Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Department

Biology

Degree Granting Department

Cell Biology, Microbiology and Molecular Biology

Major Professor

W. Douglas Cress, Ph.D.

Committee Member

Teresita Munoz-Antonia, Ph.D.

Committee Member

Srikumar P. Chellappan, Ph.D.

Committee Member

Lori A. Hazlehurst, Ph.D.

Keywords

Integrin, Lung adenocarcinoma, Osteosarcoma, pRb, STK11

Abstract

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively.

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes may account for a significant portion of RB1's potency as a tumor suppressor, a small, but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. Chapter 2 highlights pRb's role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis.

Chapter 3 defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb-dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb responsive region between -108bp to -55bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an 'integrin switch' that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis.

Lung cancer is the leading cause of cancer-related death in the U.S. and additional targeted therapies are desperately needed to treat these patients. STK11 is the third most frequently mutated gene in lung adenocarcinoma following only KRAS and TP53, yet its mutational status is not currently clinically evaluated and no therapies have been approved to specifically target its pathway. A deep understanding of the complex pathways controlled by STK11 and their alterations in cancer are required to develop effective therapies for patients with loss-of-function mutations. In Chapter 4 we present the current understanding of STK11, focusing on its molecular biology and therapeutic implications, including a compilation of studies evaluating STK11 somatic mutations in human lung cancer tissue and how the frequency of these mutations varies across histological subtypes and patient populations. Finally, we review the strategies being used to target STK11-deficient cancers at the clinical trial, pre-clinical, and basic science levels as well as proposing potential new therapies that might benefit this patient population.

STK11 is a tumor-suppressor commonly mutated in lung adenocarcinoma (LuAd). There are a number of agents that may selectively target the deregulated pathways in STK11 mutated tumors, and thus, identifying the subset of adenocarcinomas that harbor these mutations could have significant clinical benefit. In Chapter 5, we characterized a cohort of 442 adenocarcinoma patients with respect to STK11 mutation status and subset of this cohort using immunochemistry, gene expression, and western blotting. We found that measuring STK11 mutation status is complicated by the fact that many STK11 mutations lead to expression of a stable protein that is indistinguishable from wild type (WT) via immunohistochemistry. To circumvent this, we used published cell line mutation and gene expression data to derive a signature correlating with STK11 mutation status. This signature was validated in the cohort of 442 lung adenocarcinomas and strongly correlates with mutation status (ROC curve AUC = 85.29). These data suggest that STK11; mutation status may be best assessed by measuring the downstream targets included in our signature.

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