Inhibiting Phosphatidylinositol 4-Kinase IIIalpha with GSK-F1 Reduces the Plasma Membrane-Associated Phosphatidylinositol Phosphate Lipid Messenger Levels in Prostate Cancer Cells

Inhibiting phosphatidylinositol 4-kinase IIIa with GSK-F1 reduces the plasma membrane-associated phosphatidylinositol phosphate lipid messenger levels in prostate cancer cells Codrut Radoiu, Diego Sbrissa, Sreenivasa Chinni Department of Urology and Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA Background: The role of chemokine signaling in prostate cancer metastasis has demonstrated to be a promising focus of research and a potential target for abating malignant cell invasion and metastasis. CXCR4, a GPCR, is overexpressed in prostate cancer cells, with downstream signaling involved in cellular migration, proliferation and survival when it is activated by its ligand, CXCL12. Upon activation, it recruits phosphatidylinositol 4-kinase IIIa (PI4KIIIa) to the plasma membrane (PM) , which produces a pool of phosphatidylinositol 4-phosphate (PI4P). PI4P is a precursor to phosphatidylinositol 4,5-bisphosphate (PIP2), an important molecule with close implications in PI3K/AKT pathway activation and oncogenic processes. The impact of inhibiting PI4KIIIa on the on maintaining steady state levels of PI(4,5)P2 in prostate cancer cells has not been previously addressed and remains unclear. Objective: We aim to show that stable levels of PIP2 in PM depend on PI4KIIIa activity and inhibiting with a specific PI4KIIIa inhibitor, GSK-F1, will reduce the PM concentration of PIP2. Methods: PC3 cells were cultured in RPMI medium and passaged every 48 hours. Cells were transfected with PLCd1-PH-GFP plasmid DNA, as a biosensor for PIP2 using Opti-MEM and Lipofectamine 3000 reagent. 24 hours post-transfection, cells were exposed to 4 different treatments – DMSO, DMS0 + CXCL12, GSK-F1+CXCL12, GSK-F1 for approximately 20 hours. Cells were fixed and mounted on slides with antifade reagent and DAPI. Leica DMI3000 B wide-field fluorescence microscope was used for visualization. Peak area of fluorescence PLCd1-PH-GFP at plasma membrane was quantified using ImageJ software. Results: There was significantly less PLCd1-PH-GFP fluorescence at the PM in PC3 cells treated with GSK-F1 with (p=0.0087) and without (p=0.0022) CXCL12 when compared to cells treated only with CXCL12, indicating reduced availability of PIP2. Augmented fluorescence was noted in cells treated with CXCL12 alone when compared to the control group receiving only DMSO (p=0.0022), suggesting CXCR4 activation promotes PI4P through activation of PI4KIIIa and its subsequent conversion to PI(4,5)P2 through PM resident PI4PKinases. Conclusion: Inhibiting PI4KIIIa with GSK-F1 diminishes stores of PIP2 at the PM in PC3 cells, indicating the dependence on PI4KIIIa activity for maintaining stable levels of PI(4,5)P2 in prostate cancer cells and its effect on invasion and metastasis. Key words: Prostate cancer, chemokine, biosensor, metastasis, invasion, phosphatidylinosito

Targeting CXCR4 with CTCE-9908 inhibits prostate tumor metastasis

Abstract Background CXCL12/CXCR4 transactivation of epidermal growth factor family receptors in lipid raft membrane microdomains on cell surface is thought to mediate tumor growth and subsequent development of metastatic disease. CTCE-9908 is a known inhibitor of CXCR4. Herein, we tested the efficacy of CTCE-9908 in inhibiting prostate cancer cell growth, invasion, and metastasis. Methods We used a panel of in vitro assays utilizing human prostate cancer cell lines and an in vivo orthotopic prostate cancer model to assess the anti-tumoral activity of CTCE-9908. Results We demonstrated that (a) CTCE-9908 treatment resulted in no significant change in the growth of PC-3 and C4-2B cells; (b) 50 μg/ml of CTCE-9908 inhibited the invasive properties of PC-3 cells; (c) 25 mg/kg of CTCE-9908 did not alter primary tumor growth but it did significantly reduce total tumor burden in the animal including the growth of prostate and soft tissue metastases to lymph node and distant organ tissues. Histological analysis showed that CTCE-9908 treatment resulted in tumor necrosis in primary prostate tumors and no significant change in proliferation of tumor cells as measured by Ki-67 staining; (d) CTCE-9908 inhibited the tumor angiogenesis as measured by CD34 positive vessels in tumors. Conclusions These data suggest that CXCR4 inhibition by CTCE-9908 decreases the invasion potential in vitro, which then translated to a reduction of tumor spread with associated reduction in angiogenesis. Hence, CTCE-9908 may prove to be an efficacious novel agent to prevent and treat the spread of metastatic prostate cancer

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling

Abstract Introduction The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis. Methods Murine prostate epithelial cells from PTEN+/+, PTEN +/− , and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed. Results Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth. Conclusions These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth

Abstract The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4–PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4–PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis