CD133 confers cancer stem-like cell properties by stabilizing EGFR-AKT signaling in hepatocellular carcinoma

Highlights

• CD133, a CSC marker is identified in primary HCCs and HCC cell lines.

• The expression of EGFR is correlated with The expression of CD133 in HCC cells.

• CD133 induces CSCs properties through the EGFR stabilization.

• The stabilization of EGFR by CD133 activates EGFR-AKT signaling in HCCs.

• CD133 might be a novel target to sensitize HCC cells that manifest resistance to EGFR-targeted therapy.

Abstract

Hepatocellular carcinoma (HCC) is the seventh most common malignant tumor and the third leading cause of cancer-related death in the world. Cancer stem cells (CSCs) are small subpopulation of cells within tumors that drive chemoresistance and tumor recurrence in various cancers. We characterized CSCs in primary HCC and identified CD133 as a CSC surface marker. CD133⁺ HCC cells displayed more stem cell-like properties, tumor spheroid-forming ability, chemoresistance, migration ability, and tumorigenic capacity than CD133^- HCC cells. The biological function and molecular mechanism of CD133 remain unclear. HCC cell lines with a high level of CD133 expression overexpressed EGFR, which is overexpressed in approximately 70% of conventional HCCs. CD133 depletion destabilized EGFR by augmenting EGFR internalization and thus inhibited EGFR-AKT signaling. CD133 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Furthermore, EGFR-deficient CD133⁺ HCC cells manifested greater sensitivity to anticancer drugs and less spheroid-formation capacity than control CD133⁺ HCC cells. Our results strongly indicate that CD133 facilitates CSC-like properties by stabilizing EGFR-AKT signaling in HCC. It might therefore be feasible to use CD133 as a novel target to sensitize HCC cells that manifest resistance to EGFR-targeted therapy.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies and is the third leading cause of cancer-related death in the world [1]. Although treatments for HCC have been developed, the prognosis remains poor because of a >70% tumor recurrence rate after surgical resection, arterial embolization, chemotherapy, and radiotherapy [2], [3]. Previous studies found that liver cancer is derived from liver stem cells, which regenerate in normal liver tissue. While the cellular origin of HCC recurrence is still unclear, it is the best strategy to target HCC recurrence. Recently, cancer stem cells (CSCs), which are defined by tumor-initiation specificity, have been discovered in many types of cancer. There is a growing body of evidence that CSCs are responsible for tumor growth, resistance to therapy, metastasis, and recurrence [4], [5], [6]. Therefore, recent studies of CSCs have increasingly focused on the identification of CSC-specific markers [7], [8], [9], [10].

CD133 is a 5-transmembrane glycoprotein expressed by a subpopulation of the hematopoietic stem cells originating from fetal liver and bone marrow [11], [12]. It is also commonly known in humans and rodents as Prominin 1 (PROM1) [12]. CD133 is considered a surface marker of CSCs in cancers of the brain, colon, pancreas, prostate, and liver. Previous work showed that CD133 is a highly expressed marker of liver cancer stem cells (LCSCs) [13], [14]. A high level of CD133 expression was an independent prognostic indicator for survival and tumor recurrence in patients with HCC [15]. Interestingly, CD133⁺ HCC cells exhibited chemoresistance to fluorouracil and doxorubicin through activation of the AKT/PKB and Bcl-2 pathway [16]. Despite extensive research efforts, the specific signaling pathway and mechanism by which CD133⁺ cells are able to evade conventional therapies in HCC and other cancers remains largely unknown.

The epidermal growth factor receptor (EGFR) is one of the receptor tyrosine kinases (RTKs), which play roles in the control of cell proliferation, migration, and differentiation in various types of cancer [17]. Because of the specification of EGFR, the dysregulation of EGFR is involved in many cancer types. EGFR overexpression, which occurs in 40–70% of human HCC, is related to tumorigenesis [18]. Therefore, many studies attempt to target EGFR, and EGFR antagonists have been developed to treat patients with HCC. EGFR antagonists have shown effects on human and rat HCC cells; however, the EGFR inhibitors gefitinib, cetuximab, and erlotinib have not shown any meaningful effects in patients with advanced HCC [19], [20].

Although some mechanisms of EGFR function in cancer are well known, therapies targeting EGFR are still limited. Moreover, the treatment strategies targeting CSCs are also insufficient, although much recent research has focused on CSCs.

We found that CD133⁺ HCC cell lines and primary CD133⁺ HCC cells derived from patients with liver cancer have CSC-like capability. Furthermore, the levels of CD133 expression and EGFR expression are directly proportional in several HCC cell lines and tissues of patients with liver cancer. We demonstrated that CD133 confers CSC-like properties by stabilizing EGFR expression in HCC. Based on those results, we hypothesize that the inhibition of CD133 expression can magnify the therapeutic efficacy of conventional anticancer drugs against EGFR-overexpressing HCC cells.