Abstract
The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9+ cells were capable of self-renewal and differentiation into SOX9− cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9+ cells reproduced, differentiated into SOX9− cells, and generated tumors at a high frequency in vivo. Moreover, SOX9+ cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9− patients, SOX9+ patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.
Highlights
SOX9 expression is confined to the bile duct, while hepatocytes do not express SOX9 during embryogenesis; this expression pattern persists in adulthood[28]
Previously reported hepatocellular carcinoma (HCC)-cancer stem cells (CSCs) markers such as epithelial cell adhesion molecule (EpCAM), CD90, CD133, CD24, CD13, sal-like protein 4 (SALL4), and keratin 19 (K19) are all expressed in the embryonic liver, while their expressions are not normally detected in adult hepatocytes
Considering the results of two independent lineage tracing studies, which demonstrated that embryonic SOX9+ cells possess an ability to differentiate into hepatocytes[29,30], we hypothesized that SOX9 is an excellent candidate CSC marker in human HCC
Summary
SOX9 expression is confined to the bile duct, while hepatocytes do not express SOX9 during embryogenesis; this expression pattern persists in adulthood[28]. Considering the results of two independent lineage tracing studies, which demonstrated that embryonic SOX9+ cells possess an ability to differentiate into hepatocytes[29,30], we hypothesized that SOX9 is an excellent candidate CSC marker in human HCC. To test this hypothesis, we sorted SOX9+ cell population from several human HCC cell lines and analyzed their CSC characteristics. We provide evidence that osteopontin (OPN), another downstream target of the Wnt/beta-catenin pathway[32], is regulated by SOX9 and highly expressed in the blood samples from the patients bearing SOX9+ HCC, putting OPN as a surrogate marker of SOX9 in human HCCs
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