Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer

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Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer

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Title: Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer
Author: Subedee, Ashim ORCID  0000-0002-1208-5182
Citation: Subedee, Ashim. 2015. Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
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Abstract: Breast cancer is a highly heterogeneous disease with differences in histopathological and biological characteristics, variable prognoses, and response to therapy. Clinically, breast
tumors are classified based on the expression of hormone receptors (ER and PR) and HER2 as hormone receptor positive (ER+, PR+), HER2+, and triple negative (ER-, PR-, HER2-). Based
on gene expression profiling, breast cancers have been classified into luminal (luminal A and B), HER2+, basal-like and claudin-low subtypes. Knowledge of the molecular properties of luminal and HER2+ subtypes has led to the development of endocrine and HER2-targeted therapies. However, the molecular determinants and transcriptional regulators of basal-like tumors that constitute the majority of triple negative breast cancer (TNBC) are poorly understood. In this dissertation, we have defined some of the molecular characteristics of the basal-like breast cancer phenotype and also identified multiple transcriptional regulators specific to TNBCs.

By using three different reprogramming approaches – somatic cell fusion, nuclear
reprogramming, and transcription factor transduction, we showed that the basal-like breast cancer phenotype is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. We found that luminal breast cancers share a common core epigenetic program, whereas basal-like breast cancers are highly heterogeneous. We demonstrated that protein extracts of basal-like breast cancer cells can reprogram a subset of
luminal breast cancer cells to a basal-like state. Additionally, we identified three transcription factors, EN1, TBX18, and TCF4, the overexpression of which induced the repression of some
luminal features in luminal breast cancer cells.

We also performed a targeted cellular viability screen for selected transcription factors differentially expressed between triple negative and other breast cancer subtypes and identified multiple factors essential for TNBCs including EN1 and TRIP13. We found that downregulation of EN1 and TRIP13 preferentially and significantly reduce cellular viability, colony formation, and in vivo tumorigenicity of TNBC cell lines. We demonstrated that downregulation of EN1 induces an arrest in the G1 phase of the cell cycle and apoptosis. By analyzing the gene expression and histone H3 lysine 27 acetylation (H3K27ac) profiles of TNBC cell lines following downregulation of EN1, we found that EN1 regulates genes involved in angiogenesis, neurogenesis, cell matrix interactions, and WNT signaling pathways. We also performed ChIP-seq for exogenously expressed HA-tagged EN1 to identify its genomic targets. Lastly, we showed that the expression of EN1 correlates with shorter overall survival among patients with basal-like breast tumors. Similarly, by analyzing the gene expression profiles of TNBC cell lines following downregulation of TRIP13, we found that TRIP13 regulates genes involved in IL6 signaling, cell proliferation, and angiogenesis; in line with this we confirmed reduced levels of JAK2 and phospho-STAT3 following TRIP13 downregulation.

In summary, we have unraveled some of the molecular mechanisms of basal-like and luminal breast cancer cell phenotypes and identified factors that might repress luminal differentiation programs in basal-like breast tumors. We have also identified multiple triple negative breast cancer specific transcription regulators. We believe these studies have increased our molecular understanding of basal-like and triple negative breast cancers and have provided potential therapeutic targets for these breast tumors.
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