Epigenetic Mechanisms in Gastric Cancer: Potential New Therapeutic Opportunities

: Gastric cancer (GC) is one of the deadliest malignancies worldwide. Complex disease heterogeneity, late diagnosis, and suboptimal therapies result in the poor prognosis of patients. Besides genetic alterations and environmental factors, it has been demonstrated that alterations of the epigenetic machinery guide cancer onset and progression, representing a hallmark of gastric malignancies. Moreover, epigenetic mechanisms undergo an intricate crosstalk, and distinct epigenomic profiles can be shaped under different microenvironmental contexts. In this scenario, targeting epigenetic mechanisms could be an interesting therapeutic strategy to overcome gastric cancer heterogeneity, and the efforts conducted to date are delivering promising results. In this review, we summarize the key epigenetic events involved in gastric cancer development. We conclude with a discussion of new promising epigenetic strategies for gastric cancer treatment

Caspase-2 in liver disease and hepatocellular carcinoma

Caspases are key factors in the regulation of the apoptotic and/or inflammatory responses, both crucial in the pathogenesis of diverse diseases. Caspase-2 is the most evolutionary conserved albeit functionally poorly defined member of the caspase family. The precise role of caspase-2 as an initiator or effector caspase is still unknown, but it has been involved in a wide variety of functions, from apoptosis to genomic stability, oxidative stress, metabolism, and cancer. However, many conflicting results render the exact function of this protease still unresolved. Although caspase-2 has several hundred substrates, the activation, processing, and activity on specific substrates remain poorly described. Recent evidence indicates that caspase-2 has a role in metabolic homeostasis and is required for lipotoxicity-induced apoptosis in hepatocytes, contributing to non-alcoholic steatohepatitis (NASH) progression towards hepatocellular carcinoma (HCC). Caspase-2 protein expression strongly localizes to injured/ballooned hepatocytes, correlating with NASH severity. Also, mice lacking caspase-2 showed protection from western diet-induced obesity, dyslipidemia, and insulin resistance. Although there are no effective therapies for NASH and HCC, the evaluation of a pan-caspase inhibitor has reached a phase I/II in clinical trials for advanced liver disease. Nevertheless, a better understanding of caspase functions with the identification of specific proteolytic substrates is essential for future therapeutic developments. Bearing in mind the pressing need to identify new targets for NASH-HCC and its metabolic-related comorbidities, and the favorable effect of caspase-2 genetic inhibition in animal models, pharmacological caspase-2 inhibition arises as a promising strategy that should be further investigated

Epigenetic mechanisms in gastric cancer: potential new therapeutic opportunities

Gastric cancer (GC) is one of the deadliest malignancies worldwide. Complex disease heterogeneity, late diagnosis, and suboptimal therapies result in the poor prognosis of patients. Besides genetic alterations and environmental factors, it has been demonstrated that alterations of the epigenetic machinery guide cancer onset and progression, representing a hallmark of gastric malignancies. Moreover, epigenetic mechanisms undergo an intricate crosstalk, and distinct epigenomic profiles can be shaped under different microenvironmental contexts. In this scenario, targeting epigenetic mechanisms could be an interesting therapeutic strategy to overcome gastric cancer heterogeneity, and the efforts conducted to date are delivering promising results. In this review, we summarize the key epigenetic events involved in gastric cancer development. We conclude with a discussion of new promising epigenetic strategies for gastric cancer treatment

Novel AKT1-GLI3-VMP1 pathway mediates KRAS oncogene-induced autophagy in cancer cells

Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents. It has been suggested that autophagy plays a role in tumor promotion and progression downstream oncogenic pathways; however, the molecular mechanisms underlying this phenomenon have not been elucidated. Here, we provide both in vitro and in vivo evidence of a novel signaling pathway whereby the oncogene KRAS induces the expression of VMP1, a molecule needed for the formation of the authophagosome and capable of inducing autophagy, even under nutrient-replete conditions. RNAi experiments demonstrated that KRAS requires VMP1 to induce autophagy. Analysis of the mechanisms identified GLI3, a transcription factor regulated by the Hedgehog pathway, as an effector of KRAS signaling. GLI3 regulates autophagy as well as the expression and promoter activity of VMP1 in a Hedgehog-independent manner. Chromatin immunoprecipitation assays demonstrated that GLI3 binds to the VMP1 promoter and complexes with the histone acetyltransferase p300 to regulate promoter activity. Knockdown of p300 impaired KRAS- and GLI3-induced activation of this promoter. Finally, we identified the PI3K-AKT1 pathway as the signaling pathway mediating the expression and promoter activity ofVMP1upstream of the GLI3-p300 complex. Together, these data provide evidence of a new regulatory mechanism involved in autophagy that integrates this cellular process into the molecular network of events regulating oncogene-induced autophagy.Fil: Lo Ré, Andrea Emilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Fernández Barrena, Maite G.. No especifíca;Fil: Almada, Luciana L.. No especifíca;Fil: Mills, Lisa D.. No especifíca;Fil: Elsawa, Sherine F.. No especifíca;Fil: Lund, George. No especifíca;Fil: Ropolo, Alejandro Javier. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Molejon, Maria Ines. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vaccaro, Maria Ines. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Zapico, Martin Ernesto. No especifíca

Metabolic-associated fatty liver disease: from simple steatosis towards liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, endorsed by the Spanish Association for the Study of the Liver (AEEH)

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).S

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7) mice undergoing chronic (CCl) and acute (acetaminophen) injury. SLU7 expression was restored in CCl-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7 mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.Supported by MINECO/AEI/FEDER (UE SAF2016-75972-R, PID2019-104265RB-I00/AEI/10.13039/501100011033, and PID2019-104878RB-100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES-2017-079883, to M.R.); a Ramón y Cajal Program contract (RYC2018-024475-1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila

Splicing factor SLU7 prevents oxidative stress-mediated hepatocyte nuclear factor 4α degradation, preserving hepatic differentiation and protecting from liver damage

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7+/−) mice undergoing chronic (CCl4) and acute (acetaminophen) injury. SLU7 expression was restored in CCl4-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7+/− mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.Supported by MINECO/AEI/FEDER (UE SAF2016‐75972‐R, PID2019‐104265RB‐I00/AEI/10.13039/501100011033, and PID2019‐104878RB‐100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES‐2017‐079883, to M.R.); a Ramón y Cajal Program contract (RYC2018‐024475‐1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila

Oral Methylthioadenosine Administration Attenuates Fibrosis and Chronic Liver Disease Progression in Mdr2−/− Mice

BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

[EN] Background & Aims: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. Methods: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Followup RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. Results: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. Conclusions: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. Lay summary: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.A.V. was supported by ADA of the University of Navarra, Spain, O.E. by FSE; MINECO; FJCI-2017-34233, Spain, R.E. by a donation from Mauge Burgos de la Iglesia’s family, Spain, and P. Olaizola by the Basque Government (PRE_2016_1_0269), Basque Country, Spain. M.J.P. was funded by ISCIII [FIS PI14; 00399, PI17; 00022] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC-2015-17755), Spain. M.A.A was funded by La Caixa Foundation, HEPACARE project, Spain, ISCIII FIS PI16/01126 cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain, and “Fundación Científica de la Asociación Española Contra el Cáncer’’ (AECC Scientific Foundation) Rare Cancers 2017, Spain. J.M.B. was funded by the Spanish Carlos III Health Institute (ISCIII) (FIS PI15; 01132, PI18; 01075 and Miguel Servet Program CON14; 00129 and CPII19; 00008), Spain, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; “Euskadi RIS3” (2019222054) and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15; CA; 016; BD), Basque Country, Spain; “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation) Rare Cancers 2017, Spain. S.V. was supported by FEDER; MINECO (SAF2017-89944-R), Spain, by the Government of Navarra-Health Research Department (58; 2018), Navarra, Spain, by La Caixa and Caja Navarra Foundation-CIMA agreement, Spain. None of the funding sources were involved in the decision to submit the article for publication. This article is based upon work from COST Action CA18122 European Cholangiocarcinoma Network, supported by COST (European Cooperation in Science and Technology). COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks (www.cost.eu)

Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures

Objective: despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). Design: a prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. Results: an initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. Conclusion: implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.Funding: we thank the financial support of CIBERehd; grants PI16/01126 and PI19/00163 from Instituto de Salud Carlos III (ISCIII) cofinanced by ’Fondo Europeo de Desarrollo Regional’ (FEDER) ’Una manera de hacer Europa’; grants 58/2017 and 55/2018 from Gobierno de Navarra Salud; grant 0011-1411-2020-000010 from AGATA Strategic Project from Gobierno de Navarra; grant 2020/101 from Euroregion Nouvelle Aquitaine-Euskadi-Navarra; Fundación Eugenio Rodríguez Pascual; Fundación Mario Losantos, Fundación M Torres; grant 2018/117 from AMMF, the Cholangiocarcinoma Charity; the COST Action CA181122 Euro-cholangio-Net; POSTD18014AREC postdoctoral fellowship from AECC to MA; and Ramón y Cajal Program contracts RYC-2014-15242 and RYC-2018-024475-1 to FJC and MGFB