Sprouty in breast cancer

SPROUTY IN BREAST CANCER LO TING LING B.SC. (PHARMACY) WITH HONOURS NATIONAL UNIVERSITY OF SINGAPORE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 I ACKNOWLEDGEMENTS My gratitude and special thanks extend to: Associate Prof. Graeme R. Guy for his excellent mentorship and guidance. His constant support and encouragement have been instrumental towards the progress of my Ph.D. research. I am also thankful to the following Ph.D. review committee: Associate Prof. Uttam Surana Associate Prof. Low Boon Chuan for their constructive suggestions and scientific discussions. The following scientists who helped me in various ways: Dr. Permeen Yusoff for assistance in immunohistochemistry and in situ hybridization experiments. Dr. Fong Chee Wai for scientific guidance in my first year of PhD. Dr. Rebecca Jackson and Chow Soah Yee for proof reading this thesis. Dr. Jormay Lim, Dr. Lao Dieu Hung, Sumana Chandramouli and Chow Soah Yee for discussions and sharing of reagents. All past and present lab members and all people who have helped me in one way another. Heartfelt appreciation goes to my parents and my husband for their unwavering love, support and patience throughout my Ph.D. candidature. Ting Ling Lo April 2007 II TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF FIGURES VIII LIST OF TABLES XI ABBREVIATIONS XII SUMMARY XVII CHAPTER Introduction 1.1 Mammalian cell signalling 1.2 Receptor tyrosine kinase (RTK) signalling pathways 1.3 Ras/ERK pathway 1.3.1 Introduction 1.3.2 Adapter and scaffold proteins 1.3.3 Ras 1.3.4 Raf 1.3.5 MEK 1.3.6 ERK 1.3.7 Modulators of the strength and duration of ERK signalling 1.4 Sprouty family of proteins 10 1.4.1 Discovery of Sprouty 10 1.4.2 Structure of Sproutys 13 1.4.3 Sproutys and their mechanisms of inhibition of RTK signalling 13 1.4.4 Regulation of the activity of Sproutys 16 1.4.4.1 Post-translational modifications of Sproutys 16 1.4.4.1.1 Tyrosine phosphorylation of Sproutys 1.4.4.1.2 Serine phosphorylation and palmitoylation of Sproutys III 1.4.5 1.5 1.5.2 1.5.3 1.5.4 1.7 18 Physiological functions of Sproutys 20 1.4.5.1 Vertebrate development 20 1.4.5.2 Proliferation and differentiation 22 1.4.5.3 Cell motility 22 Cancer 1.5.1 1.6 1.4.4.2 Regulation of Sprouty levels 23 Alterations of three types of genes are responsible for tumourigenesis: oncogenes, tumour suppressor genes and stability genes 23 Mechanisms of alterations in cancer susceptibility genes 26 1.5.2.1 Genetic mechanisms 26 1.5.2.2 Epigenetic mechanisms 27 1.5.2.3 Transcriptional mechanisms 29 Implications of alterations in cancer susceptibility genes 30 1.5.3.1 RTK as oncogenes 32 1.5.3.2 Oncogenes in Ras-Raf-MEK-ERK pathway 32 1.5.3.2.1 Ras 33 1.5.3.2.2 Raf 33 1.5.3.3 Tumour suppressors 35 Tumour markers 38 Polyoma Middle T antigen (PyMT) as a cancer model 40 1.6.1 Introduction to Polyoma virus 40 1.6.2 PyMT as a model to study transformation 40 1.6.3 General structure of PyMT 41 1.6.4 PyMT signalling pathways 42 Aims of research CHAPTER 2.1 46 Materials and Methods Chemicals and reagents 47 IV 2.2 DNA Methodology 47 2.2.1 General DNA manipulations 47 2.2.2 Preparation of electro-competent cells 48 2.2.3 Purification and analysis of DNA 48 2.2.4 Plasmid constructions 49 2.2.5 Reverse transcription-polymerase chain reaction (RT-PCR) analysis 50 2.2.6 Microarray gene expression data analysis 50 2.2.7 Quantitative real-time PCR 51 2.2.8 DNA sequencing 52 2.2.9 Synthesis of [α-32P] dCTP-labeled cDNA 52 2.2.10 Hybridization of cDNA probes to Cancer Profiling Array 52 2.2.11 Loss of heterozygosity (LOH) assay 53 2.3 RNA extraction and reverse transcription 54 2.4 Histological analysis 54 2.5 RNA in situ hybridization analysis 54 2.6 Immunohistochemistry 55 2.7 Cell culture 56 2.7.1 Transfection of mammalian cells 56 2.7.2 Treatment of cells with 5-aza-deoxycytidine and trichostatin 56 2.7.3 Stable transfection 57 2.7.4 Proliferation assay 57 2.7.5 Transformation assays 58 2.7.6 Staining for foci formation 59 2.7.7 Soft agar colony formation 59 2.7.8 In vivo tumour formation 59 2.7.9 siRNA knock down 60 2.7.10 Adenovirus System 60 Protein methodology 60 2.8 V 2.8.1 Preparation of extracts from cells 60 2.8.2 Analysis of proteins 61 2.8.3 Antibodies and immunoprecipitation reagents 61 2.8.4 Immunoprecipitations 62 2.8.5 Western transfer and immunodetection 62 CHAPTER 3.1 Down-regulation of Sproutys in breast cancer hSpry1 and expression is down-regulated in breast cancer 63 3.1.1 Microarray database 63 3.1.2 Matched normal and tumour tissue cDNA array studies 65 3.1.3 Quantitative real-time PCR studies 73 3.2 Expression of mSpry1 and mSpry2 in developing mouse mammary gland 76 3.3 In situ analysis of human breast tissues showed the down-regulation of Spry isoforms in breast cancer 84 Immunohistochemical analysis of human breast tissues showed the down-regulation of Spry isoforms in breast cancer 87 3.5 Expression of Spry2 in breast cancer cell lines 89 3.6 Down-regulation of hSpry2 in breast cancer is not due to epigenetic silencing 91 Inhibiting Spry’s function in MCF-7 cells results in cells proliferating faster and exhibiting anchorage-independent growth 97 Inhibiting Spry’s function in MCF-7 cells results in the formation of larger tumours 100 Discussion 103 3.4 3.7 3.8 3.9 CHAPTER Down-regulation of Sprouty2 in liver cancer 4.1 Introduction to Hepatocellular carcinoma (HCC) 107 4.2 Spry2 transcript is down-regulated in Hepatocellular carcinoma (HCC) 108 4.2.1 Microarray studies 108 4.2.2 Quantitative real-time PCR studies 111 VI 4.2.3 In situ and immunohistochemistry studies showed that Spry2 is differentially expressed in normal, cirrhotic and HCC liver tissues 111 4.3 Down-regulation of Spry2 in HCC is not due to loss of heterozygosity 115 4.4 Hypermethylation of promoter is not responsible for down-regulation of Spry2 117 Spry2 inhibits HGF-stimulated ERK and exerts an antiproliferative effect in the hepatoma cell line SNU449 119 Knocking down Spry2 levels transforms NIH3T3 cells in the presence of FGF stimulation 119 Discussion 123 4.5 4.6 4.7 CHAPTER Evidence of roles of Sprouty in cancers 5.1 Down-regulation of Sprys in prostate cancer 127 5.2 Evidence for Sprouty isoforms as tumour suppressors 130 5.3 Sprouty as a potential tumour marker 131 5.3.1 Melanoma 131 5.3.2 Gastrointestinal stromal tumours (GISTs) 133 5.3.3 clear cell Renal Cell Carcinomas (ccRCC) 134 5.3.4 Lung tumours induced by K-Ras 134 5.4 Summary CHAPTER 6.1 6.2 135 Sprouty inhibits Ras/ERK activation and transformation downstream of Polyoma Middle T antigen (PyMT) PyMT as a model to study transformation 137 6.1.1 Membrane binding 137 6.1.2 Binding to PP2A 139 6.1.3 Src binding 140 6.1.4 Crucial PyMT-stimulated signalling pathways that mediate transformation of cells 141 Spry2 inhibits PyMT-induced transformation in NIH3T3 cells 144 VII 6.3 Elucidation of Spry2's mechanism of inhibition of PyMTinduced transformation of cells 144 6.3.1 Spry1, and inhibit Ras/ERK signalling downstream of the PyMT 148 6.3.2 Comparison of the protein interactions of Spry isoforms in PyMT versus FGF signalling 150 6.3.2.1 Spry2 does not interact with c-Cbl in the presence of PyMT 153 6.3.2.2 Spry2's inhibition of ERK is not mediated by Grb2 sequestration 153 6.3.2.3 Conserved N-terminal tyrosine in Spry isoforms is crucial for the ability of Spry isoforms to inhibit the Ras/ERK signalling downstream of PyMT 158 6.3.2.4 Spry isoforms are not phosphorylated in the presence of PyMT 158 Asn53 and Tyr55 residues in the aa 50-60 region of Spry2 are crucial for mediating Spry2's ability to inhibit Ras/ERK signalling downstream of PyMT 158 Spry isoforms not inhibit the interaction between PyMT and its associated signaling proteins 160 6.3.3 6.3.4 6.3.5 6.4 Spry isoforms not inhibit PI3 kinase signalling downstream of PyMT 163 Discussion CHAPTER 165 Summary and future perspectives 7.1 Sprys expression is deregulated in various cancers 168 7.2 The mechanism of down-regulation of Sprys in different cancers is cancer- specific 168 Sprys as a tumour suppressors and molecular markers? 169 7.3 References 173 Publications 192 VIII LIST OF FIGURES 1.3.1 The Ras/ERK pathway. 1.3.3 Ras proteins function as regulated GDP–GTP binary switches. 1.4.1 Various isoforms of Sprouty proteins and their respective domains. 1.5.3.1 Constitutive activation of RTKs. 1.5.3.3 Inactivation of tumour suppressor genes. 1.6.3 Schematic diagram of PyMT. 1.6.4 A diagrammatic representation of Shc and PI3-kinase (PI3K) pathways stimulated by membrane-bound PyMT. 3.1.1 Spry2 is down-regulated in breast and uterine cancer. 3.1.2 (A) Layout of the commercially available cDNA array (Cancer Profiling Array) (BD Clontech). (B) hSpry1 and hSpry2 cDNA probes are specific for their respective Spry isoforms. (C) hSpry2 is down-regulated in breast cancer. (D) hSpry1 is down-regulated in breast cancer. (E) Expression levels of ErbB2 in breast tumours demonstrate that the sample population of breast tumours in the blot are a true representation of the population of breast cancers. (F) The cDNA samples in the Cancer Profiling Array are equally loaded. 3.1.3 (A), (B) Spry1 and are down-regulated in breast tumours. (C) Expression levels of ErbB2 were analyzed to show that the sample population of breast tumours used were from a representative breast cancer patient population. 3.2.1 Mouse mammary gland development during puberty, pregnancy and lactation. 3.2.2 Phases of mammary gland development. 3.2.3 (A) mSpry2 is highly expressed in developing mammary ducts in and week old females and appeared to be confined specifically to the epithelial lining of the mammary ducts. (B) mSpry2's localization in week old male mouse mammary gland was found to be similar to that observed in week old female mouse mammary gland. IX (C) In pregnant mice, the expression of mSpry2 becomes elevated again in actively proliferating alveoli. mSpry2 expression then becomes diminished in the lactating female and appears to be totally absent during the involution phase. (D) A bar chart representation of the qualitative levels of Spry2 during the various stages of mammary gland development, based on the in situ hybridization data in Fig. 3.2.3A and Fig. 3.2.3C. (E) mSpry2 sense probe control demonstrates a lack of non-specific staining. (F) Co-localization of the two Spry isoforms, specifically in the luminal epithelial cells of the mammary ducts of a pregnant mouse. 3.3.1 hSpry2 and hFgf8 are co-localized in the epithelial lining of normal breast ducts. 3.3.2 (A), (B) Spry1 and are down-regulated in human breast cancer tissues. 3.4 (A) Immunohistochemical analysis of human breast tissue showed that Spry2 localizes specifically in the epithelial lining of breast ducts. (B) Immunohistochemical analysis of human breast tissue showed that Spry2 expression is down-regulated in breast cancer. 3.5 Spry2 mRNA expression in various normal and tumour breast cell lines. 3.6.1 (A) Schematic representation of the hSPRY2 gene structure highlighting the positions of the two CpG islands (CpG I and CpG II) relative to the transcription start site. (B) Epigenetic silencing is not responsible for the down-regulation of Spry2 expression in breast tumours. 3.62 WT1, an upstream transcriptional effector of Spry1, is not the cause of downregulation of Spry1 in breast cancer. 3.7 Inhibiting Spry’s function causes MCF-7 cells to proliferate faster and exhibit anchorage independent growth. 3.8 (A), (B) Inhibiting Spry’s function causes MCF-7 cells to have greater in vivo tumourigenic potential. (C) Expression of hSpry2Y55F in the xenograft tumours excised from the nude mice. 4.2.1 Spry2 transcript is down-regulated in Hepatocellular carcinoma (HCC). 4.2.2 Spry2 but not Spry1 is significantly down-regulated in HCC. 4.2.3 Spry2 expression is relatively higher in normal and cirrhotic liver compared to HCC liver. 4.3 Loss of Heterozygosity (LOH) does not cause of the down-regulation of Spry2 HCC. in 177 El-Serag HB, Hampel H, Yeh C, Rabeneck L. Extrahepatic manifestations of hepatitis C among United States male veterans. 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J Biol Chem. 2002; 277(5):3195-201. Guy GR, Wong ES, Yusoff P, Chandramouli S, Lo TL, Lim J, Fong CW. Sprouty: how does the branch manager work? J Cell Sci. 2003; 116(Pt 15):3061-8. Review. [...]... guanine methyl transferase min minute MKP MAP kinase phosphatase ml milliliter MLH1 mutL homolog 1 mM millimolar Mnk MAPK interacting serine/threonine kinase Mnk1 MAPK interacting serine/threonine kinase 1 MP-1 MEK partner 1 N or Asn asparagine NaCl sodium chloride XV NF1 neurofibromin1 N-terminal amino (NH2)-terminal OD optical density P or Pro proline PBS phosphate-buffered saline PCR polymerase chain... 1995), which in turn function as docking sites for cytoplasmic signalling proteins containing Src homology 2 (SH2)- and phosphotyrosine-binding (PTB) domain containing proteins such as Shc, Grb2, Src, Cbl or phospholipase Cγ (PLCγ) These proteins then recruit additional effector molecules containing SH2, SH3, PTB and pleckstrinhomology (PH) domains to the activated receptor, which results in the assembly... represented in red, that mediates binding to Raf1 (Raf1-binding domain, RBD) The N-terminal half of the Spry proteins is more divergent, however, except for the presence of an invariant tyrosine residue (Y) located in a short, conserved motif, indicated in yellow Many of the inhibitory functions of the Spry proteins are dependent on this residue (B) Amino acid sequence alignment of regions containing the invariant... and scaffold proteins Adaptor and scaffolding proteins are important components of Ras/ERK signalling in eukaryotic cells These proteins play a role in intracellular signalling by both recruiting various proteins to specific locations and by assembling networks of proteins particular to a cascade Adaptor proteins, through protein-protein interactions via specific motifs, provide a link between molecules... PDVF polyvinylidene difluoride PGR progesterone receptor PH pleckstrin homology PI3K phosphatidylinositol 3-kinase PKC protein kinase C PLCγ phospholipase Cγ PP2A protein phosphatase 2A PTB phosphotyrosine-binding PtdIns (4) P phosphatidylinositol 4-triphosphate PtdIns (4,5) P phosphatidylinositol 4,5-triphosphate PtdIns (3,4,5) P phosphatidylinositol 3,4,5-triphosphate PTP1B protein tyrosine phosphatase... minute XVI RPMI Roswell Park Memorial Institute RSK ribosomal protein S6 kinase RTK receptor tyrosine kinase S or Ser serine SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sef similar expression to FGF SEM standard error of mean SGK serum/glucocorticoid inducible kinase SH2 src homology2 SH3 src homology3 Shc SH2 domain-containing transforming protein C Shp2 SH2 domain-containing... phosphorylated tyrosine Q or Gln glutamine R or Arg arginine Raf rapidly growing fibrosarcoma RalGDS ral GDP dissociation stimulator RALT/Mig-6 receptor-associated late transducer/mitogen inducible gene-6 RAR retinoic acid receptor Ras rat sarcoma RASSF1 ras association (ralGDS/AF-6) domain family 1 RB retinoblastoma RBD raf binding domain RIN ras and rab interactor 1 RKIP raf kinase inhibitor protein rpm revolutions... invariant N-terminal tyrosine residues of mouse Sprouty proteins Conserved residues are indicated in red and boxed (Figure adapted from Mason et al., 2006) 13 1.4.2 Structure of Sproutys Drosophila Spry is a 591-amino-acid (63-kDa) protein with a unique 124-residue cysteinerich C-terminal domain, flanked by cysteine-free regions that contain many stretches of repeated or alternating amino acids (Hacohen... independent of Ras Therefore, Spry is able to inhibit ERK activity through a Ras-independent pathway Spry4 binds to the catalytic domain of Raf1 through its carboxyl-terminal cysteine-rich domain (Raf-binding domain) and this binding is necessary for inhibitory activity of Spry4 Lee et al (2001) also discovered that Spry4 inhibited FGF and VEGF-induced MAPK phosphorylation in HUVEC (endothelial) cells However,... of a signalling cascade and proteins such as RTKs These adapter proteins often contain a variety of motifs that mediate protein-protein interactions An example of an adapter protein is Grb2 It contains both SH2 and SH3 sequences and its function is to recruit cytoplasmic proteins via its SH3 domain to an activated RTK via SH2 domains-binding to phosphorylated residues of the receptors In the Ras/ERK . MAPK interacting serine/threonine kinase Mnk1 MAPK interacting serine/threonine kinase 1 MP-1 MEK partner 1 N or Asn asparagine NaCl sodium chloride XV NF1 neurofibromin1 N-terminal. domain family 1 RB retinoblastoma RBD raf binding domain RIN ras and rab interactor 1 RKIP raf kinase inhibitor protein rpm revolutions per minute XVI RPMI Roswell Park Memorial Institute. SGK serum/glucocorticoid inducible kinase SH2 src homology2 SH3 src homology3 Shc SH2 domain-containing transforming protein C Shp2 SH2 domain-containing protein tyrosine phosphatase-2 SOS