Cripto-1 contributes to stemness in hepatocellular carcinoma by stabilizing Dishevelled-3 and activating Wnt/β-catenin pathway

Abstract

Identification and characterization of functional molecular targets conferring stemness properties in hepatocellular carcinoma (HCC) offers crucial insights to overcome the major hurdles of tumor recurrence, metastasis and chemoresistance in clinical management. In the current study, we investigated the significance of Cripto-1 in contributing to HCC stemness. Cripto-1 was upregulated in the sorafenib-resistant clones derived from HCC cell lines and patient-derived xenograft that we previously developed, suggesting an association between Cripto-1 and stemness. By in vitro experiments, Cripto-1 fostered cell proliferation, migration, and invasion. It also enhanced self-renewal ability and conferred chemoresistance of HCC cells. Consistently, silencing of Cripto-1 suppressed in vivo tumorigenicity on serial transplantation. On the downstream signaling mechanism, expression of major components of Wnt/β-catenin pathway β-catenin, AXIN2, and C-MYC, accompanied by β-catenin activity was reduced upon Cripto-1 knockdown. The suppressive effects on stemness properties with Cripto-1 knockdown in vitro and in vivo were partially rescued by forced expression of constitutively active β-catenin. Further elucidation revealed the binding of Cripto-1 to Frizzled-7 (FZD7), low-density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled-3 (DVL3) of the Wnt/β-catenin pathway and stabilized DVL3 protein. Analyses with clinical samples validated Cripto-1 overexpression in HCC tissues, as well as a positive correlation between Cripto-1 and AXIN2 expressions. High Cripto-1 level in tumor was associated with poorer disease-free survival of HCC patients. Taken together, Cripto-1 binds to FZD7/LRP6 and DVL3, stabilizes DVL3 expression and activates the Wnt/β-catenin signaling cascade to confer stemness in HCC. Our study findings substantiated the role of Cripto-1 in determining stemness phenotypes of HCC and mechanistically in modulating the Wnt/β-catenin signaling cascade, one of the most frequently deregulated pathways in liver cancer.

Deregulated GATA6 modulates stem cell‐like properties and metabolic phenotype in hepatocellular carcinoma

“…Global gene expression profiles by whole‐transcriptome sequencing (WTS) and clinicopathological parameters of the cancer genome atlas (TCGA) dataset were obtained from TCGA data portal and http://firebrowse.org (Broad Institute). Data on 50 paired HCC and nontumoral liver samples were retrieved for analysis and subsequent differential gene expression analysis was performed as we previously described . Differential gene expression analysis was carried out by means of edgeR on the raw read counts.…”

“…The assay was performed as we previously described. 20,21 Five hundred HCC cells, unless otherwise specified, were suspended in DMEM/F12 GlutaMAX™ medium (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen, Carlsbad, CA), 4 μg/ml insulin (Sigma-Aldrich), 20 ng/ml of epidermal growth factor (Sigma-Aldrich), 20 ng/ml of basic fibroblast growth factor (Sigma-Aldrich, St. Louis, MO) were seeded onto a 24-well plate, coated with 1% polyHEMA (Sigma-Aldrich, St. Louis, MO) for 8-10 days. Subsequently, the tumorspheres were photographed under the light microscope and the total number of spheres formed was counted.…”

“…Data on 50 paired HCC and nontumoral liver samples were retrieved for analysis and subsequent differential gene expression analysis was performed as we previously described. 21 Differential gene expression analysis was carried out by means of edgeR on the raw read counts. Negative binomial models were used to capture variance dispersion for WTS read count data, empirical Bayes estimation was adopted for gene-specific variation, and generalized linear models were applicable to general experiments.…”

A Multidisciplinary Review of the Roles of Cripto in the Scientific Literature Through a Bibliometric Analysis of its Biological Roles

“…In hepatocellular carcinoma (HCC), it was shown that Cripto promotes stemness by stabilizing Dishevelled-3 and activating the Wnt/β-catenin signaling cascade. The knock down of Cripto also reduced the activity of components of the Wnt/β-catenin pathway, such as β-catenin, AXIN2, and C-MYC [90]. The invasion potential of HCC cells has been shown to be regulated by NANOG, which increased the expression of Nodal and Cripto to promote SMAD3 phosphorylation and SNAIL expression and EMT progression [91].…”

Glycochenodeoxycholic acid induces stemness and chemoresistance via the STAT3 signaling pathway in hepatocellular carcinoma cells

“…Enhanced expression of several CSC-related markers, such as epithelial cell adhesion molecule (EpCAM), CD44, CD133, and Nanog, is associated with poor prognosis of HCC [ 4 – 7 ]. Similarly, alterations in stemness-related signaling pathways, such as Wnt/β-catenin, Notch, and Hedgehog pathways, resulted in chemoresistance and recurrence of HCC [ 8 – 10 ]. Therefore, CSCs and their associated pathways are becoming the focus of potential therapies for HCC.…”