Epidemiological studies indicate that statins, cholesterol-lowering drugs, prevent aggressive prostate cancer and other types of cancer. Employing essentially non-prostate cell lines, we previously showed that statins rapidly downregulate nuclear levels of phosphorylated Akt via P2X7, a purinergic receptor recently implicated in invasive growth. Here, we present studies on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-positive prostatic cells. We document an involvement of EH domain-binding protein 1 (EHBP1), previously associated with aggressive prostate cancer and insulin-stimulated trafficking and cell migration, in P2X7 signaling. We also show that EHBP1 is essential for an anti-invasive effect of atorvastatin. Furthermore, EHBP1 interacted with P-Rex1, a guanine nucleotide exchange factor previously implicated in invasive growth. Mevalonate did not prevent this anti-invasive effect of atorvastatin. These data indicate that atorvastatin modulates invasiveness via P2X7, EHBP1 and P-Rex1. Interestingly, the interaction between EHBP1 and P-Rex1 was not induced by extracellular adenosine triphosphate (ATP), the endogenous P2X7 ligand, and statins counteracted invasiveness stimulated by extracellular ATP. In support of these experimental data, a population-based genetic analysis showed that a loss of function allele in the P2X7 gene (rs3751143) associated with non-aggressive cancer, and the common allele with aggressive cancer. Our data indicate a novel signaling pathway that inhibits invasiveness and that is druggable. Statins may reduce the risk of aggressive prostate cancer via P2X7 and by counteracting invasive effects of extracellular ATP.

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNAp21 cip1 complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.The serine/threonine kinase Akt is a central regulator of apoptosis and cell growth and is activated by insulin, growth factors, and cellular stress. Many studies indicate a key role for Akt in carcinogenesis and as a target for therapeutic agents (1-4). 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, have anticancer properties (5), and we have shown that statins decrease levels of phosphorylated Akt (pAkt) 3 in lung and pancreatic cancer cells (6 -9). A conspicuous finding was that dephosphorylation of nuclear Akt was induced within minutes and that this effect was associated with inhibited cell proliferation. Extracellular ATP also induced nuclear pAkt depletion, and several lines of evidence indicated that the rapid pAkt depletion was mediated by the purinergic receptor P2X7 (7,8).A family of protein phosphatases, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP), was identified as a phosphatase for Akt (10). Two isoforms, PHLPP1 and PHLPP2, have been shown to dephosphorylate distinct Akt isoforms, at one (Ser 473 ) of two phosphorylation sites required for activation (11). This dephosphorylation is regulated by the immunophilin FKBP51 (FK506-binding protein 51), which acts as scaffolding protein for Akt and PHLPP (12). Other phosphatases shown to dephosphoryl...

Endometriosis is an estrogen-dependent inflammatory disease that affects approximately 10% of women. Debilitating pelvic or abdominal pain is one of its major clinical features. Current animal models of endometriosis-associated pain require surgery either to implant tissue or to remove the ovaries. Moreover, existing models do not induce spontaneous pain, which is the primary symptom of patients with chronic pain, including endometriosis. A lack of models that accurately recapitulate the disease phenotype must contribute to the high failure rate of clinical trials for analgesic drugs directed at chronic pain, including those for endometriosis. We set out to establish a murine model of endometriosis-associated pain. Endometriosis was induced nonsurgically by injecting a dissociated uterine horn into a recipient mouse. The induced lesions exhibited histological features that resemble human lesions along with an increase in proinflammatory cytokines and recruitment of immune cells. We also observed the presence of calcitonin gene–related peptide–, TRPA1-, and TRPV1-expressing nerve fibers in the lesions. This model induced mechanical allodynia, spontaneous abdominal pain, and changes in thermal selection behavior that indicate discomfort. These behavioral changes were reduced by drugs used clinically for endometriosis, specifically letrozole (aromatase inhibitor) and danazol (androgen). Endometriosis also induced neuronal changes as evidenced by activation of the NF-κB signaling pathway in TRPA1- and TRPV1-expressing dorsal root ganglion neurons. In conclusion, we have established a model of endometriosis-associated pain that responds to clinically active drugs and can, therefore, be used to identify novel therapies.

Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFb1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKTinhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer.