permeability. Mol Cell 4: 91524. Elliott BE, Ekblom P, Pross H, Niemann A, Rubin K (1994). Anti -beta 1 integrin IgG inhibits pulmonary macrometastasis and the siz e of micrometastases from a murine mammary carcinoma. Cell Adhes Commun 1: 31932. Ellis LM, Staley CA, Liu W, Fleming RY, Parikh NU, Bucana CD et al (1998). Downregulation of vascular endothelial growth factor in a human colon carcinoma cell line transfe cted with an antisense expression vector specific for c-src. J Biol Chem 273: 10527. Emaduddin M, Bicknell DC, Bodmer WF, Feller SM (2008). Cell growth, global phosphotyrosine elevation, and c Met phosphorylation through Src family kinases in colorectal cancer cells. Proc Natl Acad Sci U S A 105: 235862. Fabian MA, Biggs WH, 3rd, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG et al (2005). A small molecule kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 23: 329 36. Felsenfeld D P, Schwartzberg PL, Venegas A, Tse R, Sheetz MP (1999). Selective regulation of integrin --cytoskeleton interactions by the tyrosine kinase Src. Nat Cell Biol 1: 2006.

PAGE 113

113 Folkman J (1990). What is the evidence that tumors are angiogenesis dependent? J Natl Ca ncer Inst 82: 4 6. Frame MC (2002). Src in cancer: deregulation and consequences for cell behaviour. Biochim Biophys Acta 1602: 114 30. Francis JL, Carty N, Amirkhosravi M, Loizidou M, Cooper A, Taylor I (1992). The effect of Warfarin and factor VII on tis sue procoagulant activity and pulmonary seeding. Br J Cancer 65: 329 34. Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A et al (1991). E -cadherin -mediated cell -cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 113: 17385. Garcia R, Parikh NU, Saya H, Gallick GE (1991). Effect of herbimycin A on growth and pp60c src activity in human colon tumor cell lines. Oncogene 6: 19839. Gentile A, Trusolino L, Comoglio PM (2008). The Met tyrosine kinase receptor in development and cancer. Cancer Metastasis Rev 27: 8594. Gomes N, Vassy J, Lebos C, Arbeille B, Legrand C, Fauvel Lafeve F (2004). Breast adenocarcinoma cell adhesion to the vascular subendothelium in whole blood and under flow conditions: effects of alphavbeta3 and alphaIIb beta3 antagonists. Clin Exp Metastasis 21: 55361. Hauck CR, Hsia DA, Puente XS, Cheresh DA, Schlaepfer DD (2002). FRNK blocks v-Src stimulated invasion and experimental metastases without effects on cell motility or growth. Embo J 21: 6289302. Hennequin LF, Allen J, Breed J, Curwen J, Fennell M, Green TP et al (2006). N (5 -chloro 1,3 benzodioxol 4 yl) 7 -[2 (4 -methylpiperazin 1 yl)ethoxy] 5 (tetrahydro 2H -pyran 4 yloxy)quinazolin 4 amine, a novel, highly selective, orally available, dual -specific c Src/Ab l kinase inhibitor. J Med Chem 49: 646588. Henriquez NV, van Overveld PG, Que I, Buijs JT, Bachelier R, Kaijzel EL et al (2007). Advances in optical imaging and novel model systems for cancer metastasis research. Clin Exp Metastasis 24: 699705. Herynk MH Zhang J, Parikh NU, Gallick GE (2007). Activation of Src by c -Met overexpression mediates metastatic properties of colorectal carcinoma cells. J Exp Ther Oncol 6: 20517. Hiscox S, Morgan L, Green TP, Barrow D, Gee J, Nicholson RI (2006). Elevated Src ac tivity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells. Breast Cancer Res Treat 97: 26374. Hsu MY, Meier FE, Nesbit M, Hsu JY, Van Belle P, Elder DE et al (2000). E -cadherin expression in melanoma cells restores keratino cyte -mediated growth control and down regulates expression of invasion related adhesion receptors. Am J Pathol 156: 151525.

PAGE 114

114 Iiizumi M, Mohinta S, Bandyopadhyay S, Watabe K (2007). Tumor -endothelial cell interactions: therapeutic potential. Microvasc Res 7 4: 11420. Isayeva T, Kumar S, Ponnazhagan S (2004). Anti angiogenic gene therapy for cancer (review). Int J Oncol 25: 33543. Ito A, Watabe K, Koma Y, Kitamura Y (2002). An attempt to isolate genes responsible for spontaneous and experimental metastasis i n the mouse model. Histol Histopathol 17: 9519. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ (2009). Cancer statistics, 2009. CA Cancer J Clin 59: 22549. Jenkins DE, Hornig YS, Oei Y, Dusich J, Purchio T (2005). Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice. Breast Cancer Res 7: R44454. Jenkins DE, Yu SF, Hornig YS, Purchio T, Contag PR (2003). In vivo monitoring of tumor relapse and me tastasis using bioluminescent PC 3M -luc C6 cells in murine models of human prostate cancer. Clin Exp Metastasis 20: 74556. Johnson LN, Lewis RJ (2001). Structural basis for control by phosphorylation. Chem Rev 101: 220942. Kadono Y, Okada Y, Namiki M, Se iki M, Sato H (1998). Transformation of epithelial MadinDarby canine kidney cells with p60(v -src) induces expression of membrane -type 1 matrix metalloproteinase and invasiveness. Cancer Res 58: 22404. Kallman RF, Silini G, Van Putten LM (1967). Factors i nfluencing the quantitative estimation of the in vivo survival of cells from solid tumors. J Natl Cancer Inst 39: 53949. Kaplan KB, Swedlow JR, Morgan DO, Varmus HE (1995). c -Src enhances the spreading of src / fibroblasts on fibronectin by a kinase inde pendent mechanism. Genes Dev 9: 150517. Krstein Mseide TKaRPH (2005). Microenvironmental effects on tumour progression and metastasis vol. 15. Springer Netherlands, 22pp. Khwaja A, Rodriguez -Viciana P, Wennstrom S, Warne PH, Downward J (1997). Matrix adhesion and Ras transformation both activate a phosphoinositide 3 OH kinase and protein kinase B/Akt cellular survival pathway. Embo J 16: 278393. Kilarski WW, Jura N, Gerwins P (2003). Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton. Exp Cell Res 291: 7082. Kohn EC, Liotta LA (1990). L651582: a novel ant iproliferative and antimetastasis agent. J Natl Cancer Inst 82: 5460.

PAGE 115

115 Koppikar P, Choi SH, Egloff AM, Cai Q, Suzuki S, Freilino M et al (2008). Combined inhibition of c -Src and epidermal growth factor receptor abrogates growth and invasion of head and nec k squamous cell carcinoma. Clin Cancer Res 14: 428491. Kumar P, Amin MA, Harlow LA, Polverini PJ, Koch AE (2003). Src and phosphatidylinositol 3 kinase mediate soluble E -selectin -induced angiogenesis. Blood 101: 39608. Lance A. Liotta and Elise C. Kohn. Cancer Medicine Invasion and Metastases http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed6&part=A2597 (accessed October 2009). Lee D, Gautschi O (2006). Clinical development of SRC tyrosine kinase inhibitors in lung cancer. Clin Lung Cancer 7: 3814. Luo J, Lubaroff DM, Hendrix MJ (1999). Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E -cadherin transfection. Cancer Res 5 9: 35526. Lutz MP, Esser IB, Flossmann -Kast BB, Vogelmann R, Luhrs H, Friess H et al (1998). Overexpression and activation of the tyrosine kinase Src in human pancreatic carcinoma. Biochem Biophys Res Commun 243: 503 8. Lyons SK (2005). Advances in imagin g mouse tumour models in vivo. J Pathol 205: 194205. Maekawa R, Maki H, Wada T, Yoshida H, Nishida Nishimoto K, Okamoto H et al (2000). Anti metastatic efficacy and safety of MMI 166, a selective matrix metalloproteinase inhibitor. Clin Exp Metastasis 18: 616. Mahabeleshwar GH, Byzova TV (2007). Angiogenesis in melanoma. Semin Oncol 34: 55565. Martin GS (2001). The hunting of the Src. Nat Rev Mol Cell Biol 2: 46775. Marx M, Warren SL, Madri JA (2001). pp60(c -src) modulates microvascular endothelial phenotype and in vitro angiogenesis. Exp Mol Pathol 70: 20113. Matsumoto S, Tanaka F, Sato K, Kimura S, Maekawa T, Hasegawa S et al (2009). Monitoring with a noninvasive bioluminescent in vivo imaging system of pleural metastasis of lung carcinoma. Lung Canc er 66: 759. McLean GW, Fincham VJ, Frame MC (2000). v-Src induces tyrosine phosphorylation of focal adhesion kinase independently of tyrosine 397 and formation of a complex with Src. J Biol Chem 275: 233339. Mitra SK, Schlaepfer DD (2006). Integrin regul ated FAK -Src signaling in normal and cancer cells. Curr Opin Cell Biol 18: 51623. Moasser MM, Srethapakdi M, Sachar KS, Kraker AJ, Rosen N (1999). Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest. Cancer Res 59: 6145 52.

PAGE 116

116 Morabito A, De Maio E, Di Maio M, Normanno N, Perrone F (2006). Tyrosine kinase inhibitors of vascular endothelial growth factor receptors in clinical trials: current status and future directions. Oncologist 11: 75364. Mueller KL, Hunter LA, Ethier SP, Boerner JL (2008). Met and c Src cooperate to compensate for loss of epidermal growth factor receptor kinase activity in breast cancer cells. Cancer Res 68: 331422. Neil J. Gallagher, Andrew J. Lockton, Merran Macpherson, Anna Marshall, Glen Clack (2005). A phase I multiple ascending dose study to assess the safety, tolerability and pharmacokinetics of AZD0530, a highly selective, orally available, dual -specific Src-Abl kinase inhibitor. J Clin Oncol 23, 3125. Newton SA, E. J. Reeves, H. A. Gralnick, S Mohla, K. M. Yamada, K. Olden, and S. K. Akiyama (1995). Role of integrin fibronectin receptor in metastasis of human breast carcinoma cells in athymic nude mice. Int. J. Oncol. 6: 10631070. Palframan R, Airey M, Moore A, Vugler A, Nesbitt A (2009). Use of biofluorescence imaging to compare the distribution of certolizumab pegol, adalimumab, and infliximab in the inflamed paws of mice with collagen -induced arthritis. J Immunol Methods 348: 3641. Park SI, Shah AN, Zhang J, Gallick GE (2007). Regulation of angiogenesis and vascular permeability by Src family kinases: opportunities for therapeutic treatment of solid tumors. Expert Opin Ther Targets 11: 120717. Parsons JT, Parsons SJ (1997). Src family protein tyrosine kinases: cooperating with growth facto r and adhesion signaling pathways. Curr Opin Cell Biol 9: 18792. Playford MP, Schaller MD (2004). The interplay between Src and integrins in normal and tumor biology. Oncogene 23: 792846. Pongchairerk U, Guan JL, Leardkamolkarn V (2005). Focal adhesion kinase and Src phosphorylations in HGF induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA 1. World J Gastroenterol 11: 584552. Pulaski BA, Ostrand Rosenberg S (2001). Mouse 4T1 breast tumor model. Curr Protoc Immunol Chapter 20 : Unit 20 2. Purnell PR, Mack PC, Tepper CG, Evans CP, Green TP, Gumerlock PH et al (2009). The Src inhibitor AZD0530 blocks invasion and may act as a radiosensitizer in lung cancer cells. J Thorac Oncol 4: 448 54. Roche S, Fumagalli S, Courtneidge SA (1995). Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division. Science 269: 15679. Rucci N, Susa M, Teti A (2008). Inhibition of protein kinase c -Src as a therapeutic approach for cancer and bone metastases. Anticancer Agents Med Chem 8: 3429.

PAGE 117

117 Saad F (2009). Src as a therapeutic target in men with prostate cancer and bone metastases. BJU Int 103: 43440. Scatena CD, Hepner MA, Oei YA, Dusich JM, Yu SF, Purchio T et al (2004). Imaging of bioluminescent LNCaP luc M6 tumors: a ne w animal model for the study of metastatic human prostate cancer. Prostate 59: 292303. Schenone S, Manetti F, Botta M (2007). SRC inhibitors and angiogenesis. Curr Pharm Des 13: 211828. Schlaepfer DD, Mitra SK, Ilic D (2004). Control of motile and invasi ve cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692: 77102. Schneider MR, Schirner M, Lichtner RB, Graf H (1996). Antimetastatic action of the prostacyclin analogue cicaprost in experimental mammary tumors. Breast Cancer Res Treat 38: 1 3341. Schwartzberg PL, Xing L, Hoffmann O, Lowell CA, Garrett L, Boyce BF et al (1997). Rescue of osteoclast function by transgenic expression of kinase -deficient Src in src / mutant mice. Genes Dev 11: 283544. Schweppe RE, Kerege AA, French JD, Sharma V, Grzywa RL, Haugen BR (2009). Inhibition of Src with AZD0530 reveals the Src -Focal Adhesion kinase complex as a novel therapeutic target in papillary and anaplastic thyroid cancer. J Clin Endocrinol Metab 94: 2199203. Siemann DW, Mulcahy RT (1984). Char acterization of growth and radiation response of KHT tumor cells metastatic from lung to ovary and kidney. Clin Exp Metastasis 2: 7381. Soriano P, Montgomery C, Geske R, Bradley A (1991). Targeted disruption of the c -src proto oncogene leads to osteopetrosis in mice. Cell 64: 693702. Takahashi Y (1999). [Experimental model for cancer metastasis]. Gan To Kagaku Ryoho 26: 4017. Tavoloni N, Inoue H, Sabe H, Hanafusa H (1994). v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorag e independent growth involves heterogeneity of primary cultures. J Cell Biol 126: 47583. Taylor SJ, Shalloway D (1996). Src and the control of cell division. Bioessays 18: 9 11. Termuhlen PM, Curley SA, Talamonti MS, Saboorian MH, Gallick GE (1993). Site -specific differences in pp60c -src activity in human colorectal metastases. J Surg Res 54: 2938. The Cancer Cure Foundation Cancer statistics from NCI/ACS http://www.cancure.org/statistics.htm (acces sed October 2009). Thomas SM, Brugge JS (1997). Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13: 513609.

PAGE 118

118 Timpson P, Jones GE, Frame MC, Brunton VG (2001). Coordination of cell polarization and migration by the Rho family GTPa ses requires Src tyrosine kinase activity. Curr Biol 11: 183646. Toyoshima K, Yamamoto T, Kawai S, Yoshida M (1987). Viral oncogenes, vyes and v -erbB, and their cellular counterparts. Adv Virus Res 32: 97127. Underiner TL, Ruggeri B, Gingrich DE (2004). Development of vascular endothelial growth factor receptor (VEGFR) kinase inhibitors as anti angiogenic agents in cancer therapy. Curr Med Chem 11: 73145. Urquidi V, Sloan D, Kawai K, Agarwal D, Woodman AC, Tarin D et al (2002). Contrasting expression of thrombospondin1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis. Clin Cancer Res 8: 6174. Vleminckx K, Vakaet L, Jr., Mareel M, Fiers W, van Roy F (1991). Genetic manipulation of E cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 66: 10719. Wang HH, Qiu H, Qi K, Orr FW (2005). Current views concerning the influences of murine hepatic endothelial adhesive and cytotoxic pro perties on interactions between metastatic tumor cells and the liver. Comp Hepatol 4: 8. Wei L, Yang Y, Zhang X, Yu Q (2004). Altered regulation of Src upon cell detachment protects human lung adenocarcinoma cells from anoikis. Oncogene 23: 905261. Whites ell L, Mimnaugh EG, De Costa B, Myers CE, Neckers LM (1994). Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci U S A 91: 83248. Widel MS, Widel M (2006). [Mechanisms of metastasis and molecular markers of malignant tumor progression. I. Colorectal cancer]. Postepy Hig Med Dosw (Online) 60: 45370. Wiener JR, Windham TC, Estrella VC, Parikh NU, Thall PF, Deavers MT et al (2003). Activated SRC protein tyrosine kinase is overexpressed in late -stage human ovarian cancers. Gynecol Oncol 88: 739. Wittekind C, Neid M (2005). Cancer invasion and metastasis. Oncology 69 Suppl 1: 146. Wu CY, Hsieh HL, Sun CC, Tseng CP, Yang C M (2008). IL 1 beta induces proMMP 9 expression via c Src -dependent PDGFR/PI3K/Akt/p300 cascade in rat brain astrocytes. J Neurochem 105: 1499512. Yeatman TJ (2004). A renaissance for SRC. Nat Rev Cancer 4: 47080.

PAGE 119

119 Zacharski LR, Henderson WG, Rickles FR, Forman WB, Cornell CJ, Jr., Forcier RJ et al (1984). Effect of warfarin anticoagulation on survival in carcinoma of the lung, colon, head and neck, and prostate. Final report of VA Cooperative Study #75. Cancer 53: 204652. Zacharski LR, Meehan KR, Algarr a SM, Calvo FA (1992). Clinical trials with anticoagulant and antiplatelet therapies. Cancer Metastasis Rev 11: 42131. Zhang J, Yang PL, Gray NS (2009). Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 9: 2839. Zheng R, Yano S, Mats umori Y, Nakataki E, Muguruma H, Yoshizumi M et al (2005). SRC tyrosine kinase inhibitor, m475271, suppresses subcutaneous growth and production of lung metastasis via inhibition of proliferation, invasion, and vascularization of human lung adenocarcinoma cells. Clin Exp Metastasis 22: 195204.

PAGE 120

120 BIOGRAPHICAL SKETCH Meiyu Dong was born in a small city in Northern China and was moved with her parents when she was still a kid to a big city Tianjin. She spent her childhood, middle school, high school and un iversity life in that same city. She was raised in a big happy family. Her father used to work as a professor, which provides her an environment of curiosity and research. After graduating from Tianjin Medical University with a masters degree from medi cal school, she decided to leave Tianjin, where she spent about 25 years, and pursue her Ph.D. at the University of Florida in 2004. She was accepted in IDP program and joined the Department of Pharmacology and Therapeutics under the mentorship of Dr. Die tmar W. Siemann in 2005. Luckily, she met her husband Zhihong Hu on UF campus and got married in 2006. What is more, she had her first born son, Robert Hu in 2008.