Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer

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.Medical Science

Novel Candidate Virulence Factors in Rice Pathogen Xanthomonas oryzae pv. oryzicola as Revealed by Mutational Analysis▿

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice. Transposon-mediated mutational analysis of the pathogen with a quantitative assay revealed candidate virulence factors including genes involved in the pathogenesis of other phytopathogenic bacteria, virulence factors of animal pathogens, and genes not previously associated with virulence

Emerging oncology drug targets in the 21st century: An FDA analysis

Abstract only e14067 Background: Traditional chemotherapy agents are cytotoxic and typically act on all rapidly dividing cells. In contrast, targeted cancer therapies take advantage of unique genetic or proteomic susceptibility in cancer cells, their microenvironment, or the immune system to selectively target tumor tissue or stimulate immune activity. We catalogued oncology drug approvals in the U.S. in the 21 st century to study the evolution of molecular targets over the last twenty years. Methods: We used internal FDA databases and data repositories to generate a dataset of all oncology approvals granted 1/1/2000-12/31/2019. This dataset was curated through a comprehensive survey of drug labels, drug reviews, and published literature to include target gene(s), mechanism of action, drug class, and approved indications for each drug. Approvals were grouped into five-year periods for trend analyses. Results: There has been an increase in the number of oncology approvals over the last twenty years, the vast majority of which are targeted therapies. There have been considerable recent advances in some disease sites. Three disease sites (ovary, bladder, and liver) had many approvals in the last five years after few to none 2000-2014; lung cancer and melanoma have seen substantial advances in the last ten years. Additionally, the first site-agnostic approvals occurred in the last five years. Kinase inhibitors are the most common drug class by both number of drugs and indications. Immune checkpoint inhibitors (ICI) are the second most common class by number of indications, despite having only entered the market in 2011 and being the sixth most common class by number of drugs. The target gene PD-1 has the highest number of approved indications, followed by EGFR and the BCR-ABL fusion gene. The number of novel genes targeted by drugs approved in 2010-2019 is nearly double that of 2000-2009, with the most novel gene targets in 2019 (n = 7). Conclusions: This analysis captures the landscape of targeted drug approvals in the 21 st century, including the continued dominance of kinase inhibitors despite the dramatic impact of ICIs on cancer care since their introduction in 2011. The promise of targeted therapy in oncology is especially evident from the introduction of site-agnostic indications, new approvals in cancer types with limited therapy options, and ICIs with many indications due to targeting tumors indirectly via the immune system. The continual introduction of novel gene targets provides a snapshot of ongoing novel drug development, and it will be interesting to see whether this trend continues over the next twenty years

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells

EN1 Is a Transcriptional Dependency in Triple-Negative Breast Cancer Associated with Brain Metastasis.

To define transcriptional dependencies of triple-negative breast cancer (TNBC), we identified transcription factors highly and specifically expressed in primary TNBCs and tested their requirement for cell growth in a panel of breast cancer cell lines. We found that EN1 (engrailed 1) is overexpressed in TNBCs and its downregulation preferentially and significantly reduced viability and tumorigenicity in TNBC cell lines. By integrating gene expression changes after EN1 downregulation with EN1 chromatin binding patterns, we identified genes involved in WNT and Hedgehog signaling, neurogenesis, and axonal guidance as direct EN1 transcriptional targets. Quantitative proteomic analyses of EN1-bound chromatin complexes revealed association with transcriptional repressors and coactivators including TLE3, TRIM24, TRIM28, and TRIM33. High expression of EN1 correlated with short overall survival and increased risk of developing brain metastases in patients with TNBC. Thus, EN1 is a prognostic marker and a potential therapeutic target in TNBC. SIGNIFICANCE: These findings show that the EN1 transcription factor regulates neurogenesis-related genes and is associated with brain metastasis in triple-negative breast cancer