RESUMEN
BACKGROUND: A balanced protein homeostasis network helps cholangiocarcinoma (CCA) maintain their oncogenic growth, and disrupting proteostasis therapeutically will induce proteotoxic stress. Phosphatase and tensin homolog (PTEN) have been reported to be involved in proteostasis, and PTEN-associated pathways are commonly altered in CCA. Celastrol, a triterpene from plants, exhibits cytotoxic effects in various types of cancer. However, the underlying mechanisms remain unclear. PURPOSE: We investigated the therapeutic effect of celastrol in CCA and identified the molecular characteristics of tumors that were sensitive to celastrol. The target of celastrol was explored. We then evaluated the candidate combination therapeutic strategy to increase the effectiveness of celastrol in celastrol-insensitive CCA tumors. METHODS: Various CCA cells were categorized as either celastrol-sensitive or celastrol-insensitive based on their response to celastrol. The molecular characteristics of cells from different groups were determined by RNA-seq. PTEN status and its role in proteasome activity in CCA cells were investigated. The CMAP analysis, molecular docking, and functional assay were performed to explore the effect of celastrol on proteasome activities. The correlation between PTEN status and clinical outcomes, as well as proteasomal activity, were measured in CCA patients. The synergistic therapeutic effect of autophagy inhibitors on celastrol-insensitive CCA cells were measured. RESULTS: Diverse responses to celastrol were observed in CCA cells. PTEN expression varied among different CCA cells, and its status could impact cell sensitivity to celastrol: PTENhigh tumor cells were resistant to celastrol, while PTENlow cells were more sensitive. Celastrol induced proteasomal dysregulation in CCA cells by directly targeting PSMB5. Cells with low PTEN status transcriptionally promoted proteasome subunit expression in an AKT-dependent manner, making these cells more reliant on proteasomal activities to maintain proteostasis. This caused the PTENlow CCA cells sensitive to celastrol. A negative correlation was found between PTEN levels and the proteasome signature in CCA patients. Moreover, celastrol treatment could induce autophagy in PTENhigh CCA cells. Disrupting the autophagic pathway in PTENhigh CCA cells enhanced the cytotoxic effect of celastrol. CONCLUSION: PTEN status in CCA cells determines their sensitivity to celastrol, and autophagy inhibitors could enhance the anti-tumor effect in PTENhigh CCA.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Fosfohidrolasa PTEN , Triterpenos Pentacíclicos , Triterpenos , Colangiocarcinoma/tratamiento farmacológico , Triterpenos Pentacíclicos/farmacología , Fosfohidrolasa PTEN/metabolismo , Humanos , Línea Celular Tumoral , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Triterpenos/farmacología , Simulación del Acoplamiento Molecular , Tripterygium/química , Antineoplásicos Fitogénicos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Autofagia/efectos de los fármacos , Bortezomib/farmacologíaRESUMEN
The treatment of hepatocellular carcinoma (HCC) is particularly challenging due to the inherent tumoral heterogeneity and easy resistance towards chemotherapy and immunotherapy. Arsenic trioxide (ATO) has emerged as a cytotoxic agent effective for treating solid tumors, including advanced HCC. However, its effectiveness in HCC treatment remains limited, and the underlying mechanisms are still uncertain. Therefore, this study aimed to characterize the effects and mechanisms of ATO in HCC. By evaluating the susceptibilities of human and murine HCC cell lines to ATO treatment, we discovered that HCC cells exhibited a range of sensitivity to ATO treatment, highlighting their inherent heterogeneity. A gene signature comprising 265 genes was identified to distinguish ATO-sensitive from ATO-insensitive cells. According to this signature, HCC patients have also been classified and exhibited differential features of ATO response. Our results showed that ATO treatment induced reactive oxygen species (ROS) accumulation and the activation of multiple cell death modalities, including necroptosis and ferroptosis, in ATO-sensitive HCC cells. Meanwhile, elevated tumoral immunogenicity was also observed in ATO-sensitive HCC cells. Similar effects were not observed in ATO-insensitive cells. We reported that ATO treatment induced mitochondrial injury and mtDNA release into the cytoplasm in ATO-sensitive HCC tumors. This subsequently activated the cGAS-STING-IFN axis, facilitating CD8+ T cell infiltration and activation. However, we found that the IFN pathway also induced tumoral PD-L1 expression, potentially antagonizing ATO-mediated immune attack. Additional anti-PD1 therapy promoted the anti-tumor response of ATO in ATO-sensitive HCC tumors. In summary, our data indicate that heterogeneous ATO responses exist in HCC tumors, and ATO treatment significantly induces immunogenic cell death (ICD) and activates the tumor-derived mtDNA-STING-IFN axis. These findings may offer a new perspective on the clinical treatment of HCC and warrant further study.
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Trióxido de Arsénico , Carcinoma Hepatocelular , Muerte Celular Inmunogénica , Neoplasias Hepáticas , Proteínas de la Membrana , Nucleotidiltransferasas , Trióxido de Arsénico/farmacología , Trióxido de Arsénico/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Humanos , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Muerte Celular Inmunogénica/efectos de los fármacos , Línea Celular Tumoral , Interferones/metabolismo , Transducción de Señal/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ratones Endogámicos C57BLRESUMEN
Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line-derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Humanos , Fosforilación , Gemcitabina , Nucleósidos , Conductos Biliares Intrahepáticos , Fosfohidrolasa PTENRESUMEN
BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Colangiocarcinoma , Exosomas , Fosfohidrolasa PTEN , Animales , Humanos , Ratones , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Colangiocarcinoma/metabolismo , Modelos Animales de Enfermedad , Exosomas/metabolismo , Lisosomas/fisiología , Complejo de la Endopetidasa Proteasomal , Fosfohidrolasa PTEN/metabolismo , Estudios RetrospectivosRESUMEN
Oncogene-derived metabolic reprogramming is important for anabolic growth of cancer cells, which is now considered to be not simply rely on glycolysis. Pentose phosphate pathway and tricarboxylic acid cycle also play pivotal roles in helping cancer cells to meet their anabolic and energy demands. The present work focused on gankyrin, a relatively specific oncogene in hepatocellular carcinoma (HCC), and its impact on glycolysis and mitochondrial homeostasis. Metabolomics, RNA-seq analysis, and subsequent conjoint analysis illustrated that gankyrin regulated the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and mitochondrial function and homeostasis, which play pivotal roles in tumor development. Mechanistically, gankyrin was found to modulate HCC metabolic reprogramming via TIGAR. Gankyrin positively regulated the transcription of TIGAR through Nrf2, which bound to the antioxidant response elements (AREs) in the promoter of TIGAR. Interestingly, TIGAR feedback regulated the transcription of Nrf2 and subsequently gankyrin by promoting nuclear importation of PGC1α. The loop between gankyrin, Nrf2, and TIGAR accelerated glucose metabolism toward the PPP and TCA cycle, which provided vital building blocks, such as NADPH, ATP, and ribose of tumor and further facilitated the progression of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Ciclo del Ácido Cítrico , Neoplasias Hepáticas/patología , Glucólisis , Glucosa/metabolismoRESUMEN
Gallbladder cancer (GBC) is an aggressive malignancy of biliary tract with poor prognosis. Although several studies have shown the frequency of relevant genetic alterations, there are few genetic models or translational studies that really benefit for GBC treatment in the era of precision medicine. By targeted sequencing and immunohistochemistry staining, we identified that phosphate and tension homology deleted on chromosome ten (PTEN) was frequently altered in GBC specimens, and loss of PTEN expression was independently correlated with poor survival outcomes. Further drug screening assays revealed proteasome inhibitor bortezomib as a promising agent for GBC treatment, and knockdown of PTEN increased bortezomib efficacy both in vivo and in vitro. Therapeutic evaluation of patient derived xenografts (PDXs) strongly supported the utilization of bortezomib in PTEN deficient GBC. Mechanically, functional PTEN inhibited ARE-dependent transcriptional activity, the same machinery regulating the transcription of proteasome subunits, thus PTEN suppressed proteasome activity and bortezomib sensitivity. Through siRNA screening, we identified the ARE-related transcriptional suppressor BACH1 involved in PTEN-mediated proteasome inhibition and regulated by PTEN-AKT1 axis. In summary, our study indicates that proteasome activity represents a prime therapeutic target in PTEN-deficient GBC tumors, which is worthy of further clinical validation.
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Bortezomib/administración & dosificación , Regulación hacia Abajo , Neoplasias de la Vesícula Biliar/tratamiento farmacológico , Mutación , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Adulto , Anciano , Animales , Bortezomib/farmacología , Línea Celular Tumoral , Femenino , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Patient-derived xenografts (PDXs) and PDX-derived cells (PDCs) are useful in preclinical research. We performed a drug screening assay using PDCs and identified proteasome inhibitors as promising drugs for cholangiocarcinoma (CCA) treatment. Furthermore, we determined that phosphate and tensin homology deleted on chromosome ten (PTEN) deficiency promotes protein synthesis and proteasome subunit expression and proteolytic activity, creating a dependency on the proteasome for cancer cell growth and survival. Thus, targeting the proteasome machinery with the inhibitor bortezomib inhibited the proliferation and survival of CCA cells lacking functional PTEN. Therapeutic evaluation of PDXs, autochthonous mouse models, and patients confirmed this dependency on the proteasome. Mechanistically, we found that PTEN promoted the nuclear translocation of FOXO1, resulting in the increased expression of BACH1 and MAFF BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element, which is present in genes encoding proteasome subunits. PTEN induced the accumulation and nuclear translocation of these proteins, which directly repressed the transcription of genes encoding proteasome subunits. We revealed that the PTEN-proteasome axis is a potential target for therapy in PTEN-deficient CCA and other PTEN-deficient cancers.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Animales , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Conductos Biliares Intrahepáticos , Bortezomib/farmacología , Bortezomib/uso terapéutico , Línea Celular Tumoral , Colangiocarcinoma/tratamiento farmacológico , Humanos , Ratones , Fosfohidrolasa PTEN/genética , Complejo de la Endopetidasa ProteasomalRESUMEN
BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.
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Carcinogénesis , Colangiocarcinoma/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Línea Celular , Transformación Celular Neoplásica/metabolismo , Colangiocarcinoma/patología , Resistencia a Antineoplásicos , Humanos , Ratones , Estrés OxidativoRESUMEN
BACKGROUND: Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma (HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics. METHODS: The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus (PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2. RESULTS: In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival (OS) and disease-free survival (DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-ß induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines. CONCLUSIONS: Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-ß/Smad signal pathway.
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Carcinoma Hepatocelular/metabolismo , Transición Epitelial-Mesenquimal , Proteínas de Homeodominio/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , Carga Tumoral , Regulación hacia ArribaRESUMEN
Kras mutations are among the most common genetic abnormalities in human neoplasms, including cholangiocarcinomas, pancreatic cancer and colon cancer. PTEN has previously been associated with cholangiocarcinoma development in murine models. Here, we have established novel mouse models of neoplasms by liver-specific and biliary-pancreatic Kras activation and PTEN deletion. By liver-specific disruption of PTEN and activation of Kras in mice caused rapid development of intrahepatic biliary epithelial proliferative lesions (Intrahepatic cholangiocarcinoma, ICC), which progress through dysplasia to invasive carcinoma. In contrast, Kras activation in combination with heterozygous PTEN deletion induced mixed carcinomas of liver (both ICC and hepatocellular carcinoma, HCC), whereas Kras activation alone did not induce biliary tract neoplasm. Use of Sox9-Cre-LoxP-based approach to coordinately delete PTEN and activate Kras in the adult mouse resulted in not only development of low-grade biliary lesions (ICC and extrahepatic bile duct carcinoma, ECC) but also pancreatic carcinomas. Our data provide a functional link between PTEN gene status, hepatobiliary cell fate, and HCC, biliary carcinoma, pancreatic cancer pathogenesis, and present novel genetically engineered mouse models of PTEN loss-driven malignancy.