AKT2 Loss Attenuated the SOX9-Regulated Mixed Hepatocarcinoma-Cholangiocarcinoma Development In Mice Lacking PTEN in the Liver

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA

Background: Primary liver carcinoma, which has been mainly classified into hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA), ranks as the 3^rd global leading cause of mortality among all cancer types. Although HCC has a higher incidence, tumors involve with CCA behave more aggressively and are more likely to relapse after surgical resection. As liver malignancies tend to exhibit a high degree of heterogeneity and consist of poorly differentiated cells, determining the cell-of-origin of liver tumor and how tumor-initiating cells (TICs) promote tumorigenesis may help develop treatments for liver cancer. PTEN is a well-known tumor suppressor gene that negatively regulates the PI3K/AKT pathway. Our previous work showed that mice specifically lacking PTEN in albumin-containing liver cells (PM, PTEN^loxP/loxP; Alb-Cre⁺) imitated the nature of liver tumor progression. Liver tumors developed in mice lacking PTEN lead to heterogeneous liver tumor development from TICs.

Methods: To investigate the mechanism of PTEN-regulated liver TICs, we generated a double mutant mouse model (DM, PTEN^loxP/loxP; AKT2^loxP/loxP; Alb-Cre⁺) by deleting AKT2, the most abundant AKT isoform in the albumin-expressing liver hepatocytes. H&E and tumor staining were examined to screen the phenotype throughout the PM, DM, and wild-type (WT) mice liver sections. We further explored the role of AKT2 and SOX9 in TICs using immunostaining and immunoblotting in both mouse and human samples.

Results: HCC, CCA, and mixed-lineage tumors were observed after lipid-induced liver injury in the PM mice. In these mice, we observed a significantly upregulated expression of SOX9, a transcriptional factor that plays a vital role in the embryonic development and regeneration of liver parenchyma following chronic injury. Clinical data demonstrated the expression of SOX9 is upregulated in liver cancer, particularly CCA patients, and is negatively correlated with the overall poorer survival rate. Here, we demonstrated that the downregulation of SOX9 attenuated the ability of Huh7 cells to form spheres in suspension culture, suggesting that SOX9 plays an important role in promoting the stemness and self- renewal of TICs. PTEN loss unequivocally leads to the activation of AKT. We showed here that phosphorylation of AKT at both serine and threonine sites is induced in the PM livers and liver cells isolated from the PM livers. T address the role of AKT2 in this PTEN-regulated TIC induction, we first compared the tumor spectrum in the DM mice vs. the PM mice. Our data suggested that only PM mice formed tumors starting at 6 months of age along with a moderate accumulation of reactive ductal cells and the formation of Von Meyenbury complex (VMC), a benign liver condition that can develop into a malignancy. DM mice also presented with VMC conditions starting from 9 months of age with minimal steatosis. There were no tumors detected in these mice through 15 months of age. A significant abatement of phosphorylated-AKT and SOX9 expression was observed in the DM livers compared with the PM livers.

Conclusions: Our data indicated that deletion of AKT2 not only arrests activated AKT signaling and SOX9 overexpression, but also suppresses the progression of PTEN-driven tumors at the pre-malignancy level.