Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Cancer stem cells (CSCs) have attracted attention as a novel therapeutic target for cancer because they play important roles in the development and aggravation of cancer. CD44 is expressed as a standard isoform (CD44s) and several variant isoforms. CD44v is a major isoform expressed on CSCs of a variety of tumors and has been extensively studied. However, HCC tissues dominantly express CD44s, whose function in CSCs remains unclear. In the present study, we investigated the roles of CD44s in CSCs of HCC. Knock‐out of the CD44 gene in HuH7 HCC cells on which only CD44s is expressed resulted in decreased spheroid formation and increased drug sensitivity. The expression of CSC marker genes, including CD133 and EpCAM, was significantly downregulated in the spheroids of CD44‐deficient cells compared with those in the spheroids of HuH7 cells. In addition, CD44 deficiency impaired antioxidant capacity, concomitant with downregulation of glutathione peroxidase 1 (GPX1) and thioredoxin. Because GPX1 uses the reduced form of glutathione (GSH) to regenerate oxidized cellular components, GSH levels were significantly increased in the CD44‐deficient cells. We also found that NOTCH3 and its target genes were downregulated in the spheroids of CD44‐deficient cells. NOTCH3 expression in HCC tissues was significantly increased compared with that in adjacent nontumor liver tissues and was correlated with CD44 expression. These results suggest that CD44s is involved in maintenance of CSCs in a HCC cell line, possibly through the NOTCH3 signaling pathway.
Highlights
Primary liver cancer accounts for more than 30 000 deaths each year in Japan and is one of the leading causes of cancer death worldwide.[1]
We have previously reported that CD44 is expressed as a standard isoform (CD44s) expression in Hepatocellular carcinoma (HCC) tissues, which is assessed by an antibody against total CD44, was significantly associated with poor prognosis while no significant association was observed between the prognosis and CD44v expression, which is assessed by a CD44v‐specific antibody.[23]
It has been reported that NANOG‐deficient prostate cancer cells generated by the CRISPR/Cas[9] system showed a significant decrease in tumorigenicity, compared with parental cells.[27]
Summary
Primary liver cancer accounts for more than 30 000 deaths each year in Japan and is one of the leading causes of cancer death worldwide.[1]. Accumulating evidence suggests that recurrence and metastasis in many cancers can be attributed to cancer stem cells (CSCs).[4,5] Because CSCs have the ability of self‐renewal and differentiation, CSCs are indispensable for initiating and maintaining tumor phenotypes while other cancer cells (non‐ CSCs) do not have such properties.[6] The existence of CSCs was first demonstrated in acute myeloid leukemia.[7] Since using specific CSC markers, the existence of CSCs has been proven in various tumors such as brain, breast, lung, colon, and liver.[8,9,10,11,12] Several studies reported that liver CSCs express several markers such as epithelial adhesion molecule (EpCAM), CD13, CD44, and/or CD133 and that the expression of these molecules on HCC cells is correlated with poor prognosis.[12,13,14] Because CSCs display the features of tumorigenicity and resistance to conventional chemotherapy and radiotherapy, it is suggested that surviving CSCs eventually cause tumor recurrence and metastasis conventional therapies could eliminate non‐CSCs.[4,5] the removal of CSCs is important to cure cancer completely. We established CD44‐knocked out (CD44‐ KO) cells from human HuH7 HCC cells, in which only CD44s is expressed, by means of the CRISPR/Cas[9] system, and investigated the phenotypic changes in CSC properties of CD44‐KO cells
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