Author ORCID Identifier

0000-0003-0511-1888

Date of Graduation

8-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Don L. Gibbons, M.D., Ph.D.

Committee Member

Jonathan M. Kurie, M.D.

Committee Member

Faye Johnson, M.D., Ph.D.

Committee Member

Randy Johnson, Ph.D.

Committee Member

Li Ma, Ph.D.

Committee Member

Yang Xia, M.D., Ph.D.

Abstract

Lung cancer is the leading cause of cancer-related deaths due to conventional therapy resistance and metastatic disease, therefore understanding the mechanisms governing these biological functions is vital for improving patient survival. Approximately 30% of patients with the adenocarcinoma histologic subset of lung cancer possess an activating KRAS mutation, characterized by a lack of response to chemotherapies with a poor overall 5-year survival rate. Despite the mutational frequency, KRAS remains a challenge to pharmacologically inhibit and current drugs undergoing clinical trials that target specific downstream effector proteins of KRAS, such as MEK inhibitors, have failed to produce significant clinical benefits. Previous studies by our group on the metastatic process revealed that malignant lung cancer cells undergo an epithelial-to-mesenchymal transition (EMT) that is regulated by a double-negative feedback loop between the transcription factor Zeb1 and the microRNA-200 family (miR-200). Furthermore, these studies demonstrated that mesenchymal lung cancer cell invasion and metastasis are dependent on interaction with the extracellular matrix (ECM). In addition to metastasis, EMT has been implicated in resistance to radiation and chemotherapies as well as resistance to certain targeted drug treatments. Thus, the comprehensive objective of this study was to: (1) elucidate the detailed mechanisms of metastasis by investigating the collaborative effect of EMT and ECM on KRAS mutant lung cancer metastasis, and (2) delineate the mechanism of EMT in promoting MEK inhibitor resistance in KRAS mutant lung cancers. Our findings reveal increased collagen deposition in mesenchymal tumor tissues due to amplification of collagen gene expression in Zeb1-driven mesenchymal lung cancer cells. Additionally, collagen fibers in the metastatic tumor tissues exhibit greater linearity and organization, correlating with direct Zeb1-upregulation of the collagen crosslinking enzyme LOXL2. Coordinated expression of LOXL2 with collagen increases insoluble collagen crosslinking and deposition in the tumor microenvironment, resulting in activated Fak/Src signaling to promote metastasis. Using functional in vivo shRNA screens coupled with proteomic profiling, we identified Zeb1-driven epithelial-to-mesenchymal transition (EMT) as a regulator of MAPK-dependent signaling activity and tumor maintenance in lung cancer cells. Mechanistic studies in novel cell line and animal models further demonstrated that the Ras-Raf-Mek-Erk MAPK signaling cascade in epithelial cells is activated through upregulation of the scaffold protein Il17rd, which is directly repressed by Zeb1 during EMT. Translationally, we observe that poorly-differentiated, mesenchymal lung cancer cells within the heterogeneous lung tumor tissue of in vivo models are resistant to MEK inhibitors and identified Zeb1 and Il17rd as potential biomarkers from large patient datasets. Reversion of functional EMT by genetic expression of miR-200 or treatment with the class I HDAC inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and significantly reduced tumor growth. This study establishes the mechanisms of metastasis and MEK inhibitor resistance in KRAS mutant lung cancers, identifies multiple potential prognostic markers of metastasis and resistance, and provides pre-clinical evidence for a promising combinatorial therapy against lung cancer progression.

Keywords

Lung cancer, EMT, drug resistance, metastasis, LOXL2, ECM, collagen, MAPK, Mek inhibitor

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