Role of intracellular calcium on phenotypic differentiation of hepatocellular carcinoma cell lines

Besides physiological processes in the body, spatio-temporal changes in intracellular Ca2+ signaling is known to play a crucial role in carcinogenesis. Store-operated Ca2+ Entry (SOCE) is one of the major Ca2+ entry pathways in non-excitable cells including hepatocellular carcinoma (HCC) cells to regulate intracellular Ca2+ homeostasis. In recent studies, the main reason of the reoccurrence of cancer after treatments has been shown to be the presence of the tumor initiating cells (TICs) in the heterogeneous tumor cell population. Therefore, the purpose of this study is to delineate the role of SOCE in phenotypical differentiation of TICs found in Huh7 HCC cell line expressing specific cell surface markers, EpCAM (Epithelial Cell Adhesion Molecule) and CD133. For this purpose, we investigated the Ca2+ homeostasis mechanism and performed target validation on functional SOCE in these cell subpopulations (EpCAM+CD133+ or TICs). We initially separated the TICs from parental cells via a flow cell sorter cytometer (Fluorescent Activated Cell Sorter, FACS) by the help of fluorophore-conjugated specific antibodies targeting their cell surface antigens of interest. We, secondly, have the cells overexpress the two functionally important components of SOCE, an endoplasmic reticulum (ER) integral membrane protein of endoplasmic reticulum (ER) that senses intra luminal Ca2+ content, stromal interaction molecule (STIM1) and a membrane Ca2+ influx protein (Orai1), in these cells. We, then, investigated their role in real time changes in intracellular Ca2+ dynamics, cellular proliferation, drug resistance (MDR1 gene expression, P-glycoprotein), EMT marker mRNA expression, and cellular invasion-migration properties as well as their capability of in vitro carcinogenesis. Based on our data, ER Ca2+ release and SOCE were found to be higher in TICs compared to the EpCAM-CD133- cells. This may explain how TICs avoid apoptosis, possibly buffering the excess amount of Ca2+ in ER in to prevent mitochondrial Ca2+ uptake which is necessary for triggering apoptosis. Slow proliferation rates of TICs comparable to that of the parental cells, might be the resulted of Ca2+ buffering mechanism that holds these cells in an undifferentiated state. Overexpressing (OE) the SOCE components, STIM1 and Orai1, in TICs led to an effective SOCE in EpCAM+CD133+ STIM1+Orai1- OE cells. This observation suggested us that there might be a stoichiometric relationship between STIM1 and Orai1 expressions also described by others. In multidrug resistance (MDR) experiments, among STIM1- OE, Orai1- OE and STIM1+Orai1-OE constructs STIM1+Orai1- OE has the highest expression level of MDR1 gene suggesting a causal relationship between SOCE and MDR development in TICs. In the epithelial to mesenchymal transition (EMT) experiments, we evaluated the transcription levels of EMT markers, E-cadherin (epithelial), N-cadherin and vimentin (mesenchymal), in STIM1-OE and STIM1+Orai1 OE TICs. In this regard, elevation of both epithelial and mesenchymal markers suggested us the presence of partial EMT, an interphase between EMT and complete EMT. Based on our functional and genomic analyses, STIM1 and Orai1 would be important for ER Ca2+ buffering capacity and SOCE entry operation mechanism, respectively, crucial upstream events for cellular survival. Targeting the TICs for SOCE components STIM1-Orai1 and their related signaling pathways as well as manipulating related miRNAs can support development of specific anticancer strategies.