Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) levels have been considered a potent inducer of chronic inflammation. In the present study, the synergistic role of high salt with interleukin (IL)-17 towards induction of the inflammatory and angiogenic stress factor vascular endothelial growth factor (VEGF)-A was investigated. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/ml) enhanced the expression of VEGF-A (2.9 and 2.6-fold, respectively, P<0.05) compared with untreated cells. Furthermore, co-treatment with both high salt and sub-minimal IL-17 led to a 5.9-fold increase in VEGF-A expression (P<0.01), thus suggesting a synergistic role of these factors. VEGF-A promoter analysis and specific small interfering RNA knock-down of transcription factors revealed that high salt induced VEGF-A expression through nuclear factor of activated T-cells (NFAT)5, while IL-17 induced VEGF-A expression via signal transducer and activator of transcription (STAT)3 signaling mechanisms. Treatment of normal human aortic endothelial cells with the supernatant of activated MCF-7 cells enhanced cell migration and induced expression of migration-specific factors, including vascular cell adhesion protein, β1 integrin and cluster of differentiation 31. These data suggest that high salt levels synergize with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low-salt diet, anti-NFAT5 and anti-STAT3 therapies may provide novel avenues for enhanced efficiency of the current cancer therapy.

Abstract. Prostratin, a phorbol ester natural plant compound, has been demonstrated to exert an anti-retroviral effect through activation of latent cluster of differentiation (CD)4+T lymphocytes and inhibition of viral entry into the cell through downregulation of chemokine receptor type 4 (CXCR4) expression. However, the potential effect of prostratin on cancer is yet to be defined. As CXCR4 is well known to induce cancer migration, it was hypothesized that prostratin induces an anti-cancer effect through inhibition of CXCR4 expression. The authors previously demonstrated that high stimulating conditions (sub-minimal IL-17, 0.1 ng/ml, synergized with high salt, Δ0.05 M NaCl) promote breast cancer cell proliferation and CXCR4 expression through upregulation of salt-inducible kinase (SIK)-3. The present study demonstrated that prostratin selectively exerted increased cytotoxicity (IC50 of 7 µM) when breast cancer cells were cultured in high stimulating conditions, compared with regular basal culture conditions (IC50 of 35 µM). Furthermore, the cytotoxic potential of prostratin was increased seven-fold in the four breast cancer cell lines (MCF-7, MDA-MB-231, BT-20 and AU-565) compared with the non-malignant MCF10A breast epithelial cell line. This suggested that prostratin specifically targets cancer cells over normal cells. Mechanistic studies revealed that prostratin inhibited CXCR4 expression in breast cancer cells through downregulation of SIK3 expression. Overall, the data suggest that prostratin is a novel drug target for the pro-oncogenic factor SIK3. These studies could form a basis for further research to evaluate the anticancer effect of prostratin in a combinatorial chemotherapeutic regimen. IntroductionChronic inflammation is a well-established hallmark of cancer proliferation (1). The cellular stress caused by inflammation induces release of cytokine and chemokine factors which induce tumor progression and metastasis (2). Several agents have been suggested to induce chronic inflammation (3). Recent evidence from our lab have demonstrated that breast cancer cells cultured under high salt conditions (Δ0.05 M NaCl, 50% above basal culture conditions) were able to upregulate reactive nitrogen species (4,5). Importantly, sodium-MRI studies in breast cancer patients have demonstrated an increased sodium content, of up to 63% above the surrounding soft tissue, in the breast tumors (6,7). These support a notion that high salt exerts an effector role on tumor progression, either working individually or synergistically to enhance an inflammatory tumor microenvironment. Phospho-proteomic based studies from our laboratory have demonstrated that high salt (Δ0.05 M) synergized with sub-minimal stimulation of IL-17 (0.1 ng/ml) induced upregulation of SIK-3, salt inducible kinase-3, a serine-specific protein kinase in breast cancer cells (8). Mechanistic studies have demonstrated that SIK3 played a crucial role in induction of G1/S-phase release of cell cycle, along with enhanced expression of metastasis specific che...

Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) has been considered a potent inducer of chronic inflammation. In this study, we investigated the synergistic role of high salt with IL-17 towards induction of inflammatory and angiogenic stress factor VEGF. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/mL) enhanced the expression of VEGF-A (2.9 and 2.6 fold, respectively p&lt;0.05) compared to untreated cells. Further, co-treatment with both high salt and sub-minimal IL-17 demonstrated a 5.9 fold increase in VEGF-A expression (p&lt;0.01), thus suggesting a synergistic role of these both factors. VEGF-A promoter analysis and specific siRNA knock-down of transcription factors revealed that high salt induces VEGF-A through NFAT5, while IL-17 induced VEGF-A via STAT-3 signaling mechanisms. Treatment of the supernatant from activated MCF-7 cells on normalized human aortic endothelial cells induced enhanced cell migration and expression of migration specific factors VCAM, β1-integrin and CD31. Based on these data, we conclude that high salt synergizes with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low salt diet, anti-NFAT5 and anti-STAT3 therapies might provide novel venues for enhanced efficiency of current cancer therapy. Citation Format: Suneetha Amara, Dalal Alotaibi, Venkataswarup Tiriveedhi. NFAT-5/STAT-3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4404.