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Clusterin (CLU) is a chaperone‐like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo‐ or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′‐kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine‐rich repeat‐containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR‐190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR‐190‐dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation. In human prostate cells, clusterin (CLU) overexpression was found to enhance the phosphorylation of AKT restricted to isoform 2. Mechanistically, this was explained by the finding that the phosphatase PHLPP1, known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR‐190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that PTEN was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. Remarkably, we also demonstrated that by keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotype, namely PNT1A and PC3 cells. [ABSTRACT FROM AUTHOR]

Negative crosstalk between PHLPP and PTEN in TGFβ-1 activated stem cells and cancer cells. A schematic illustration depicting factors involved in the down-regulation of PTEN in response to PHLPP overexpression. A Similar scenario involving miR190 is proposed for PHLPP down-regulation in response to PTEN overexpression. • Crosstalk between PHLPPs and PTEN in cancer cells, but not in normal cells. • The crosstalk promotes invasiveness in cancer cells and TGF-β1 activated stem cells. • The crosstalk is mediated by purinergic P2X4 receptor. Akt kinase regulates several cellular processes, among them growth, proliferation and survival, and has been correlated to neoplastic disease. We report here crosstalk between several Akt regulatory phosphatases that controls the level of the activated form (phosphorylated) of Akt and affects tumor cell aggressiveness. In prostate cancer cell lines, we observed that transient transfection of PTEN decreased the endogenous level of PHLPPs and in contrast, the transient transfection of PHLPPs decreased the endogenous level of PTEN. Furthermore, silencing of PTEN by siRNA resulted in increased PHLPP levels. This phenomenon was not seen in non-transformed cells or in prostate stem cells. This crosstalk promoted cancer cell invasion and was controlled by epigenetically regulated processes where activation of miRs (miR-190 and miR214), the polycomb group of proteins and DNA methylation were involved. The purinergic P2X4 receptor, which has been shown to have a role in wound healing, was identified to be the mediator of this crosstalk. We also studied prostate stem cells and found this crosstalk in the TGFβ1-activated epithelial-mesenchymal transition (EMT). The crosstalk seemed to be a natural part of EMT. In summary, we identify a crosstalk between Akt phosphatases which is not present in non-transformed prostate cells but occurs in cancer cells and stem cells transformed by TGFβ-1. This crosstalk is important for cellular invasion. Phosphatases regulate the Akt oncogene. Crosstalk between Akt phosphatases in prostate cancer cells and in TGF-β1 activated stem cells but not in non-transformed cells. This back-up mechanism facilitates invasive migration of prostate stem and cancer cells. Characterization of Akt regulation may lead to a better understanding of tumor development and to novel strategies for treatment. [ABSTRACT FROM AUTHOR]