Date of Award

Spring 3-28-2023

Embargo Period

5-31-2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Antonis Kourtidis

Second Advisor

Michael John Yost

Third Advisor

Jorge Munera

Fourth Advisor

Russell (Chip) Norris

Fifth Advisor

Robin Muise-Helmericks

Sixth Advisor

John O'Bryan

Abstract

Colon cancer is the third most common and second deadliest type of cancer. Colon cancer is broadly characterized by compromised epithelial integrity and by aberrant extracellular matrix (ECM) remodeling. However, a potential mechanistic connection between epithelial integrity and ECM remodeling that could be contributing to the disease progression, has not been explored yet. The Adherens Junction (AJ) is a cell-cell adhesion complex composed of cadherin and catenin family proteins and essential for establishing and maintaining epithelial tissue integrity. Our previous work revealed that PLEKHA7, an E-cadherin-p120 catenin partner, recruits the microprocessor and the RNA-induced silencing complex (RISC), key components of the RNAi machinery, as well as distinct sets of miRNAs and mRNAs, specifically to mature apical AJs. Through this recruitment, PLEKHA7 regulates processing and silencing activity of a set of miRNAs to suppress expression of several of their oncogenic mRNA targets. Here, to comprehensively interrogate the cellular mechanisms that are regulated by the AJ-associated RNAi machinery, we depleted PLEKHA7 from colon epithelial Caco2 cells and performed whole cell RNA sequencing, followed by pathway analysis. This analysis revealed that the top group of mRNAs that are upregulated upon PLEKHA7 depletion are those of ECM remodeling components. In particular, the master ECM remodeling regulators MEP1A, MMP1, and LOX, were among the top upregulated mRNAs upon PLEKHA7 depletion. We identified two PLEKHA7- regulated miRNAs, namely miR-24 and miR-30c, to be mediating suppression of MMP1, and LOX by PLEKHA7. PLEKHA7 knockout in Caco2 cells results in elevated MMP1 and LOX enzymatic activities, as well as in increased migration and invasion rates that depend on MMP1 and LOX activities. Corroborating the in vitro findings, Plekha7 knockout in mice results in aberrant collagen deposition in the colonic lamina propria and increased colon muscle layer thickness, both indicators of ECM remodeling and fibrosis. In turn, we also found that collagen and other ECM components differently impact junctional localization and complex formation of the AJ-associated RNAi machinery, impying for a potential negative feedback loop. Along these lines, increased ECM mechanical tension, which is typically observed under fibrotic and tumor conditions in the colon, also disrupted junctional localization of PLEKHA7 and of RNAi components. In summary, our data reveal a novel mechanism, whereby a bi-directional crosstalk between the ECM and epithelial AJs, mediated by the RNAi machinery, regulates pro-tumorigenic cell behavior in the colon.

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