Research Article: Critical role of SIK3 in mediating high salt and IL-17 synergy leading to breast cancer cell proliferation

Date Published: June 28, 2017

Publisher: Public Library of Science

Author(s): Suneetha Amara, Ciera Majors, Bipradas Roy, Salisha Hill, Kristie L. Rose, Elbert L. Myles, Venkataswarup Tiriveedhi, Aamir Ahmad.


Chronic inflammation is a well-known precursor for cancer development and proliferation. We have recently demonstrated that high salt (NaCl) synergizes with sub-effective interleukin (IL)-17 to induce breast cancer cell proliferation. However, the exact molecular mechanisms mediating this effect are unclear. In our current study, we adopted a phosphoproteomic-based approach to identify salt modulated kinase-proteome specific molecular targets. The phosphoprotemics based binary comparison between heavy labelled MCF-7 cells treated with high salt (Δ0.05 M NaCl) and light labelled MCF-7 cells cultured under basal conditions demonstrated an enhanced phosphorylation of Serine-493 of SIK3 protein. The mRNA transcript and protein expression analysis of SIK3 in MCF-7 cells demonstrated a synergistic enhancement following co-treatment with high salt and sub-effective IL-17 (0.1 ng/mL), as compared to either treatments alone. A similar increase in SIK3 expression was observed in other breast cancer cell lines, MDA-MB-231, BT20, and AU565, while non-malignant breast epithelial cell line, MCF10A, did not induce SIK3 expression under similar conditions. Biochemical studies revealed mTORC2 acted as upstream mediator of SIK3 phosphorylation. Importantly, cell cycle analysis by flow cytometry demonstrated SIK3 induced G0/G1-phase release mediated cell proliferation, while SIK3 silencing abolished this effect. Also, SIK3 induced pro-inflammatory arginine metabolism, as evidenced by upregulation of the enzymes iNOS and ASS-1, along with downregulation of anti-inflammatory enzymes, arginase-1 and ornithine decarboxylase. Furthermore, gelatin zymography analysis has demonstrated that SIK3 induced expression of tumor metastatic CXCR4 through MMP-9 activation. Taken together, our data suggests a critical role of SIK3 in mediating three important hallmarks of cancer namely, cell proliferation, inflammation and metastasis. These studies provide a mechanistic basis for the future utilization of SIK3 as a key drug discovery target to improve breast cancer therapy.

Partial Text

Chronic inflammation is a well-known precursor for cancer development and proliferation [1]. Unlike acute inflammation which exerts a beneficial pathogen or disease eliminatory function, chronic inflammation initiates a cascade of molecular events that causes malignant transformation of terminally differentiated cells and thus leading to cancer development. These smoldering chronic inflammatory events induce reactive oxygen and nitrogen species (RNS/ROS) and thus resulting in DNA damage and tumor formation. Along with this, chronic inflammation is known to induce a series of signaling transcription factors which promote uncontrolled cell division and tumor progression. The cellular stress caused by inflammation induces release of several growth factors which induce neo-vascularization to the tumor. Cancer cells metastasize through these newly formed blood vessels to various parts of the body [2].

Recent understanding has established a strong correlation between chronic inflammation and cancer [34]. A multi-factorial etiology including, chemical carcinogens, infections and diet have been suggested to play an important role in chronic inflammation. Several epidemiological studies have demonstrated that high salt (sodium chloride) diet correlated with several chronic inflammatory diseases [35, 36]. Other than in gastric cancers, to date, there is no evidence of direct correlation between high salt diet and human cancers [37]. However, importantly, sodium-MRI studies have demonstrated a sodium concentration in the breast cancer microenvironment [8]. Previous studies in our laboratory have demonstrated that high salt induced cancer cell proliferation and inflammatory reactive oxygen and nitrogen species [6]. To determine the molecular events induced by high salt in the inflammatory tumor microenvironment, we have performed phospho-proteomic studies on MCF-7 cancer cells following stimulation with high salt and sub-effective inflammatory cytokine (IL-17) stimulation. We have identified a unique salt-specific molecule, salt-inducible kinase-3 (SIK3), whose phosphorylation at Ser-493 is upregulated following stimulation under above mentioned conditions.




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