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Translational Oncology Scientist, Cancer Research Laboratory Program, London Regional Cancer Program, London Health Sciences Centre, London, Ontario
Assistant Professor: Department of Obstetrics & Gynecology, University of Western Ontario, London, Ontario
Cross Appointments: Department of Oncology, Department of Anatomy & Cell Biology
Link to my UWO website:
Translational Ovarian Cancer Research through Primary Cell Culture and Animal Modelling
Translational application of well-supported, fundamental ovarian cancer research to potential improved clinical outcome for ovarian cancer patients cannot be emphasized enough. Every year in Canada alone, about 2400 women will be diagnosed with ovarian cancer, and 1700 women will die of this disease (2007 Canadian Cancer Statistics). This is entirely due to the lack of reliable early detection methods combined with poor therapeutic management of recurrent disease. Taken together, my proposed research program will further elucidate the functional implications of altered bone morphogenetic protein (BMP) signalling in epithelial ovarian cancer (EOC) cell adhesion and metastasis and ID proto-oncogene expression in ovarian cancer initiation and progression. I will also utilize important, current techniques which are critically relevant to ovarian cancer research, including culturing and characterization of primary human ovarian surface epithelial (OSE) and EOC cells, as well as the more recently-established transgenic mouse modelling of this disease. I foresee the direct translational potential of my proposed research via the rapid initiation of key collaborative research efforts with basic and clinical scientists of the Translational Ovarian Cancer Research Group of the London Regional Cancer Program and the University of Western Ontario. More importantly, I will aid in establishing new research endeavours into the field of ovarian cancer with basic scientists of the LRCP, as well as participate in clinical research projects with clinicians of the Division of Gynaecologic Oncology.
Trevor G. Shepherd and Mark W. Nachtigal. (2007) Constitutively-active bone morphogenetic protein receptor signalling reduces ovarian cancer cell adhesion and intraperitoneal tumour formation. Manuscript in revision for Gynecologic Oncology.
Trevor G. Shepherd and Mark W. Nachtigal (2007) Distinct enhancer regions of the ID3 gene confer bone morphogenetic protein-regulated gene expression through conserved bipartite elements. Manuscript submitted to Gene.
Natasza A. Kurpios, Lesley MacNeil, Trevor G. Shepherd and John A. Hassell (2007) The Pea3 Ets transcription factor regulates differentiation of multipotent progenitor cells during mammary gland development. Submitted to Developmental Biology.
Brigitte L. Theriault, Trevor G. Shepherd, Michelle L. Mujoomdar and Mark W. Nachtigal (2007) BMP4 signalling induces EMT and promotes the aggressive phenotype of human ovarian cancer cells. Carcinogenesis, in press.
Trevor G. Shepherd, Brigitte L. Theriault, Elizabeth Campbell & Mark W. Nachtigal (2006) Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients. Nat Protocols 1: 2643-9.
Yangxin Fu, Laura M. O’Connor, Trevor G. Shepherd, Mark W. Nachtigal (2003) The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor ß (TGFß) induced Smad signaling in human ovarian cancer cells. Biochem Biophys Res Commun, 310: 391-7.
Trevor G. Shepherd and Mark W. Nachtigal (2003) Identification of a putative autocrine bone morphogenetic protein (BMP) signaling pathway in human ovarian surface epithelium (OSE) and ovarian cancer (OC) cells. Endocrinology 144: 3306-14.
Yangxin Fu, Elizabeth J. Campbell Dwyer, Trevor G. Shepherd and Mark W. Nachtigal (2003) Promoter hypermethylation and histone deacetylation contribute to reduced PACE4 gene expression in human ovarian cancer cells. Molec Cancer Res 1: 569-76.
Natasza A. Kurpios, Nancy Sabolic, Trevor G. Shepherd, Gina Fidalgo and John A. Hassell (2003) Function of PEA3 ets transcription factors in mammary gland development and oncogenesis. J Mammary Gland Biol Neoplasia. 8: 177-90.
Lesley D. Dunfield, Trevor G. Shepherd and Mark W. Nachtigal (2002) Primary culture and mRNA analysis of human ovarian cells. Biol Proced Online 4: 55-61.
Trevor G. Shepherd, Lisa Kockeritz, Michelle R. Szrajber, William J. Muller and John A. Hassell (2001) The pea3 subfamily ets genes are required for HER2/Neu-mediated mammary oncogenesis. Curr Biol 11: 1739-1748.
Trevor Shepherd and John A. Hassell (2001) Role of Ets transcription factors in mammary gland development and oncogenesis. J Mam Gland Biol Neoplasia 6: 129-140.
Nelson K. S. Khoo, John F. Bechberger, Trevor Shepherd, Shari L. Bond, Keith R. McCrae, G. Scot Hamilton, and Peeyush K. Lala (1998) SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGFbeta. Int J Cancer 77: 429-39.
London Regional Cancer Program
Cancer Research Laboratory Program
790 Commissioners Rd. E.
Canada N6A 4L6
Tel: 519.685.8500 ext. 56347
Ovarian cancer is the sixth most prevalent cancer amongst women and is the most lethal of the gynecological malignancies, yet the current knowledge of the molecular basis for this disease is still quite deficient. Thus, the discovery and thorough examination of molecules potentially implicated in ovarian cancer pathogenesis is an ongoing pursuit. To this end, I have identified that active bone morphogenetic protein (BMP)-4 signalling leads to the direct and heightened upregulation of ID1 and ID3 proto -oncogenes in human epithelial ovarian cancer (EOC) cells in comparison with normal human ovarian surface epithelial (OSE) cells. The ID genes encode helix-loop-helix proteins that bind to and inhibit the function of transcription factors; ID gene overexpression has been observed in human cancers, including ovarian cancer, and is correlated with increased metastatic potential and poor patient prognosis. My analyses addressing the mechanism controlling BMP4-mediated regulation of ID3 gene expression in EOC cells has provided additional clues explaining both the similar and differential regulation of the ID1 and ID3 proto -oncogenes that may contribute to ovarian tumorigenesis . Thus, I hypothesize that ID1 & ID3 gene overexpression contributes to human ovarian cancer pathogenesis . To directly address this research question, my studies will focus on the following:
Aim 1 : Are Id1 & Id3 proteins required for the ovarian cancer phenotype?
Aim 2 : Are Id1 & Id3 proteins involved in ovarian cancer initiation and progression?
Aim 3 : What are the important Id1- & Id3-interacting proteins in ovarian cancer cells?
Aim 4 : What key genes do Id1 & Id3 proteins regulate in ovarian cancer cells?
To carry out this research program, I will utilize several different research tools and technologies which I have developed during my postdoctoral studies. For example, ectopic overexpression of Id1 and Id3 proteins, and knockdown studies using RNA interference technology, will be performed using recombinant adenoviral vectors. I will continue to isolate and use primary human normal OSE and ascites-derived EOC cells via the establishment of collaborative efforts with the clinicians of the Division of Gynaecologic Oncology. My in vivo analyses will entail adenovirus transduction of intact mouse OSE cells by intrabursal virus administration under the ovarian bursa, as well as intraperitoneal xenografting of stably-transfected cell lines into nude mice. The results from these studies will clearly demonstrate whether Id1 and Id3 play an important role in human ovarian cancer initiation and progression. As part of the collaborative research efforts within the Translational Ovarian Cancer Research Program, transgenic mouse strains will be generated (Dr. Gabriel DiMattia as co-investigator) to develop accurate mouse models of human ovarian cancer pathogenesis.
The majority of ovarian cancer cases are first diagnosed at late-stage disease when the cancer cells have disseminated throughout the peritoneal cavity, forming metastatic lesions by adhering to mesothelial surfaces and invading ectopic tissue sites. This underscores the requirement for greater understanding of the molecular mechanisms controlling ovarian cancer cell adhesion. To this end, I have identified that active bone morphogenetic protein-4 (BMP4) signalling affects the adhesion properties of human EOC cells. BMP4 ligand secreted by EOC cells signals via the intracellular Smad transcription factor complex and upregulates ID1 and ID3 proto-oncogene expression. Natural BMP4 ligand-mediated signalling causes cell spreading and increases EOC cell adhesion; however, in marked contrast, constitutively-active mutant BMP receptor expression in human EOC cells enhances tumour cell detachment, reduces beta-integrin expression, and decreases the ability of EOC cells to adhere and form intraperitoneal tumours in nude mice. Taken together, I hypothesize that autocrine BMP4 signalling affects ovarian cancer dissemination and metastasis by modulating EOC cell adhesion. To address this research question, my studies will focus on the following:
Aim 1 : How does BMP signalling regulate adhesion molecules in EOC cells?
Aim 2 : Does Smad-dependent vs. -independent BMP signalling control EOC cell adhesion?
Aim 3 : Are BMP signalling effects on EOC cell adhesion mediated by ID1 & ID3 genes?
To carry out this research program, primary human normal OSE and ascites-derived EOC cells will be used. Activation of BMP signalling will entail treating with recombinant BMP ligands, as well as utilizing adenoviral vectors which express constitutively-active BMP type I receptors. Virus-mediated transduction of EOC cells with short-hairpin RNA expression constructs targeting ID1 and ID3 mRNA will be performed, as well as targeted knockdown of Smad4. Cell culture studies will involve both standard two-dimensional cultures as well as three-dimensional culturing of EOC cell spheroids. Modelling spheroid growth in culture will allow the direct study of BMP signalling on cell-cell interactions that may be involved in mediating EOC cell clustering that occurs in patient ascites. Most importantly, as consistently laid out in my overall ovarian cancer research program, I will use parallel cell culture and in vivo mouse modelling systems to directly address the functional role of BMP signalling on EOC cell adhesion to approximate the physiological and pathological context of ovarian cancer in patients.