732.5 - Tumor-Suppressor Role of the Caveolar α1-Na/K-ATPase Signalosome in NASH related Hepatocellular Carcinoma

Utibe-Abasi Udoh (Marshall University Joan C Edwards School of Medicine, Huntington, WV, USA), Moumita Banerjee (Marshall University Joan C Edwards School of Medicine, Huntington, WV, USA), Pradeep Rajan (Marshall University Joan C Edwards School of Medicine), Juan Sanabria (Marshall University Joan C Edwards School of Medicine), Gary Smith (Marshall University Joan C Edwards School of Medicine), Yuto Nakafuku (Marshall University Joan C Edwards School of Medicine), Komal Sodhi (Marshall University Joan C Edwards School of Medicine), Sandrine Pierre (Marshall University Joan C Edwards School of Medicine), Zijian Xie (Marshall University Joan C Edwards School of Medicine), Joseph Shapiro (Marshall University Joan C Edwards School of Medicine), Juan Sanabria (Marshall University Joan C Edwards School of Medicine)

Background 

Hepatocellular Carcinoma (HCC) is the second and fastest-growing cause of cancer- related mortality worldwide. In the Western countries, due to the epidemic of obesity, non-alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms that underlie the progression of NASH to HCC is still unclear.
Hypothesis 
We hypothesized that, the growing uncoupled metabolism during NASH progression to ESLD and HCC manifested by lower cell oxi-redox status and an apoptotic ‘switch’ activity, follows a dysregulation in the wild type Caveolar α1-Na/K-ATPase (NKA)/Src signalosome, promoting an amplification of the pSrc → PI3K→ Akt pathway, which mediates survivin overexpression driving local carcinogenesis.
Methods
Expression of Cav-1/Smac-Diablo/Survivin proteins was performed by confocal-microscopy on immuno-stained HCC cell lines (Hep3B and SNU475), and livers in both NASH and NASH-HCC rodent models, and in humans. Apoptotic activity was assayed via TUNEL. Tumor burden in rodents’ livers were evaluated using ImageJ software on Hamp;E-stained liver tissues and fibrosis via trichrome staining. Furthermore, signaling pathway studies were explored in-vitro guided by RNA sequencing. Selective blockage of Src-p at its kinase domain was performed by administration of a synthetic peptide (pNaKtide). Significant differences among groups were established at plt;0.05 using ANOVA/Turkey’s post hoc test. 
Results
Blockage of Src-p at the α1-NKA promoted apoptosis of Human HCC cell lines and tumors of disease murine models in a dose dependent manner (plt; 0.01). pNaKtide at IC50 drove the downregulation and upregulation of Survivin and SMAC expressions (plt;0.05), respectively in HCC cell lines. In-vivo, liver tumor burden and fibrosis were significantly lower in animals treated with pNaKtide vs non-treated animals (plt;0.01). Furthermore, Cav-1 and Survivin expressions were significantly higher, while SMAC protein expression was significantly lower in livers from rodents with NASH/HCC vs animals treated with pNaKtide (plt;0.01). Similar pattern of  proteins expressions was noted in tumors from patients with NASH±HCC vs liver tissue from healthy subjects or subjects with liver metastases (plt;0.05). In-vitro, Src-p at the α1-NKA activates PI3K/Akt pathway with concomitant inhibition of the FoxO cascade, favoring cell division and primary liver carcinogenesis.
Conclusions
Caveolar Src-phosphorylation at the α1-Na/K-ATPase regulated Survivin/SMAC expressions, leading to cellular “switch” from apoptosis to cell division via the activation of the PI3K/Akt  pro-survival pathway with concomitant inhibition of the FoxO cascade. Therefore, point target blockage may be explored as a treatment for tumor regression in HCC.

This work was supported by grants from the Marshall University Joan C. Edwards School of Medicine Comprehensive Cancer Center Grants to Dr Juan Ramon Sanabria.