Abstract
Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active β-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3β at Ser9. This was associated with an induction of a 48-kDa active β-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa β-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa β-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active β-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active β-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical β-catenin-dependent mechanism.
Highlights
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality and accounts for approximately 745,000 deaths per year worldwide[1]
The active, hypophosphorylated β-catenin translocates into the nucleus where it acts as a co-factor for the TCF/ LEF family of transcription factors and activates genes involved in cell proliferation, survival, stemness, invasion, and cell cycle regulation. β-catenin forms a bridge between the cytoplasmic domain of E-cadherin and the cytoskeleton, and is a constituent protein of adherens junctions critical to the establishment and maintenance of epithelial polarity[27]
When primary human hepatocytes from normal livers are cultured in Matrigel, which contains several growth factors and extracellular matrix, some cells form spheroids containing numerous Doublecortin-like kinase 1 (DCLK1) + cells[16]
Summary
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality and accounts for approximately 745,000 deaths per year worldwide[1]. We previously reported that chronic HCV infection, one of the most potent risk factors for HCC development, induces dedifferentiated phenotypes in liver cells with enhanced expression of cellular stemness-related proteins, such as DCLK1, CD133, AFP, CK19, Lgr[5], Lin[28], and c-Myc[11]. Among these tumor-related proteins, DCLK111–13 is extensively expressed in the livers of patients with cirrhosis and H CC11,14–16. DCLK1-led activation of the atypical β-catenin signaling was validated in a humanized liver mouse model and liver tissues of patients with cirrhosis and HCC
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