Abstract
The epithelial to mesenchymal transition (EMT) of malignant hepatocytes is a crucial event in hepatocellular carcinoma (HCC) progression and recurrence. We aimed to establish a human model of EMT to examine drug efficacy and specificity in HCC progression. Human HCC cell populations were characterized by immunofluorescence analysis, migration and invasion assays, array comparative genomic hybridization, whole-genome expression profiling, and promoter methylation. Therapeutic agents clinically used against HCC were examined for efficacy by determination of IC(50) values. We show that liver cancer cell lines exhibited either an epithelial or mesenchymal phenotype of which the latter showed strong migratory and invasive abilities in vitro. The common cellular origin of both cell types indicated that mesenchymal HCC cells have been derived from epithelial hepatocytes through EMT in the HCC patient. Drug exposure of mesenchymal HCC cells showed higher resistance to the targeted therapeutic agents sorafenib and erlotinib as compared to epithelial HCC cells, which were slightly more resistant to cytostatic drugs. Most remarkably, combined treatment with doxorubicin and sorafenib caused increased susceptibility of both HCC cell types resulting in enhanced drug efficacy. Taken together, this EMT model of human HCC allows the identification of molecular mechanisms and the assessment of therapeutic drug efficacy during liver cancer progression in preclinical studies.
Highlights
The epithelial to mesenchymal transition (EMT) enables carcinoma cells to invade into surrounding tissues and to form secondary tumors known as metastases
Phase contrast analysis suggested an epithelial cell type, termed 3p, and a mesenchymal cell population, designated 3sp (Fig. 1A). Both hepatocellular carcinoma (HCC) cells showed diploid DNA content (Supplementary Fig. S1) and short tandem repeat analysis verified their common genomic identity (Supplementary Table S3). qRT-Polymerase chain reaction (PCR) analysis revealed that 3p cells express epithelial markers such as E-cadherin and keratin 8, whereas 3sp cells showed a mesenchymal expression signature by upregulation of the transcription factors LEF1, SNAI1, SNAI2, and ZEB1 (Fig. 1B)
We further investigated whether 3sp cells have been derived from epithelial 3p cells through EMT in the HCC patient. Array comparative genomic hybridization (aCGH) analysis showed that changes in the genomic DNA of these HCC cell lines were widely identical (Supplementary Fig. S3), which were exemplified by the loss of genomic DNA in the TRPM3 and AXIN1 loci of both cell types (Fig. 3A and B)
Summary
The epithelial to mesenchymal transition (EMT) enables carcinoma cells to invade into surrounding tissues and to form secondary tumors known as metastases. A particular characteristic of EMT is the downregulation of E-cadherin expression, which causes disruption of cell–cell junctions and dissemination of cells from the primary tumor [1]. Dysregulation of E-cadherin is provoked by its transcriptional repressors involving Snail/SNAI1, Slug/SNAI2, ZEB1/DEF1, ZEB2/SIP1, or Twist [2]. Receptor tyrosine kinase/Ras and transforming growth factor (TGF)-b signaling as well as Wnt/b-catenin-, Notch-, Hedgehog-, and Authors' Affiliations: Departments of 1Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center, 2Obstetrics and Gynecology, Ludwig Boltzmann Institute for Gynecology and Gynecological Oncology, Medical University of Vienna, 3Austrian Institute of Technology, and 4Clinical Institute for Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria.
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