Axis inhibition protein 1 (Axin1) Deletion–Induced Hepatocarcinogenesis Requires Intact β‐Catenin but Not Notch Cascade in Mice

Abstract

Inactivating mutations of axis inhibition protein 1 (AXIN1), a negative regulator of the Wnt/β‐Catenin cascade, are among the common genetic events in human hepatocellular carcinoma (HCC), affecting approximately 10% of cases. In the present manuscript, we sought to define the genetic crosstalk between Axin1 mutants and Wnt/β‐catenin as well as Notch signaling cascades along hepatocarcinogenesis. We discovered that c‐MET activation and AXIN1 mutations occur concomitantly in ~3%‐5% of human HCC samples. Subsequently, we generated a murine HCC model by means of CRISPR/Cas9‐based gene deletion of Axin1 (sgAxin1) in combination with transposon‐based expression of c‐Met in the mouse liver (c‐Met/sgAxin1). Global gene expression analysis of mouse normal liver, HCCs induced by c‐Met/sgAxin1, and HCCs induced by c‐Met/∆N90‐β‐Catenin revealed activation of the Wnt/β‐Catenin and Notch signaling in c‐Met/sgAxin1 HCCs. However, only a few of the canonical Wnt/β‐Catenin target genes were induced in c‐Met/sgAxin1 HCC when compared with corresponding lesions from c‐Met/∆N90‐β‐Catenin mice. To study whether endogenous β‐Catenin is required for c‐Met/sgAxin1‐driven HCC development, we expressed c‐Met/sgAxin1 in liver‐specific Ctnnb1 null mice, which completely prevented HCC development. Consistently, in AXIN1 mutant or null human HCC cell lines, silencing of β‐Catenin strongly inhibited cell proliferation. In striking contrast, blocking the Notch cascade through expression of either the dominant negative form of the recombinant signal‐binding protein for immunoglobulin kappa J region (RBP‐J) or the ablation of Notch2 did not significantly affect c‐Met/sgAxin1‐driven hepatocarcinogenesis. Conclusion: We demonstrated here that loss of Axin1 cooperates with c‐Met to induce HCC in mice, in a β‐Catenin signaling–dependent but Notch cascade–independent way.

Letter to the Editor: Comment on Qiao et al.

“…We read with great interest the article entitled "Axin1 deletion induced hepatocarcinogenesis requires intact β-Catenin but not Notch cascade in mice" by Qiao et al published in the February issue of Hepatology. (1) The findings of this study corroborate our previous data that the in vivo deletion of Axin1 in hepatocytes using conditional knockout (KO) mice does not result in classical activation of the Wnt/β-catenin pathway, but leads to development of hepatocellular carcinomas (HCCs). (2) Thanks to a 23-genes signature including LGR5, we also demonstrated, using, in particular, the TGCA database, that 80% of AXIN1-mutated human tumors do not activate this Wnt/β-catenin program.…”

“…We thank Drs. Gilgenkrantz and Perret for their insightful comments on the article by Qiao et al (1) In agreement with their comments, we believe that all conclusions in our study are based on the experimental system used, including human tumor samples, cell lines, and mouse models. In our study, (1) we generated a mouse hepatocellular carcinoma (HCC) model by deleting single-guide axis inhibition protein 1 (sgAxin1) and simultaneously expressing c-Met (c-Met/sgAxin1).…”

“…Gilgenkrantz and Perret for their insightful comments on the article by Qiao et al (1) In agreement with their comments, we believe that all conclusions in our study are based on the experimental system used, including human tumor samples, cell lines, and mouse models. In our study, (1) we generated a mouse hepatocellular carcinoma (HCC) model by deleting single-guide axis inhibition protein 1 (sgAxin1) and simultaneously expressing c-Met (c-Met/sgAxin1). Using the "23 β-catenin gene signature" defined by Abitbol et al, (2) we found that c-Met/sgAxin1 HCCs did not show activation of this signature based on global gene expression analysis, although activation of the Wnt pathway was evident.…”

“…Given this very specific model and that β-catenin-and METmediated signaling pathways are known to cooperate in hepatocyte proliferation, (3) their conclusion can only apply to tumors mutated for Axin1 and induced for the c-Met pathway that represent a little bit less than half of AXIN1-mutated human HCC. (1) We agree that our data do not prove that HCCs induced by Axin1 deficiency develop independently of β-catenin because we did not use Ctnnb1 KO mice. However, the work of Qiao et al does not either "unequivocally prove that loss of Axin1-driven HCC requires an intact β-catenin."…”

Targeting Liver Cancer Stem Cells: An Alternative Therapeutic Approach for Liver Cancer

“…Usually, aberrantly activated Wnt/β-catenin signaling can result in the abnormal stabilization of positive modulators of Wnt/β-catenin such as β-catenin or loss-of-function mutations in negative modulators of the signaling such as APC and Axin [ 160 , 161 ]. Consistently, loss-of-function mutations of many positive or negative regulators of the signaling such as TP53, AXIN (axis inhibition protein), and CTNNB1/β-catenin were observed in hepatocellular tumors [ 162 , 163 ]. Other well-known direct or indirect target genes of this signaling are CD44, C-Jun, Cyclin D1, C-Myc, VEGF, and MMP-7 [ 164 , 165 ].…”