Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to poorly understood molecular basis. In the current study, we investigate two independent cohorts of 249 and 194 HCC cases for any combinatorial molecular aberrations. Specifically we assessed for simultaneous HMET expression or hMet activation and CTNNB1 mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we co-expressed hMet and β-catenin point-mutants (S33Y or S45Y) in hepatocytes using sleeping beauty (SB) transposon/transposase and hydrodynamic tail vein injection and characterized tumors for growth, signaling, gene signatures and similarity to human HCC. Missense mutations in exon-3 of CTNNB1 were identified in subsets of HCC patients. Irrespective of amino acid affected, all exon-3 mutations induced similar changes in gene expression. Concomitant HMET overexpression or hMet activation, and CTNNB1 mutations, were evident in 9-12.5% of HCCs. Co-expression of hMet and mutant-β-catenin led to notable HCC in mice. Tumors showed active Wnt and hMet signaling with evidence of glutamine synthetase and cyclin-D1 positivity and MAPK/ERK, AKT/Ras/mTOR activation. Introduction of dominant-negative TCF4 prevented tumorigenesis. The gene expression of mouse tumors in hMet-mutant-β-catenin showed high correlation with subsets of human HCC displaying concomitant hMet activation signature and CTNNB1 mutations. In conclusion, we have identified co-operation of hMet and β-catenin activation in a subset of HCC patients and modeled this human disease in mice with a significant transcriptomic intersection. This model will provide novel insight into the biology of this tumor and allow us to evaluate novel therapies as a step towards precision medicine.

S tringent response is the main strategy used by bacteria to cope with fluctuating nutrient supplies and metabolic and oxidative stresses 1,2 . This process rapidly redirects energy from cell proliferation toward stress survival by reduction of biosynthesis, conservation of ATP and blockage of GTP production 3 . The stringent response is triggered by the accumulation of the bacterial 'alarmone' (p)ppGpp (guanosine tetra-or penta-phosphate, shortened as ppGpp below) through the regulation of ppGpp synthetases and hydrolases in the RelA and SpoT homologue family 2 .Recent studies suggest that the stringent response may also function in metazoans, as metazoan genomes encode a homologue of bacterial SpoT-MESH1 (Metazoan SpoT Homologue 1, encoded by HDDC3)-that can hydrolyse ppGpp in vitro and functionally complement SpoT in Escherichia coli 4 . Furthermore, Mesh1 deletion in Drosophila displays impaired starvation resistance and extensive transcriptional reprogramming 4 . Despite these supporting lines of evidence, neither ppGpp nor its synthetase has been discovered in metazoans, thus obscuring the genuine function and the relevant substrate(s) of MESH1 in mammalian cells. Here, we have identified NADPH as an efficient substrate of MESH1. MESH1 is a cytosolic NADPH phosphatase that is induced under stress conditions, leading to the NADPH depletion and ferroptosis-a novel form of iron-dependent regulated cell death characterized by lipid peroxidation 5 . Accordingly, MESH1 removal preserves the NADPH level in stressed cells and promotes their ferroptotic survival.Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosisinducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

Recently we have shown that co-expression of hMet and mutant-β-catenin using sleeping beauty transposon/transposase leads to HCC in mice that represents around 10% of human HCC. In the current study, we investigate if Ras activation, which can occur downstream of Met signaling, is sufficient to cause HCC in association with mutant-β-catenin. We also tested therapeutic efficacy of targeting β-catenin in HCC model. We show that mutant-K-Ras (G12D), which leads to Ras activation, cooperates with β-catenin mutants (S33Y, S45Y) to yield HCC in mice. Affymetrix microarray shows >90% similarity in gene expression in mutant-K-Ras-β-catenin and Met-β-catenin HCC. K-Ras-β-catenin tumors showed upregulation of β-catenin targets like Glutamine Synthetase (GS), Lect2, Regucalcin and Cyclin-D1 and of K-Ras effectors including p-ERK, p-AKT, p-mTOR, p-EIF4E, p-4E-BP1 and p-S6 Ribosomal protein. Inclusion of dominant-negative TCF4 at the time of K-Ras-β-catenin injection prevented HCC and downstream β-catenin and Ras signaling. To address if targeting β-catenin has any benefit post-establishment of HCC, we administered K-Ras-β-catenin mice with EnCore lipid nanoparticle (LNP) loaded with a Dicer substrate siRNA targeting CTNNB1 (CTNNB1-LNP), scrambled sequence (Scr-LNP) or PBS for multiple cycles. A significant decrease in tumor burden was evident in CTNNB1-LNP group versus all controls, which was associated with dramatic decreases in β-catenin targets and some K-Ras effectors, leading to reduced tumor cell proliferation and viability. Intriguingly, in few mice, non-GS-positive tumors, which were evident as a small subset of overall tumor burden, were not affected by β-catenin suppression. In conclusion, we show that Ras activation downstream of c-Met is sufficient to induce clinically relevant HCC in cooperation with mutant β-catenin. β-Catenin suppression by a clinically relevant modality is effective in treatment of β-catenin-positive, GS-positive HCCs.

Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piRNAs, and act as RNA solvents; but, the limited availability of mutagenesis-derived alleles and their wide...