Open in another window Figure 1. Ovarian cancer cells become free-floating in the peritoneal cavity before they re-attach in the metastatic niche. Cells go through significant cytoskeletal re-modeling and withstand nourishment deprivation before they become founded at their metastatic Cisplatin supplier site. Our latest work referred to how SIK2 activation by adipocytes takes on a key part in establishing stomach metastasis. Long term function shall explore the possible part of SIK2 in the multi-step metastatic procedure. Using an in-cell gate-keeper screen we then identified SIK2-S358 as a key residue for SIK2 autophosphorylation. We extensively validated the SIK2 S358 autophosphorylation residue for use as a readout of SIK2 activity and as a biomarker of on-target activity of SIK2 inhibitors. We then established a system for the co-culture of cancer cells with adipocytes obtained from freshly excised normal omentum from women undergoing surgical staging of ovarian tumors. This system was used in combination with either an ATP-competitive SIK2 kinase inhibitor or siRNA mediated depletion of SIK2 to test the specificity of observed phenotypic effects. Co-culture of ovarian tumor cells with adipocytes led to calcium discharge in ovarian tumor cells and following activation of SIK2 as evidenced by elevated S358 phosphorylation. Significantly, SIK2 activation result in the activation from the PI3K/AKT pathway though phosphorylation of residues S154 and S541 of p85, the regulatory subunit from the PI3K complicated. The hyperlink between SIK2 activation as well as the PI3K complicated activation was further verified using rapamycin being a known solid paradoxical PI3K activator. We demonstrated that siRNA-mediated depletion of or its chemical substance inhibition significantly decreased rapamycin-induced AKT phosphorylation and sensitized ovarian tumor cells to its cytotoxic impact.6 Significantly, calcium-dependent activation of SIK2 was independent from LKB1 indicating that LKB1 isn’t a distinctive regulator of SIK2 yet that other kinases may activate it downstream of a rise in intracellular calcium with regards to the cell type and context of activation. That is similar to the previously reported activation of AMPK by CAMKK through immediate phosphorylation of T172 at the T-loop of the AMPK kinase, a site that is also phosphorylated by LKB1.7 Surprisingly, we also showed that SIK2 augments AMPK in phosphorylating ACC1. This identified SIK2 as an important activator of the fatty acid oxidation pathway.6 These studies suggest that adipocyte-mediated SIK2 phosphorylation and protein stabilization are required to activate cancer cell proliferation and fatty acid oxidation at the omental metastatic niche (Fig.?1). Future work will test whether SIK2 may cooperate with AMPK at the early stages of metastasis Cisplatin supplier when ovarian cancer Cisplatin supplier cells detach from the tumor site of origin. It is also plausible that SIK2 activation is required for cytoskeleton remodeling and re-attachment to the metastatic niche in the abdominal cavity. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.. used in combination with either an ATP-competitive SIK2 kinase inhibitor or siRNA mediated depletion of SIK2 to test the specificity of observed phenotypic effects. Co-culture of ovarian cancer cells with adipocytes resulted in calcium release in ovarian cancer cells and subsequent activation of SIK2 as evidenced by increased S358 phosphorylation. Importantly, SIK2 activation lead to the activation of the PI3K/AKT pathway though phosphorylation of residues S154 and S541 Cisplatin supplier of p85, the regulatory subunit of the PI3K complex. The link between SIK2 activation and the PI3K complex activation was further confirmed using rapamycin as a known strong paradoxical PI3K activator. We showed that siRNA-mediated depletion of or its chemical inhibition significantly reduced rapamycin-induced AKT phosphorylation and sensitized ovarian cancer cells to its cytotoxic effect.6 Importantly, calcium-dependent activation of SIK2 was independent from LKB1 indicating that LKB1 is not a unique regulator of SIK2 but Cisplatin supplier that other kinases may activate it downstream of an increase in intracellular calcium depending on the cell type and context of activation. This is reminiscent of the previously reported activation of AMPK by CAMKK through direct phosphorylation of T172 at the T-loop of the AMPK kinase, a site that is also phosphorylated by LKB1.7 Surprisingly, we also showed that SIK2 augments AMPK in phosphorylating ACC1. This identified SIK2 as an important activator of HA6116 the fatty acid oxidation pathway.6 These studies suggest that adipocyte-mediated SIK2 phosphorylation and protein stabilization are required to activate cancer cell proliferation and fatty acid oxidation at the omental metastatic niche (Fig.?1). Future work will check whether SIK2 may cooperate with AMPK at the first levels of metastasis when ovarian cancers cells detach in the tumor site of origins. Additionally it is plausible that SIK2 activation is necessary for cytoskeleton redecorating and re-attachment towards the metastatic specific niche market in the stomach cavity. Disclosure of potential issues appealing No potential issues of interest had been disclosed..