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The extracellular matrix (ECM) plays crucial roles in tissue homeostasis. Abnormalities in ECM composition are associated with pathological conditions, such as fibrosis and cancer. These ECM alterations are sensed by the epithelium and can influence its behavior through crosstalk with other mechanosensitive complexes, including the adherens junctions (AJs). We have previously shown that the AJs, through their component PLEKHA7, recruit the RNAi machinery to regulate miRNA levels and function. We have particularly shown that the junctional localization of RNAi components is critical for their function. Here, we investigated whether different ECM substrates can influence the junctional localization of RNAi complexes. To do this, we plated colon epithelial Caco2 cells on four key ECM substrates found in the colon under normal or pathogenic conditions, namely laminin, fibronectin, collagen I, and collagen IV, and we examined the subcellular distribution of PLEKHA7, and of the key RNAi components AGO2 and DROSHA. Fibronectin and collagen I negatively impacted the junctional localization of PLEKHA7, AGO2, and DROSHA when compared to laminin. Furthermore, fibronectin, collagen I, and collagen IV disrupted interactions of AGO2 and DROSHA with their essential partners GW182 and DGCR8, respectively, both at AJs and throughout the cell. Combinations of all substrates with fibronectin also negatively impacted junctional localization of PLEKHA7 and AGO2. Additionally, collagen I triggered accumulation of DROSHA at tri-cellular junctions, while both collagen I and collagen IV resulted in DROSHA accumulation at basal areas of cell–cell contact. Altogether, fibronectin and collagens I and IV, which are elevated in the stroma of fibrotic and cancerous tissues, altered localization patterns and disrupted complex formation of PLEKHA7 and RNAi components. Combined with our prior studies showing that apical junctional localization of the PLEKHA7-RNAi complex is critical for regulating tumor-suppressing miRNAs, this work points to a yet unstudied mechanism that could contribute to epithelial cell transformation. [ABSTRACT FROM AUTHOR]

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted. Here, we used immunohistochemistry of inflammatory breast cancer patient samples and analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We used viral transduction to re-express PLEKHA7 in inflammatory breast cancer cells and examined their aggressiveness in 2D and 3D cultures and in vivo. We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in most patient samples and very low expression in cell lines. Re-expressing PLEKHA7 suppressed proliferation, anchorage independent growth, spheroid viability, and tumor growth in vivo. The data indicate that PLEKHA7 is frequently deregulated and acts to suppress inflammatory breast cancer. The data also promote the need for future inquiry into the imbalance between apical and basolateral cadherin complexes as driving forces in inflammatory breast cancer. [ABSTRACT FROM AUTHOR]

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function. [ABSTRACT FROM AUTHOR]