Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

Calcium Signaling in the Thyroid: Friend and Foe.

Calcium signaling participates in a vast number of cellular processes, ranging from the regulation of muscle contraction, cell proliferation, and mitochondrial function, to the regulation of the membrane potential in cells. The actions of calcium signaling are, thus, of great physiological significance for the normal functioning of our cells. However, many of the processes that are regulated by calcium, including cell movement and proliferation, are important in the progression of cancer. In the normal thyroid, calcium signaling plays an important role, and evidence is also being gathered showing that calcium signaling participates in the progression of thyroid cancer. This review will summarize what we know in regard to calcium signaling in the normal thyroid as, well as in thyroid cancer.

Sphingosine 1-phosphate attenuates MMP2 and MMP9 in human anaplastic thyroid cancer C643 cells: Importance of S1P2.

In anaplastic thyroid cancer C643 cells, sphingosine 1-phosphate (S1P) attenuates migration by activating the S1P2 receptor and the Rho-ROCK pathway. In the present study, we show that stimulating C643 cells with S1P decreases the expression, secretion and activity of matrix metalloproteinase-2 (MMP2), and to a lesser extent MMP9. Using receptor-specific antagonists, and S1P2 siRNA, we showed that the inhibition of expression of MMP2 is mediated through S1P2. Furthermore, S1P inhibited calpain activity, and inhibiting calpain pharmacologically, inhibited the effect of S1P on MMP2 expression and activity, and on MMP9 activity. S1P treatment increased Rho activity, and by incubating cells with the Rho inhibitor C3 transferase or the ROCK inhibitor Y27632, the S1P-induced inhibition of invasion and MMP2 expression and activity was abolished. We conclude that S1P attenuates the invasion of C643 cells by activating S1P2 and the Rho-ROCK pathway, by decreasing calpain activity, and by decreasing the expression, secretion and activity of MMP2 and, to a lesser extent, MMP9. Our results thus unveil a novel function for the S1P2 receptor in attenuating thyroid cancer cell invasion.

Oxysterol-binding protein related-proteins (ORPs) 5 and 8 regulate calcium signaling at specific cell compartments.

Oxysterol-binding protein related-protein 5 and 8 (ORP5/8) localize to the membrane contact sites (MCS) of the endoplasmic reticulum (ER) and the mitochondria, as well as to the ER-plasma membrane (PM) MCS. The MCS are emerging as important regulators of cell signaling events, including calcium (Ca2+) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP2) in the PM, and to modulate mitochondrial respiration and morphology. PIP2 is the direct precursor of inositol trisphosphate (IP3), a key second messenger responsible for Ca2+-release from the intracellular Ca2+ stores. Further, mitochondrial respiration is linked to Ca2+ transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca2+ signaling in these cell compartments, and employed genetically engineered aequorin Ca2+ probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca2+. Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca2+ as well as to increased Ca2+ concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca2+. Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca2+ signaling at specific MCS foci. These local ORP5/8-mediated Ca2+ signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation.

Sphingosylphosphorylcholine regulates the Hippo signaling pathway in a dual manner.

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid which regulates many cancer-related processes, including cellular proliferation. The Hippo signaling pathway consists of a cascade of tumor suppressive kinases Mst1/2 and Lats1/2 and their downstream targets YAP and TAZ which are generally pro-proliferative transcriptional regulators. Direct phosphorylation by Lats1/2 causes inhibition or degradation of YAP/TAZ and down-regulation of their target genes. We found SPC treatment of MDA-MB-435S breast cancer cells to strongly inhibit their proliferation and to induce a sustained Lats2 protein expression (6-24h). Therefore, we hypothesized that Hippo signaling might mediate the anti-proliferative SPC response. We also saw a cell density-dependent increase in S127-phosphorylated YAP (pS127-YAP) and a decrease in mRNA levels of YAP target genes (CTGF, Cyr61) in response to long (9h) SPC treatment. Knockdown of S1P receptor 2 (S1P2) prevented the SPC-induced up-regulation of Lats2 and attenuated the anti-proliferative effect of SPC. However, while knockdown of Lats2 alone or in combination with Lats1 expectedly increased basal proliferation it did not attenuate the SPC-induced inhibition of proliferation. Exogenous expression of wild-type or kinase-dead Lats2 and knockdown of YAP/TAZ also had no effect on the anti-proliferative SPC response. It has been previously shown that activation of S1P2-G12/13 by sphingosine-1-phosphate (S1P) leads to rapid de-phosphorylation and up-regulation of YAP. Similarly, we saw a decrease in pS127-YAP and an increase in total YAP levels with short (1h) SPC treatment as well as a subsequent transient increase in YAP target gene expression. Inhibition of S1P2 prevented the SPC-induced YAP de-phosphorylation. The rapid YAP activation and subsequent up-regulation of Lats2 mRNA does not constitute a negative feedback loop as knockdown of YAP/TAZ did not inhibit SPC-induced Lats2 expression. In conclusion, in this study we show that SPC is able to regulate Hippo signaling in a dual and opposite manner, causing an initial activation of YAP followed by an inhibition. However, even the strong SPC-induced effects seen in Lats2 and YAP did not mediate the anti-proliferative SPC response.