Presentation 1:

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD), including its inflammatory subtype, steatohepatitis (MASH), affects over 30% of the world population as the highest contributor to the global burden of chronic liver diseases. MASH can lead to cirrhosis, liver failure and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer and the fastest growing cause of cancer-related death worldwide. Nevertheless, there is a gap of knowledge on both its molecular pathogenesis and effective therapy, especially for advanced disease, resulting in its dismal prognosis. Our group described the role of the α1-Na/K-ATPase (ATP1A1) as a tumor suppressor in MASH-related HCC. ATP1A1 dysregulated signaling that follows oxidative stress leads to the activation of the PI3K-Akt pro-survival pathway, conducive of upregulation of the anti-apoptotic protein survivin, a potent driver for cell division and tumorigenesis. Hypothesis: hepatocyte specific deletion of the Atp1a1 gene will induce the development of HCC emulating MASH associated protein and metabolic disturbances. 

Methods: Hepatocyte- specific ATP1A1 conditional knock out (KNO) mice were generated by Cre/LoxP methods. A two-step crossing was performed between a transgenic mouse expressing Cre recombinase under the control of the mouse albumin promoter (Albumin-Cre, Stock No: 003574, Jackson Laboratory, Bar Harbor, CA), and a mouse in which Atp1a1 alleles have been flanked (floxed)at the LoxP sites. The floxed mice were designed to have exon 15-18 excised when crossed with the Cre-expressing mice. Genotyping was confirmed by PCR and ATP1A1 expression was measured by immunofluorescence staining, confocal microscopy, and western blotting. Additionally, RNA sequencing was performed for signaling pathways. Survivin and proteins expression were elucidated by ELISA and standard immunoblotting. Liver morphology was evaluated on H&E slides.

Results: Mice present hepatocyte-specific reduction of more than 90% of ATP1A1 (Fig. 1). Additionally, the level of survivin in the liver tissues of the APT1A1 KNO mice was significantly higher when compared to wild type counterparts (p<0.01). The RNA sequencing data showed decreased expression of ATP1A1 gene, increased expression of BIRC5 gene (that encodes Survivin), Rps6kl1 (that encodes Ribosomal protein S6), and genes that play key roles in the PI3K-Akt pathway. Our histology results revealed the presence of malignancy in the livers of the ATP1A1 KNO mice at 7-9 weeks of age (Fig.1), with the presence of Mallory- Denk bodies (emulating MASH-related HCC).

Conclusion: Hepatocyte specific deletion of the Atp1a1 gene increases ATP1A1 signalling with the development of malignancy that emulates MASH-related HCC.