Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Michael Schaller

Committee Co-Chair

John M Ruppert

Committee Member

Soumit Basu

Committee Member

Laura Gibson

Committee Member

Alexey Ivanov

Committee Member

Peter Stoilov

Abstract

Kruppel-like factors (KLFs) are transcription factors that participate in numerous biological functions, mainly impacting cell fate and differentiation. KLFs 4 and 5 (KLF4/5) are two highly studied members of this family, implicated to have diverse roles in both normal and abnormal physiology. KLF4 is primarily expressed in post-mitotic, differentiated epithelium and consistent with this observation, KLF4 exerts suppressive effects on cellular growth. Paradoxically, KLF4 can promote malignant features in both squamous cell carcinoma of the skin and breast cancer. We have attempted to better understand the nature of KLF4 in cancer via its relationship with KLF5. Although the two are believed to exert opposing influences on epithelial differentiation and growth, both have important roles in breast tumorigenesis. Previously, KLFs 2, 4, and 5 were shown to function redundantly in the maintenance of the stem cell phenotype in embryonic stem cells, a context often informative to cancer. Despite individual reports detailing oncogenic effects of KLFs 4 and 5 in breast cancer, currently no relationship between the two KLFs has been described in this disease.;In Chapter 2 of this dissertation, we identified vital roles for KLF4/5 involving resistance to HER2-targeted therapies in breast cancer. Using comprehensive gene expression analysis of primary tumors from human and murine model systems, we identified a positive correlation between KLF4/5 transcript abundance, particularly in HER2-enriched tumors. Incorporating these in vivo observations into phenotypic studies revealed that KLF4/5 are critical in the cellular response to HER2 targeted therapies. The expression and activity of these KLFs was induced following HER2-inhibition, facilitating enhanced resistance to these treatments. Even in drug-naive cells, depletion of KLF4/5 resulted in reduced anoikis resistance, colony formation, and xenograft formation. These defects were attributed to diminished levels of the anti-apoptotic factors BCL-XL and MCL1in KLF-deficient cells. In the absence of drug, KLF4/5 impacted the basal levels of BCL-XL and MCL1. Similar to KLF4/5, MCL1 and BCL-XL expression was also induced following HER2-inhibition and suppression of KLF4/5 blunted this effect. Consistent with their role in drug resistance, KLF4/5 transcript levels stratify distant metastasis-free survival in patients, with elevated levels of these factors associated with poor outcome.;Although KLF4/5 have overlapping functions in drug resistance, they can exert distinct and opposing influences as well. Chapter 3 further explores this relationship in breast cancer and attempts to address how KLF4/5 interact in alternative effector pathways. Here, we demonstrate non-redundant functions for the KLFs in breast cancer. Several studies have documented opposing roles for KLF4/5 on cell proliferation, with KLF4 exerting inhibitory effects and KLF5 promoting cell growth. Here we demonstrate that the proliferative phenotype of KLF4-depleted cells requires KLF5 expression, such that reduction of KLF5 abolishes the enhanced growth of these cells. These observations partially explain how KLF4 can dually act as an oncogene despite its growth-inhibitory effects. Furthermore, we explore distinct functions of these KLFs as well. In KLF4-depleted MDA-MB-231 cells, KLF4 was the only core KLF able to rescue the specific deficits resulting from its depletion. In addition, we elaborated on a previously described association between KLF4 and Notch1 signaling in breast cancer. We found KLF4 to transcriptionally participate in canonical Notch1 signaling, binding to and upregulating several target genes of this pathway including Notch1, Hes5 and Hey1. Moreover, KLF5 did not significantly contribute to the Notch1 pathway in this setting, further supporting non-redundant roles for the KLFs in certain effector pathways. Expanding on this relationship between KLF4 and Notch1, we compared ChIP-seq datasets for these respective genes in a stem cell context finding a significant overlap in many signaling cascades known to be important in tumorigenesis such as Wnt and Ras.;In summary, the relationship between KLFs KLF4/5 in breast carcinoma contributes to our understanding on how the genes participate in response to therapy and the cancer process. In future functional and translational studies, tandem analysis of both genes may provide additional insight than investigating these genes individually.

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