CtBP makes a variety of interactions with both sequence-specific transcription factors and co-regulators that are targeted to selected chromatin locales by other sequence specific transcription factors. Through its dimerization, it is able to interact and effectively recruit specific sets of chromatin modifying factors to alter the chromatin landscape. Many of these functional CtBP Cargo contain a consensus PXDLS CtBP-binding motif that directly mediates this interaction. Others do not, and may require additional interactions that could be either direct or indirect. A major focus of this project is to define the molecular rules that dictate how and when these CtBP mediated complexes form and how they influence chromatin and gene expression. A chief aspect of this approach will be to examine how changes in the occupancy of CtBP and its functional cargo correlate with changes in those cellular attributes associated with more aggressive behavior in breast cancer. The variety of cargo carried by CtBP and the numerous complexes CtBP is able to form present a significant challenge for understanding the molecular and cellular biology of CtBP during tumor initiation and tumor progression. A central goal of this project is to demonstrate how the molecular concepts and tenets we define for CtBP function, using various cellular systems, unfold in the larger biological context of disease. This is being accomplished through the combined use of rodent animal model systems with specific emphasis on how conclusions drawn from these pre-clinical models can be extrapolated and tested in human breast cancer samples and mammary tissues. We have successfully constructed and validated 12 human and murine cell lines that have been engineered for inducible over-expression of CtBP1, or CtBP2 or inducible depletion of both CtBP1 and CtBP2 in the human breast cancer cell lines MCF-7 (estrogen receptor positive luminal cell line), MDA-MB-231 (triple negative basal-like cell line) and MCF10A (non tumorigenic breast epithelial cell line) and 4T1 (highly metastatic mesenchymal murine breast cancer cell line). These cells have been validated for expression in gene and protein expression assays and functionally validated for cell migration, cell invasion, and degree of epithelial differentiation. The role of CtBP in invasion and metastasis is being investigated using tumor implantation models in collaboration with Lalage Wakefield (collaborator), Head Cancer Biology of TGF-beta Section, Laboratory of Cancer Biology and Genetics. Xenograft studies will be conducted under conditions of nutrient excess and depletion. One of the most challenging aspects of this proposal is to define how CtBP participates in breast cancer risk and clinical outcome in patients and patient populations and how its function may be influenced by patient metabolic status. To do this we have assembled an independent cohort of formalin fixed and paraffin embedded breast cancer patient samples from 900 patients through an IRB-approved protocol in collaboration with the Brody School of Medicine, East Carolina University. We have procured survival and clinicopathological data on the patients with a median follow-up of 7.1 years. The cohort is 40% women of African ancestry and 60% women of European ancestry. Clinico-pathological information includes BMI. We have completed a tissue microarray (TMA) of 600 patients of this cohort in collaboration with Stephen Hewitt, Laboratory of pathology. To date, we have quantitatively profiled the expression of CtBP1, CtBP2 and 22 other antigens know to participate in CtBP-dependent gene regulatory pathways that influence cellular differentiation, cell migration, genome stability and self-renewal properties. To assist in this study, we have developed histological surrogates for obesity using correlation between the cross-sectional area of adipocytes in patient samples and their reported BMI. These surrogates for obesity have been used to show correlation with metabolomic features of breast tumor samples in collaboration with Stephan Ambs (Laboratory of Human Carcinogenesis). Approximately 151 of the 600 patient cohort has been analyzed by RNA-Seq, exome sequencing, and copy number variation by exome sequencing. The genomic data has been used in combination with proteomic data derived from the TMA to make correlation between gene expression and protein expression to infer the genomic influences of gene expression. Most notably CtBP expression is highly associated with poor survival in this cohort. In addition CtBP levels (above or below median expression) is highly predictive of poor survival in both patient with lymph node negative and lymph node positive disease. Furthermore, high protein expression of CtBP2 is associated with increase genomic instability based on copy number variation. Moreover this diverse cohort has been studied by new computational approaches that apply algorithms of conditional lethality to interpret both proteomic and genomic trends in the data and whether or not the trends vary by race, ethnicity and BMI. Finally, in collaboration with Marc Nicklaus (Chemical Biology Laboratory), we have identified 4 lead compounds that function as small molecular inhibitors of CtBP function. The compounds are small (300 da), water soluble, and have low cytotoxicity. In functional assays these compounds reactivate genes know to be repressed by CtBP at both the mRNA and protein level, decrease cell migration and cell invasion, and increase the level of DNA repair. In summary, we are actively engage in an ongoing ambitious, multifaceted and highly integrated research plan that represents a major transition for my group to place more focus on the translation of basic concepts of chromatin-based gene regulation to understanding the biology of breast cancer risk and disease progression. It marks a unique turn in the research in the lab to begin to gain perspective on modifiable risk factors for breast cancer. The association with metabolism has broad implications for understanding the role of lifestyle and how cancer risk can only be understood from population based perspectives.
Showing the most recent 10 out of 22 publications
