Caveolin-1 promotes the tumor suppressor properties of oncogene-induced cellular senescence.

Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1-null mice. A lack of K-RasG12D-induced premature senescence in caveolin-1-null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1-null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer.

TMEM16A/ANO1 suppression improves response to antibody-mediated targeted therapy of EGFR and HER2/ERBB2.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

Sulforaphane Inhibits c-Myc-Mediated Prostate Cancer Stem-Like Traits.

Preventive and therapeutic efficiencies of dietary sulforaphane (SFN) against human prostate cancer have been demonstrated in vivo, but the underlying mechanism(s) by which this occurs is poorly understood. Here, we show that the prostate cancer stem cell (pCSC)-like traits, such as accelerated activity of aldehyde dehydrogenase 1 (ALDH1), enrichment of CD49f+ fraction, and sphere forming efficiency, are attenuated by SFN treatment. Interestingly, the expression of c-Myc, an oncogenic transcription factor that is frequently deregulated in prostate cancer cells, was markedly suppressed by SFN both in vitro and in vivo. This is biologically relevant, because the lessening of pCSC-like phenotypes mediated by SFN was attenuated when c-Myc was overexpressed. Naturally occurring thio, sulfinyl, and sulfonyl analogs of SFN were also effective in causing suppression of c-Myc protein level. However, basal glycolysis, a basic metabolic pathway that can also be promoted by c-Myc overexpression, was not largely suppressed by SFN, implying that, in addition to c-Myc, there might be another SFN-sensitive cellular factor, which is not directly involved in basal glycolysis, but cooperates with c-Myc to sustain pCSC-like phenotypes. Our study suggests that oncogenic c-Myc is a target of SFN to prevent and eliminate the onset of human prostate cancer. J. Cell. Biochem. 117: 2482-2495, 2016. © 2016 Wiley Periodicals, Inc.

CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells.

Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 µmol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma.

Functional relevance of D,L-sulforaphane-mediated induction of vimentin and plasminogen activator inhibitor-1 in human prostate cancer cells.

PURPOSE: D,L-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo efficacy against prostate cancer in experimental rodents. This study was undertaken to determine the role of vimentin and plasminogen activator inhibitor-1 (PAI-1) in anticancer effects of SFN. METHODS: Effect of SFN on levels of different proteins was determined by Western blotting or immunofluorescence microscopy. RNA interference of vimentin and PAI-1 was achieved by transient transfection. Apoptosis was quantified by flow cytometry. Transwell chambers were used to determine cell migration. RESULTS: Exposure of PC-3 and DU145 human prostate cancer cells to SFN resulted in induction of vimentin protein, which was accompanied by down-regulation of E-cadherin protein expression. The SFN-mediated induction of vimentin was also observed in a normal human prostate epithelial cell line. RNA interference of vimentin did not have any appreciable effect on early or late apoptosis resulting from SFN exposure. On the other hand, SFN-mediated inhibition of PC-3 and DU145 cell migration was significantly augmented by knockdown of the vimentin protein. Knockdown of vimentin itself was inhibitory against cell migration. The SFN-treated cells also exhibited induction of PAI-1, which is an endogenous inhibitor of urokinase-type plasminogen activator system. Similar to vimentin, PAI-1 knockdown resulted in a modest augmentation of PC-3 cell migration inhibition by SFN. Tumors from SFN-treated transgenic adenocarcinoma of mouse prostate mice showed a 1.7-fold increase in vimentin protein level compared with control tumors. CONCLUSION: The present study indicates that vimentin and PAI-1 inductions confer modest protection against SFN-mediated inhibition of prostate cancer cell migration.

Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone.

The plants used in Ayurvedic medicine, which has been practiced in India for thousands of years for the treatment of a variety of disorders, are rich in chemicals potentially useful for prevention and treatment of cancer. Withania somnifera (commonly known as Ashwagandha in Ayurvedic medicine) is one such medicinal plant whose anticancer value was realized over four decades ago after isolation of a crystalline steroidal compound (withaferin A) from the leaves of this shrub. The root and leaf extracts of W. somnifera are shown to confer protection against chemically-induced cancers in experimental rodents, and retard tumor xenograft growth in athymic mice. Anticancer effect of W. somnifera is generally attributable to steroidal lactones collectively referred to as withanolides. Withaferin A (WA) appears most active against cancer among structurally divergent withanolides isolated from the root or leaf of W. somnifera. Cancer-protective role for WA has now been established using chemically-induced and oncogene-driven rodent cancer models. This review summarizes the key in vivo preclinical studies demonstrating anticancer effects of WA. Molecular targets and mechanisms likely contributing to the anticancer effects of WA are also discussed. Finally, challenges in clinical development of WA for the prevention and treatment of cancer are highlighted.

Chemoprevention of prostate cancer by d,l-sulforaphane is augmented by pharmacological inhibition of autophagy.

There is a preclinical evidence that the oral administration of d,l-sulforaphane (SFN) can decrease the incidence or burden of early-stage prostate cancer [prostatic intraepithelial neoplasia (PIN)] and well-differentiated cancer (WDC) but not late-stage poorly differentiated cancer (PDC). Because SFN treatment induces cytoprotective autophagy in cultured human prostate cancer cells, the present study tested the hypothesis that chemopreventive efficacy of SFN could be augmented by the pharmacologic inhibition of autophagy using chloroquine (CQ). Incidence of PDC characterized by prostate weight of more than 1 g was significantly lower in the SFN + CQ group than in control (P = 0.004), CQ group (P = 0.026), or SFN group (P = 0.002 by Fisher exact test). Average size of the metastatic lymph node was lower by about 42% in the SFN + CQ group than in control (P = 0.043 by Wilcoxon test). On the other hand, the SFN + CQ combination was not superior to SFN alone with respect to inhibition of incidence or burden of microscopic PIN or WDC. SFN treatment caused in vivo autophagy as evidenced by transmission electron microscopy. Mechanistic studies showed that prevention of prostate cancer and metastasis by the SFN + CQ combination was associated with decreased cell proliferation, increased apoptosis, alterations in protein levels of autophagy regulators Atg5 and phospho-mTOR, and suppression of biochemical features of epithelial-mesenchymal transition. Plasma proteomics identified protein expression signature that may serve as biomarker of SFN + CQ exposure/response. This study offers a novel combination regimen for future clinical investigations for prevention of prostate cancer in humans.