Abstract
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.
Highlights
Chronic inflammation is a well-known precursor for cancer development and proliferation [1]
We have previously performed a dose-response study with Sodium chloride/salt (NaCl) (Δ0 to Δ200 mM) [6, 13] and IL-17 (0–1000 ng/mL) [6, 14] and demonstrated that high salt at 50 mM above basal concentration (Δ0.05 M) along with sub-effective IL-17 (0.1 nM) is known to induce breast cancer cell proliferation and promote inflammatory responses
Our phospho-proteomic analysis (S1 Table) identified a unique salt sensitive kinase salt-inducible Kinase-3 (SIK3), salt inducible kinase-3 (Q9Y2K2; NCBI protein Id: NP_079440.2), a 1263 amino acid serine/threonine kinase, which has shown a specific phosphorylation at site ser-493 following co-treatment with NaCl (Δ0.05)
Summary
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 leading to cancer development. These smoldering chronic inflammatory events induce reactive oxygen and nitrogen species (RNS/ ROS) and resulting in DNA damage and tumor formation. Chronic inflammation is known to induce a series of signaling transcription factors which promote uncontrolled cell division and tumor progression. Cancer cells metastasize through these newly formed blood vessels to various parts of the body [2]
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