Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44+ and CD44+CD133+ cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.

BackgroundAccumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis.Methodology/Principal FindingsCD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues.Conclusions/SignificanceThe identified genes, such as GPC3 that are distinctly expressed in liver CD90+CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.

BackgroundCD26, dipeptidyl peptidase IV, was discovered firstly as a membrane-associated peptidase on the surface of leukocyte. We previously demonstrated that a subpopulation of CD26+ cells were associated with the development of distant metastasis, enhanced invasiveness and chemoresistance in colorectal cancer (CRC). In order to understand the clinical impact of CD26, the expression was investigated in CRC patient's specimens. This study investigated the prognostic significance of tumour CD26 expression in patients with CRC. Examination of CD26+ cells has significant clinical impact for the prediction of distant metastasis development in colorectal cancer, and could be used as a selection criterion for further therapy.MethodsTumour CD26 expression levels were studied by immunohistochemistry using Formalin-fixed paraffin embedded (FFPE) tissues in 143 patients with CRC. Tumour CD26 expression levels were correlated with clinicopathological features of the CRC patients. The prognostic significance of tumour tissue CD26 expression levels was assessed by univariate and multivariate analyses.ResultCD26 expression levels in CRC patients with distant metastasis were significantly higher than those in non-metastatic. High expression levels of CD26 were significantly associated with advanced tumour staging. Patients with a high CD26 expression level had significantly worse overall survival than those with a lower level (p<0.001).ConclusionsThe expression of CD26 was positively associated with clinicopathological correlation such as TNM staging, degree of differentiation and development of metastasis. A high CD26 expression level is a predictor of poor outcome after resection of CRC. CD26 may be a useful prognostic marker in patients with CRC.