Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA-mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib-induced ferroptosis. Mechanistically, in a TEAD-dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib-induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ-based rewiring strategies as potential approaches to overcome HCC therapy resistance.

Fibroblast activation protein α (FAP) plays an important role in tissue remodeling and helps tumor cells invade surrounding tissue. We sought to investigate FAP as a prognostic molecular marker in colorectal cancer (CRC) using immunohistochemical and transcriptomic data. FAP expression and clinicopathological information were obtained from The Cancer Genome Atlas data set. The association of FAP expression and tissue cellular heterogeneity landscape was explored using the xCell method. We evaluated FAP protein expression in a cohort of 92 CRCs and 19 non-tumoral tissues. We observed that FAP was upregulated in tumors both at the mRNA and protein levels, and its expression was associated with advanced stages, poor survival, and consensus molecular subtype 4. FAP expression was also associated with angiogenesis and collagen degradation. We observed an enrichment in immune-cell process-related genes associated with FAP overexpression. Colorectal cancers with high FAP expression display an inflamed phenotype enriched for macrophages and monocytes. Those tumors showed enrichment for regulatory T cell populations and depletion of T H 1 and natural killer T cells, pointing to an immunosuppressive environment. Colorectal cancers with high levels of stromal FAP are associated with aggressive disease progression and survival. Our results suggest that FAP plays additional roles in tumor progression such as modulation of angiogenesis and immunoregulation in the tumor microenvironment.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome.

We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β-catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs.

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/β-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.

Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro , ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.

Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine.