A Novel Function for KLF4 in Modulating the De-differentiation of EpCAM-/CD133- nonStem Cells into EpCAM+/CD133+ Liver Cancer Stem Cells in HCC Cell Line HuH7.

The complex and heterogeneous nature of hepatocellular carcinoma (HCC) hampers the identification of effective therapeutic strategies. Cancer stem cells (CSCs) represent a fraction of cells within tumors with the ability to self-renew and differentiate, and thus significantly contribute to the formation and maintenance of heterogeneous tumor mass. Increasing evidence indicates high plasticity in tumor cells, suggesting that non-CSCs could acquire stem cell properties through de-differentiation or reprogramming processes. In this paper, we reveal KLF4 as a transcription factor that can induce a CSC-like phenotype in non-CSCs through upregulating the EpCAM and E-CAD expression. Our studies indicated that KLF4 could directly bind to the promoter of EpCAM and increase the number of EpCAM+/CD133+ liver cancer stem cells (LCSCs) in the HuH7 HCC cell line. When KLF4 was overexpressed in EpCAM-/CD133- non-stem cells, the expressions of hepatic stem/progenitor cell genes such as CK19, EpCAM and LGR5 were significantly increased. KLF4 overexpressing non-stem cells exhibited greater cell viability upon sorafenib treatment, while the cell migration and invasion capabilities of these cells were suppressed. Importantly, we detected an increased membranous expression and colocalization of ß-CAT, E-CAD and EpCAM in the KLF4-overexpressing EpCAM-/CD133- non-stem cells, suggesting that this complex might be required for the cancer stem cell phenotype. Moreover, our in vivo xenograft studies demonstrated that with a KLF4 overexpression, EpCAM-/CD133- non-stem cells attained an in vivo tumor forming ability comparable to EpCAM+/CD133+ LCSCs, and the tumor specimens from KLF4-overexpressing xenografts had increased levels of both the KLF4 and EpCAM proteins. Additionally, we identified a correlation between the KLF4 and EpCAM protein expressions in human HCC tissues independent of the tumor stage and differentiation status. Collectively, our data suggest a novel function for KLF4 in modulating the de-differentiation of tumor cells and the induction of EpCAM+/CD133+ LCSCs in HuH7 HCC cells.

Antiproliferative activity of (R)-4'-methylklavuzon on hepatocellular carcinoma cells and EpCAM+/CD133+ cancer stem cells via SIRT1 and Exportin-1 (CRM1) inhibition.

Cytotoxic effects of (R)-4'-methylklavuzon were investigated on hepatocellular carcinoma cells (HuH-7 and HepG2) and HuH-7 EpCAM+/CD133+ cancer stem cells. IC50 of (R)-4'-methylklavuzon was found as 1.25 µM for HuH-7 parental cells while it was found as 2.50 µM for HuH-7 EpCAM+/CD133+ cancer stem cells. (R)-4'-methylklavuzon tended to show more efficient in vitro cytotoxicity with its lower IC50 values on hepatocellular carcinoma cell lines compared to its lead molecule, goniothalamin and FDA-approved drugs, sorafenib and regorafenib. Cell-based Sirtuin/HDAC enzyme activity measurements revealed that endogenous Sirtuin/HDAC enzymes were reduced by 40% compared to control. SIRT1 protein levels were upregulated indicating triggered DNA repair mechanism. p53 was overexpressed in HepG2 cells. (R)-4'-methylklavuzon inhibited CRM1 protein providing increased retention of p53 and RIOK2 protein in the nucleus. HuH-7 parental and EpCAM+/CD133+ cancer stem cell spheroids lost intact morphology. 3D HepG2 spheroid viabilities were decreased in a correlation with upregulation in p53 protein levels.

Effect of adipocyte-secreted factors on EpCAM+/CD133+ hepatic stem cell population.

Recent epidemiological studies have associated obesity with a variety of cancer types including HCC. However, the tumor initiating role of obesity in hepatocarcinogenesis is still unknown. The objective of this paper is to investigate the effect of adipocyte-secreted factors on EpCAM+/CD133+ cancer stem cells and to identify which factors play a role in modulating hepatic cancer stem cell behavior. Our results demonstrated that adipocyte-secreted factors affect motility and drug resistance of EpCAM+/CD133+ cells. When incubated with adipocyte conditioned media, EpCAM+/CD133+ cells exhibited augmented motility and reduced sorafenib-induced apoptosis. Using array-based system, we identified secretion of several cytokines such as IL6, IL8 and MCP1 by cultured adipocytes and activation of c-Met, STAT3 and ERK1/2 signaling pathways in EpCAM+/CD133+ cells incubated with adipocyte conditioned media. Treating EpCAM+/CD133+ cancer stem cells with IL6 receptor blocking antibody or c-Met inhibitor SU11274 both reduced the increase in motility; however SU11274 had greater effect on relieving protection from sorafenib-induced apoptosis. These results indicate that adipocyte-secreted factors might regulate cancer stem cell behavior through several signaling molecules including c-Met, STAT3 and ERK1/2 and inhibition of these signaling pathways offer novel strategies in targeting the effect of adipose-derived cytokines in cancer.

Heparin treatment increases thioredoxin interacting protein expression in hepatocellular carcinoma cells.

Heparins play an important role in cell growth, differentiation, migration and invasion. However, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. To determine the effect of heparin on gene expression, we performed a cDNA microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. In this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. We determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. We showed the importance of heparin mediated histone modifications and down-regulation of Enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxin-interacting protein. When we tested biological significance of these data; we observed that cells overexpressing thioredoxin-interacting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. Interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. In conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. This study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis.