FUNCTIONAL GENOMICS ANALYSIS OF HEPATOCELLULAR TUMOR-INITIATING CELLS

Bilge Karacicek, Yasemin Erac and Metiner Tosun

Faculty of Medicine, Izmir University of Economics, Izmir, Turkey

Abstract

Based on our earlier observations in TRPC1 gene knock-down studies, store-operated Ca²⁺ entry (SOCE) appeared to be central role in survival and proliferation of Huh-7 hepatocellular cancer cell lines. This study investigates further the role of SOCE mediated by stromal interactive molecule (STIM1), a Ca²⁺ sensor of endoplasmic reticulum (ER) and Orai1, Ca²⁺ release-activated Ca²⁺ ion channel, in differentiation of hepatocellular tumor-initiating cells (TICs). Both functional and genome-wide expression analyses were performed in TICs which these proteins were overexpressed to elucidate the key determinants of the phenotypical switch. First, TICs were separated from Huh-7 hepatocellular carcinoma (HCC) cell lines via fluorescence-accelerated cell sorter by using fluorescently-labeled antibodies recognizing specific cell adhesion molecules, CD133 and EpCAM, expressed on this TIC subpopulation. STIM1 and/or Orai1 were overexpressed by plasmid vector transfection. Changes in ER Ca²⁺ release and SOCE dynamics were monitored in fura-2-loaded cells seeded on circular coverslips via front-surface spectrofluorimetry. Transcriptome profiling was performed by using HumanHT-12_v4_BeadChip (Illumina). MicroRNA profile was determined by a whole-genome sequencing platform (NextSeq 500, Illumina). mRNAs targeted by these miRNAs were searched through a public database, MiRBase. Our data showed that Orai1 expression is higher in TICs while no significant difference observed for STIM1. In addition, SOCE and ER stress-induced Ca²⁺ release was significantly higher in TICs compared to that of parental HCC cells. Ca²⁺ release following the ER stress was doubled in STIM1-overexpressing TICs. Epithelial marker E-cadherin expression was increased 3 fold in TICs overexpressing STIM1 and/or Orai1. MDR1 gene expression was also twice as much in TICs and it was doubled by inducing either STIM1 or Orai1 expression. Migration/invasion and in vitro tumorigenesis profiles were also assessed in TICs overexpressing STIM1 and/or Orai1. Briefly, potential anticancer targets related with Ca²⁺ homeostasis in HCC will be discussed based on the data obtained from both functional and genomic analyses. This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, 113S399 to MT).