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NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation.

ABSTRACT: Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin alpha4. Suppressing NG2 expression or overexpressing alpha4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCalpha expression, but overexpressing integrin alpha4 enhanced FAK phosphorylation independently of PKCalpha. Cotransfection with NG2 cDNA and integrin alpha4 siRNA did not lower PKCalpha and pFAK levels more than transfection with either alone. PKCalpha was upstream of FAK phosphorylation, as silencing PKCalpha decreased FAK phosphorylation. PKCalpha overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin alpha4 oppositely regulate anoikis in fibroblasts. NG2 and integrin alpha4 regulate FAK phosphorylation by PKCalpha-dependent and -independent pathways, respectively.

SUBMITTER: Joo NE

PROVIDER: S-EPMC3940336 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation.

Joo N E NE Watanabe T T Chen C C Chekenya M M Stallcup W B WB Kapila Y L YL

Cell death and differentiation 20080222 5

Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found ...[more]

PMID: 18292781

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Project description:miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.

Project description:BackgroundDespite effective radiotherapy for the initial stages of cancer, several studies have reported the recurrence of various cancers, including medulloblastoma. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by comparing the extracellular protease activity and the expression pattern of molecules, known to be involved in cell adhesion, migration and invasion, between non-irradiated and irradiated cells.Methodology/principal findingsWe identified an increase in invasion and migration of irradiated compared to non-irradiated medulloblastoma cells. RT-PCR analysis confirmed increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin and integrin subunits (e.g., ?3, ?5 and ?1) in irradiated cells. Furthermore, we noticed a ?2-fold increase in tyrosine phosphorylation of FAK in irradiated cells. Immunoprecipitation studies confirmed increased interaction of integrin ?1 and FAK in irradiated cells. In addition, our results show that overexpression of uPAR in cancer cells can mimic radiation-induced activation of FAK signaling. Moreover, by inhibiting FAK phosphorylation, we were able to reduce the radiation-induced invasiveness of the cancer cells. In this vein, we studied the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42. As observed from the immunoprecipitation studies, uPAR knockdown reduced interaction among the focal adhesion molecules, such as FAK, Paxillin and p130Cas, which are known to play key roles in cancer metastasis. Pretreatment with uPAR shRNA expressing construct reduced uPAR and phospho FAK expression levels in pre-established medulloblastoma in nude mice.Conclusion/significanceTaken together, our results show that radiation enhances uPAR-mediated FAK signaling and by targeting uPAR we can inhibit radiation-activated cell adhesion and migration both in vitro and in vivo.

Dataset Information

NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation.

Publications

NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation.

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