Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

J Michael Ruppert

Committee Co-Chair

Erik A Bey

Committee Member

Karen H Martin

Committee Member

Elena N Pugacheva

Committee Member

Mohamad A Salkeni

Committee Member

Scott A Weed

Abstract

Transcription factors are crucial to the normal and pathologic biology of cells. Aberrant genetic regulation underlies all facets of cancer, including metastasis, therapeutic resistance, and other clinically relevant manifestations. Kruppel-like factor 4 (KLF4) and GLI1, transcription factors that interact with DNA through conserved zinc finger domains, are implicated in human malignancy. The stability of GLI1 protein is a central determinant of Hedgehog pathway signaling output, and is regulated through interaction with the F-box protein beta-TrCP. In pancreatic ductal adenocarcinoma, GLI1 may be activated by oncogenic mechanisms. While KLF4 is not specific to any discrete signal pathway, KLF4 is well-characterized in numerous biological processes, including pluripotency and stress response. The functions of KLF4 are highly context-dependent, and the role of KLF4 in metastasis and other emergent phenotypes remains unclear. This dissertation addresses two distinct topics: Mechanisms underlying GLI1 stabilization in pancreatic cancer, and the function of KLF4 in the metastasis of breast cancer cells.;As an effector protein of the Hedgehog (Hh) signal pathway, GLI1 is implicated in pancreatic ductal adenocarcinoma (PDA) and other cancer types. Like other GLI proteins, GLI1 stability is regulated by the ubiquitin-proteasome system through the E3 ligase SKP1/CUL1/F-box (SCF) protein SCFbeta-TrCP . While Hh signaling is known to induce expression of the high mobility group box protein SOX9 in several contexts, the function of this signaling mechanism in PDA is poorly understood. In Chapter 2, we report the stabilization of GLI1 protein by SOX9 in PDA cells, finding that SOX9 inhibited SCF beta-TrCP-mediated protein degradation. In the presence of SOX9, the association of GLI1 and beta-TrCP was reduced. The discovery that SOX9 interacts with the F-box domain of beta-TrCP led us to question whether SOX9 could inhibit SCFbeta-TrCP complex assembly, as this region is known to function in binding the SKP1 adaptor protein. Indeed, we observed enhanced association between SKP1 and beta-TrCP upon suppression of SOX9, whereas overexpression of SOX9 led to drastically reduced SKP1--TrCP interaction. Additionally, SOX9 functioned to promote the nuclear tethering and degradation of beta-TrCP. In PDA cells, deficiency of SOX9 resulted in loss of malignant properties and cancer stem cell (CSC) traits, effects that could be rescued by suppression of beta-TrCP. We also provide evidence that additional substrates of beta-TrCP may be similarly regulated by SOX9. These results identify a positive feedback mechanism of GLI1 stability in PDA cells, revealing that SOX9 can inhibit SCFbeta-TrCP activity to suppress degradation of oncoproteins.;While KLF4 inhibits cell proliferation and is downregulated in some tumor types, KLF4 promotes survival and therapeutic resistance in breast cancer cells. Metastasis remains a significant clinical problem, yet a comprehensive understanding of the metastatic process remains elusive. In Chapter 3, we address the function of KLF4 in a mouse model of breast cancer metastasis. In triple-negative breast cancer cell lines, stable suppression of KLF4 resulted in enhanced spontaneous metastasis to the lungs and liver. Similarly, we observed fewer metastases in association with KLF4 overexpression. KLF4 had minimal impact on primary tumor initiation and growth. Although increased circulating tumor cells (CTCs) arose from KLF4-knockdown tumors, this effect was not predicted by function of KLF4 in 2D assessments of motility and invasion. Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase associated with metastasis. We found that KLF4 suppresses expression of DDR1. Accordingly, suppression of KLF4 resulted in greater expression of DDR1 mRNA and protein, and increased adhesion to collagen. We propose that KLF4 inhibits metastasis of breast cancer cells through downregulation of DDR1.

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