Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

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.

The epithelial adherens junction component PLEKHA7 regulates ECM remodeling and cell behavior through miRNA-mediated regulation of MMP1 and LOX.

Epithelial adherens junctions (AJs) are cell-cell adhesion complexes that are influenced by tissue mechanics, such as those emanating from the extracellular matrix (ECM). Here, we introduce a mechanism whereby epithelial AJs can also regulate the ECM. We show that the AJ component PLEKHA7 regulates levels and activity of the key ECM remodeling components MMP1 and LOX in well-differentiated colon epithelial cells, through the miR-24 and miR-30c miRNAs. PLEKHA7 depletion in epithelial cells results in LOX-dependent ECM remodeling in culture and in the colonic mucosal lamina propria in mice. Furthermore, PLEKHA7-depleted cells exhibit increased migration and invasion rates that are MMP1- and LOX- dependent, and form colonies in 3D cultures that are larger in size and acquire aberrant morphologies in stiffer matrices. These results reveal an AJ-mediated mechanism, through which epithelial cells drive ECM remodeling to modulate their behavior, including acquisition of phenotypes that are hallmarks of conditions such as fibrosis and tumorigenesis.

Actin-dependent recruitment of AGO2 to the zonula adherens.

Adherens junctions are cadherin-based structures critical for cellular architecture. E-cadherin junctions in mature epithelial cell monolayers tether to an apical actomyosin ring to form the zonula adherens (ZA). We have previously shown that the adherens junction protein PLEKHA7 associates with and regulates the function of the core RNA interference (RNAi) component AGO2 specifically at the ZA. However, the mechanism mediating AGO2 recruitment to the ZA remained unexplored. Here, we reveal that this ZA-specific recruitment of AGO2 depends on both the structural and tensile integrity of the actomyosin cytoskeleton. We found that depletion of not only PLEKHA7, but also either of the three PLEKHA7-interacting, LIM-domain family proteins, namely LMO7, LIMCH1, and PDLIM1, results in disruption of actomyosin organization and tension, as well as disruption of AGO2 junctional localization and of its miRNA-binding ability. We also show that AGO2 binds Myosin IIB and that PLEKHA7, LMO7, LIMCH1, and PDLIM1 all disrupt interaction of AGO2 with Myosin IIB at the ZA. These results demonstrate that recruitment of AGO2 to the ZA is sensitive to actomyosin perturbations, introducing the concept of mechanosensitive RNAi machinery, with potential implications in tissue remodeling and in disease.

Simultaneous E-cadherin and PLEKHA7 expression negatively affects E-cadherin/EGFR mediated ovarian cancer cell growth.

BACKGROUND: The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied. METHODS: Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets. RESULTS: We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome. CONCLUSIONS: These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels.

The neuropilin 2 isoform NRP2b uniquely supports TGFß-mediated progression in lung cancer.

Neuropilins (NRP1 and NRP2) are co-receptors for heparin-binding growth factors and class 3 semaphorins. Different isoforms of NRP1 and NRP2 are produced by alternative splicing. We found that in non-small cell lung cancer (NSCLC) cell lines, transforming growth factor-ß (TGFß) signaling preferentially increased the abundance of NRP2b. NRP2b and NRP2a differ only in their carboxyl-terminal regions. Although the presence of NRP2b inhibited cultured cell proliferation and primary tumor growth, NRP2b enhanced cellular migration, invasion into Matrigel, and tumorsphere formation in cultured cells in response to TGFß signaling and promoted metastasis in xenograft mouse models. These effects of overexpressed NRP2b contrast with the effects of overexpressed NRP2a. Hepatocyte growth factor (HGF)-induced phosphorylation of the kinase AKT was specifically promoted by NRP2b, whereas inhibiting the HGF receptor MET attenuated NRP2b-dependent cell migration. Unlike NRP2a, NRP2b did not bind the PDZ domain scaffolding protein GAIP carboxyl terminus-interacting protein (GIPC1) and only weakly recruited phosphatase and tensin homolog (PTEN), potentially explaining the difference between NRP2b-mediated and NRP2a-mediated effects. Analysis of NSCLC patient tumors showed that NRP2b abundance correlated with that of the immune cell checkpoint receptor ligand PD-L1 as well as with epithelial-to-mesenchymal transition (EMT) phenotypes in the tumors, acquired resistance to epidermal growth factor receptor (EGFR) inhibitors, disease progression, and poor survival in patients. NRP2b knockdown attenuated the acquisition of resistance to the EGFR inhibitor gefitinib in cultured NSCLC cells. Thus, in NSCLC, NRP2b contributed to the oncogenic response to TGFß and correlated with tumor progression in patients.

Effects of 4-tert-octylphenol given in drinking water for 4 months on the male reproductive system of Fischer 344 rats.

The environmental pollutant 4-tert-octylphenol (OP) is both toxic and estrogenic to mammalian cells, and injection of OP into adult male rats has devastating effects on their reproductive system. We now report the effects of OP in drinking water ( 1 x 10(-5), 1 x 10(-7) or 1 x 10 (-9) M) on the male reproductive system. Exposure of adult male rats for 4 months to any tested dose of OP had no significant effect on water or food consumption; body weight gain; hematocrit; reproductive organ weights; mean serum LH, FSH or testosterone concentrations; germ cell yield or relative numbers of different classes of testicular cells; or testicular sperm number. In contrast, all doses of OP caused an increase in epididymal sperm with tail abnormalities that would interfere with sperm motility, and the highest dose decreased epididymal sperm number. Our findings raise the possibility that consumption of OP in drinking water may adversely influence male reproductive fertility.

Differential effects of TGFbeta and vitreous on the transformation of retinal pigment epithelial cells.

PURPOSE: In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-beta (TGFbeta) have been implicated in this EMT. The role of TGFbeta in the vitreous-mediated transformation of low-passage human RPE cells was investigated. METHODS: Cells were treated with vitreous or TGFbeta2. SB431542 was used to inhibit TGFbeta signaling. Morphology was investigated using phase-contrast or confocal microscopy. Motility was measured using a monolayer-wounding assay. Invasion was determined using basement membrane matrix-based assays. Gene expression was measured by quantitative PCR, immunohistochemistry, or immunoblotting. RESULTS: Changes in phosphorylation or cellular localization of Smad -2, -3, or -4 indicated a TGFbeta-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFbeta treatment, but peripheral actin aggregates were seen in vitreous-treated cells. SB431542 did not block the morphologic change induced by vitreous. Vitreous-treated cells exhibited increased motility and invasion, whereas TGFbeta-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (alpha-SMA and CTGF) were increased by treatment with TGFbeta but suppressed by vitreous. TGFbeta or vitreous caused increased expression of Snail1. CONCLUSIONS: Vitreous or TGFbeta caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFbeta activity in vitreous was necessary but not sufficient for the vitreous-induced motile, invasive phenotype. However, differences in the cytoskeletal organization and in the expression of CTGF and alpha-SMA suggested that TGFbeta-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.