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2005 Scientific Report

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Laboratory of Signal

Laboratory of Signal Regulation and Cancer Sara A. Courtneidge, Ph.D. Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research in London. She began her career in the basic sciences in 1978 as a postdoctoral fellow in the laboratory of J. Michael Bishop at the University of California School of Medicine. She later joined her alma mater as a member of the scientific staff. In 1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as group leader and in 1991 was appointed a senior scientist with tenure. She joined Sugen in 1994 as Vice President of Research, later becoming Senior Vice President of Research and then Chief Scientist. Dr. Courtneidge joined VARI in January 2001 as a Distinguished Scientific Investigator. Staff Eduardo Azucena, Ph.D. Paul Bromann, Ph.D. Hasan Korkaya, Ph.D. Laboratory Members Ian Pass, Ph.D. Darren Seals, Ph.D. Laila Al-Duwaisan Research Interests Our laboratory wants to understand at the molecular level how proliferation is controlled in normal cells and by what mechanisms these controls are subverted in tumor cells. We largely focus on a family of oncogenic tyrosine kinases, the Src family. The prototype of the family, vSrc, originally discovered as the transforming protein of Rous sarcoma virus, is a mutated and activated version of a normal cellular gene product, cSrc. The activity of all members of the Src family is normally under strict control; however the enzymes are frequently activated or overexpressed, or both, in human tumors. In normal cells, Src family kinases (SFKs) have been implicated in signaling from many types of receptors, including receptor tyrosine kinases, integrin receptors, and G protein–coupled receptors. Signals generated by SFKs are thought to play roles in cell cycle entry, cytoskeletal rearrangements, cell migration, and cell division. In tumor cells, Src may play a role in growth factor–independent proliferation or in invasiveness. Furthermore, some evidence points to a role for SFKs in angiogenesis. Some of the current projects in the laboratory are outlined below. The role of the Src substrate Tks5/Fish in tumorigenesis Tks5/Fish is an adaptor protein which has five SH3 domains and a phox homology (PX) domain. Tks5/Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells after treatment with any of several growth factors. We recently found that in Src-transformed cells, Tks5/Fish is localized to specialized regions of the plasma membrane called podosomes (sometimes referred to as invadopodia). These actin-rich protrusions from the plasma membrane are sites of matrix invasion and locomotion. The PX domain of Tks5/Fish associates with phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, and it is required for targeting Tks5/Fish to podosomes. The fifth SH3 domain of Tks5/Fish mediates its association with members of the ADAMs family of membrane metalloproteases, which in Srctransformed cells are also localized to podosomes. We have begun to dissect the role of Tks5/Fish in transformation. Src-transformed cells with reduced Tks5/Fish levels no longer form podosomes and are poorly invasive. We detected Tks5/Fish expression in podosomes in invasive human cancer cell lines, as well as in tissue samples from human breast cancer and melanoma. Tks5/Fish expression was also required for invasion of human cancer cells. Furthermore, we have developed an assay to generate podosomes upon expression of Tks5/Fish, which will allow us to dissect the requirements for podosome formation in more detail. We are also investigating the potential of both Tks5/Fish and its binding proteins as markers of invasive disease and as potential therapeutic targets. 18

The role of Src family kinases in mitogenic signaling pathways We have previously shown that Src family kinases are required for both Myc induction and DNA synthesis in response to platelet-derived growth factor (PDGF) stimulation of NIH3T3 fibroblasts. We have also previously identified and characterized a small-molecule inhibitor of Src family kinases called SU6656. We wanted to address whether there is a discrete SFK-specific pathway leading to enhanced gene expression, or whether SFKs act to generally enhance PDGFstimulated gene expression. To do this, we treated quiescent NIH3T3 cells with PDGF in the presence or absence of SU6656 and analyzed global patterns of gene expression. We determined that a discrete set of immediate early genes was induced by PDGF and inhibited by SU6656. We further determined that SFKs did not stimulate the rate of transcription of these genes, but rather promoted mRNA stabilization. We are currently exploring how SFKs signal gene expression by enhancing mRNA stability. Breast cancer Increased Src activity can be demonstrated in the majority of breast cancers, both estrogendependent and estrogen-independent, yet the role of Src in breast tumorigenesis has not been fully established. We have been characterizing the role of Src in estrogen-stimulated signal transduction pathways in breast cancer cell lines. We have shown that Src family kinase activity is required for estrogen to stimulate mitogenesis in MCF7 cells. Furthermore, inhibition of Src prevents estrogen stimulation both of Myc and of MAP kinase activity. We are currently dissecting which Src signaling pathways are necessary for estrogenstimulated growth, as well as how Src activity results in the activation of MAP kinase and in the production of Myc. Recent Publications Bromann, Paul A., Hasan Korkaya, Craig P. Webb, Jeremy Miller, Tammy L. Calvin, and Sara A. Courtneidge. 2005. Platelet-derived growth factor stimulates Src-dependent mRNA stabilization of specific early genes in fibroblasts. Journal of Biological Chemistry 280(11): 10253–10263. Seals, Darren F., Eduardo F. Azucena, Jr., Ian Pass, Lia Tesfay, Rebecca Gordon, Melissa Woodrow, James H. Resau, and Sara A. Courtneidge. 2005. The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell 7(2): 155–165. Bromann, Paul A., Hasan Korkaya, and Sara A. Courtneidge. 2004. The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 23(48): 7957–7968. From left to right: Azucena, Seals, Pass, Al-Duwaisan, Bromann 19

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