Revistas
Revista:
JOURNAL OF HEPATOLOGY
ISSN:
1600-0641
Año:
2023
Vol.:
79
N°:
4
Págs.:
989 - 1005
BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models.
METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n= 72) and tumoral (n= 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed.
RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of beta-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming.
CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients.
IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.
Autores:
Chen, C. B.; Wu, H. H.; Ye, H.; et al.
Revista:
CANCERS
ISSN:
2072-6694
Año:
2022
Vol.:
14
N°:
1
Págs.:
78
Polycystic liver disease (PLD) is a group of rare disorders that result from structural changes in the biliary tree development in the liver. In the present work, we studied alterations in molecular mechanisms and signaling pathways that might be responsible for these pathologies. We found that activation of the unfolded protein response, a process that occurs in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum, as well as the scarring of the liver tissue, contribute to the pathogenesis of PLD and the development of cancer. As a preclinical animal model we have used mutant mice of a specific signaling pathway, the c-Jun N-terminal kinase 1/2 (Jnk1/2). These mice resemble a perfect model for the study of PLD and early cancer development.
Revista:
CANCERS
ISSN:
2072-6694
Año:
2022
Vol.:
14
N°:
9
Págs.:
2048
Simple Summary Hepatocarcinogenesis is a long process which implies the loss of hepatic functions. Our effort is to understand the mechanisms implicated in this pathological process in order to contribute to the development of new diagnostic markers and therapeutic targets. In this study we have identified a set of lncRNAs significantly downregulated in hepatocellular carcinoma (HCC) in correlation with the grade of tumor dedifferentiation and patients' worse prognosis. Mechanistically, our results show that they are related with hepatic differentiation and at least a subset of those lncRNAs are essential to ensure the expression of other hepato-specific genes required for liver function. Moreover, we demonstrate that the expression of these lncRNAs in HCC is silenced by DNA methylation. All in all, we uncover connected epigenetic alterations involved in the progression of liver cancer and identify potential new biomarkers. Background: Long noncoding RNAs (lncRNAs) are emerging as key players in cancer, including hepatocellular carcinoma (HCC). Here we identify the mechanism implicated in the HCC inhibition of a set of lncRNAs, and their contribution to the process of hepatocarcinogenesis. Methods and Results: The top-ranked 35 lncRNAs downregulated in HCC (Top35 LNDH) were validated in several human HCC cohorts. We demonstrate that their inhibition is associated with promoter hypermethylation in HCC compared to control tissue, and in HCC human cell lines compared to primary hepatocytes. Moreover, demethylating treatment of HCC human cell lines induced the expression of these lncRNAs. The Top35 LNDH were preferentially expressed in the adult healthy liver compared to other tissues and fetal liver and were induced in well-differentiated HepaRG cells. Remarkably, their knockdown compromised the expression of other hepato-specific genes. Finally, the expression of the Top35 LNDH positively correlates with the grade of tumor differentiation and, more importantly, with a better patient prognosis. Conclusions: Our results demonstrate that the selected Top35 LNDH are not only part of the genes that compose the hepatic differentiated signature but participate in its establishment. Moreover, their downregulation through DNA methylation occurs during the process of hepatocarcinogenesis compromising hepatocellular differentiation and HCC patients' prognosis.
Revista:
Journal of hepatology
ISSN:
1600-0641
Año:
2022
Vol.:
77
N°:
6
Págs.:
1479 - 1481
Autores:
Schott, M.; Kappelmann-Fenzl, M.; Fischer, S.; et al.
Revista:
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE
ISSN:
1107-3756
Año:
2022
Vol.:
49
N°:
5
Págs.:
66
The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld-knockout (Cyld(-/-)) compared with Cyld-wild-type (Cyld(+/+)) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor-derived cell lines from Cyld(+/+) and Cyld(-/-) mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld(-/-) compared with Cyld(+/+) cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5 '-Aza-deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld(-/-) melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld-deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.
Revista:
JOURNAL OF EXPERIMENTAL AND CLINICAL CANCER RESEARCH
ISSN:
1756-9966
Año:
2022
Vol.:
41
N°:
1
Págs.:
183
Background Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk(Delta hepa) + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRAS(G12D) cells. Cell signaling, growth, gene regulation and [U-C-13]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRAS(G12D) can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRAS(G12D) promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRAS(G12D) CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.
Revista:
GUT
ISSN:
0017-5749
Año:
2022
Vol.:
71
N°:
6
Págs.:
1141 - 1151
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.
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2022
Vol.:
77
N°:
1
Págs.:
177 - 190
Background & Aims: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Methods: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1(+/-) mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. Results: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1(+/)(-) mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogenedriven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Conclusion: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA.
Autores:
Marín, J. J. G.; Prete, M. G.; Lamarca, A.; et al.
Revista:
BRITISH JOURNAL OF CANCER
ISSN:
0007-0920
Año:
2021
Vol.:
125
N°:
6
Págs.:
904 - 904
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2021
Vol.:
73
N°:
6
Págs.:
2380 - 2396
Background and Aims Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. Approach and Results Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (Jnk(Delta hepa) + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in Jnk(Delta hepa) + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. Conclusions Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.
Revista:
JOURNAL OF HEPATOLOGY
ISSN:
1600-0641
Año:
2021
Vol.:
75
N°:
2
Págs.:
363 - 376
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). Follow-up 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.
Autores:
Carotti, S.; Zingariello, M.; Francesconi, M.; et al.
Revista:
ONCOGENE
ISSN:
0950-9232
Año:
2021
Vol.:
40
N°:
23
Págs.:
4033 - 4049
Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells.
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2021
Vol.:
74
N°:
5
Págs.:
2791 - 2807
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 4 alpha (HNF4 alpha) 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 HNF4 alpha, 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 HNF4 alpha 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 HNF4 alpha 1 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.
Revista:
BIOCHEMICAL JOURNAL
ISSN:
0264-6021
Año:
2020
Vol.:
477
N°:
17
Págs.:
3131 - 3145
The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.
Revista:
CANCERS
ISSN:
2072-6694
Año:
2020
Vol.:
12
N°:
6
Págs.:
1644
Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n= 36) and malignant conditions, CCA (n= 36) or PDAC (n= 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (H-1-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n= 10) and proteins (n= 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy.
Autores:
Santos-Laso, A.; Izquierdo-Sanchez, L.; Rodrigues, P. M.; et al.
Revista:
LIVER INTERNATIONAL
ISSN:
1478-3223
Año:
2020
Vol.:
40
N°:
7
Págs.:
1670 - 1685
Background & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. Methods ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. Results The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. Conclusions Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.
Revista:
NUCLEIC ACIDS RESEARCH
ISSN:
0305-1048
Año:
2019
Vol.:
47
N°:
7
Págs.:
3450 - 3466
Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.
Autores:
Erice, O.; Munoz-Garrido, P.; Vaquero, J.; et al.
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2018
Vol.:
67
N°:
4
Págs.:
1420 - 1440
Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR-506 is overexpressed in PBC cholangiocytes and directly targets both Cl-/ HCO3-anion exchanger 2 and type III inositol 1,4,5-trisphosphate receptor, leading to cholestasis. Here, the regulation of miR-506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin-8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR-506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR-506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR-506 (1) induced dedifferentiation with down-regulation of biliary and epithelial markers together with up-regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase-3-dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR-506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion: Different proinflammatory cytokines enhance the expression of miR-506 in biliary epithelial cells; miR-506 induces PBC-like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420-1440)
Revista:
NATURE COMMUNICATIONS
ISSN:
2041-1723
Año:
2017
Vol.:
8
Págs.:
15424
The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
Revista:
DIGESTIVE DISEASES
ISSN:
0257-2753
Año:
2017
Vol.:
35
N°:
3
Págs.:
158 - 165
Background: Advanced hepatocellular carcinoma (HCC) is a neoplastic disease with a very bad prognosis and increasing worldwide incidence. HCCs are resistant to conventional chemotherapy and the multikinase inhibitor sorafenib is the only agent that has shown some clinical efficacy. It is therefore important to identify key molecular mechanisms driving hepatocarcinogenesis for the development of more efficacious therapies. However, HCCs are heterogeneous tumors and different molecular subclasses have been characterized. This heterogeneity may underlie the poor performance of most of the targeted therapies so far tested in HCC patients. The fibroblast growth factor 15/19 (FGF15/19), FGF receptor 4 (FGFR4) and beta-Klotho (KLB) correceptor signaling system, a key regulator of bile acids (BA) synthesis and intermediary metabolism, is emerging as an important player in hepatocarcinogenesis. Key Messages: Aberrant signaling through the FGF15/19-FGFR4 pathway participates in the neoplastic behavior of HCC cells, promotes HCC development in mice and its overexpression has been characterized in a subset of HCC tumors from patients with poorer prognosis. Pharmacological interference with FGF15/19-FGFR4 signaling inhibits experimental hepatocarcinogenesis, and specific FGFR4 inhibitors are currently being tested in selected HCC patients with tumoral FGF19-FGFR4/KLB expression. Conclusions: Interference with FGF19-FGFR4 signaling represents a novel strategy in HCC therapy. Selection of candidate patients based on tumoral FGF19-FGFR4/KLB levels as biomarkers may result in increased efficacy of FGFR4-targeted drugs. Nevertheless, attention should be paid to the potential on target toxic effects of FGFR4 inhibitors due to the key role of this signaling system in BA metabolism. (C) 2017 S. Karger AG, Basel
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2016
Vol.:
64
N°:
2
Págs.:
336 - 339
Revista:
ONCOGENE
ISSN:
0950-9232
Año:
2016
Vol.:
35
N°:
36
Págs.:
4719 - 4729
Resisting death is a central hallmark of cancer cells. Tumors rely on a number of genetic mechanisms to avoid apoptosis, and alterations in mRNA alternative splicing are increasingly recognized to have a role in tumorigenesis. In this study, we identify the splicing regulator SLU7 as an essential factor for the preservation of hepatocellular carcinoma (HCC) cells viability. Compared with hepatocytes, SLU7 expression is reduced in HCC cells; however, further SLU7 depletion triggered autophagy-related cellular apoptosis in association with the overproduction of reactive oxygen species. Remarkably, these responses were not observed in primary human hepatocytes or in the well-differentiated HepaRG cell line. Mechanistically, we demonstrate that SLU7 binds the C13orf25 primary transcript in which the polycistronic oncomir miR-17-92 cluster is encompassed, and is necessary for its processing and expression. SLU7 knockdown altered the splicing of the C13orf25 primary transcript, and markedly reduced the expression of its miR-17, miR-20 and miR-92a constituents. This led to the upregulation of CDKN1A (P21) and BCL2L11 (BIM) expression, two bona fide targets of the miR-17-92 cluster and recognized mediators of its pro-survival and tumorigenic activity. Interestingly, altered splicing of miR-17-92 and downregulation of miR-17 and miR-20 were not observed upon SLU7 knockdown in non-transformed hepatocytes, but was found in other (HeLa, H358) but not in all (Caco2) non-hepatic tumor cells. The functional relevance of miR-17-92 dysregulation upon SLU7 knockdown was established when oxidative stress, autophagy and apoptosis were reversed by co-transfection of HCC cells with a miR-17 mimic. Together, these findings indicate that SLU7 is co-opted by HCC cells and other tumor cell types to maintain survival, and identify this splicing regulator as a new determinant for the expression of the oncogenic miR-17-92 cluster. This novel mechanism may be exploited for the development of antitumoral strategies in cancers displaying such SLU7-miR-17-92 crosstalk.
Revista:
INTERNATIONAL JOURNAL OF CANCER
ISSN:
0020-7136
Año:
2015
Vol.:
136
N°:
10
Págs.:
2469 - 2475
Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15(+/+) and Fgf15(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf15(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15(+/+) animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis. What's new? Fibroblast growth factor-19 (FGF19), in rodents called FGF15, is a gut-derived hormone recently implicated as a driver gene in liver carcinogenesis. Here, the authors show that Fgf15(-/-) mice develop less hepatocellular carcinoma and less liver fibrosis as compared to Fgf15(+/+) littermates, underscoring the important role of the factor in liver damage and cancer development. Interestingly, Fgf15 expression is not detected in injured liver or carcinoma tissue, but is upregulated in the ileum and blood, pointing to a new gut-liver axis involved in hepatocarcinogenesis.
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2015
Vol.:
62
N°:
1
Págs.:
166 - 178
Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10-deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C-X-C chemokine receptor-4 (CXCR4), was reduced in DEN-induced MMP10-deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10-stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal-derived factor-1 (SDF1), through the extracellular signal-regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter.
CONCLUSION:
MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model.
Revista:
JOURNAL OF HEPATOLOGY
ISSN:
0168-8278
Año:
2012
Vol.:
56
N°:
2
Págs.:
367 - 373
Background & Aims Bile acids (BA) are increasingly recognized as important modulators of liver regeneration. Increased enterohepatic BA flux has been proposed to generate specific signals that activate hepatocyte proliferation after partial hepatectomy (PH). We have investigated the role of the BA membrane transporter Mrp3 (Abcc3), which is expressed in the liver and gut, in the hepatic growth response elicited by BA and in liver regeneration after PH.
Methods Liver growth and regeneration, and the expression of growth-related genes, were studied in Mrp3+/+ and Mrp3¿/¿ mice fed a cholic acid (CA) supplemented diet and after 2/3 PH. Activation of the BA receptor FXR was measured in mice after in vivo transduction of the liver with a FXR-Luciferase reporter plasmid. BA levels were measured in portal serum and liver tissue by high performance liquid chromatography-tandem mass spectrometry.
Results Liver growth elicited by CA feeding was significantly reduced in Mrp3¿/¿ mice. These animals showed reduced FXR activation in the liver after CA administration and decreased portal serum levels of BA. Liver regeneration after PH was significantly delayed in Mrp3-deficient mice. Proliferation-related gene expression and peak DNA synthesis in Mrp3¿/¿ mice occurred later than in wild types, coinciding with a retarded elevation in intra-hepatic BA levels.
Conclusions Lack of Abcc3 expression markedly impairs liver growth in response to BA and after PH. Our data suggest that Mrp3 plays a non-redundant role in the regulation of BA flux during liver regeneration.
Revista:
PLoS One
ISSN:
1932-6203
Año:
2010
Vol.:
5
N°:
12
Págs.:
e15690
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 59-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. alpha-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 TGF beta 2 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 profibrogenic 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.