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OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.
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Carcinoma , Neoplasias Colorretais , Interleucina-10 , Neoplasias Hepáticas , Receptores de Antígenos Quiméricos , Receptores de Interleucina-10 , Animais , Humanos , Camundongos , Antígeno Carcinoembrionário/imunologia , Carcinoma/imunologia , Carcinoma/secundário , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/patologia , Imunoterapia Adotiva , Interleucina-10/antagonistas & inibidores , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Interleucina-10/antagonistas & inibidores , Anticorpos Bloqueadores/imunologiaRESUMO
BACKGROUND: Robotic hepatectomy (RH) is increasingly utilized for minor and major liver resections. The IWATE criteria were developed to classify minimally invasive liver resections by difficulty. The objective of this study was to apply the IWATE criteria in RH and to describe perioperative and oncologic outcomes of RH over the last decade at our institution. METHODS: Perioperative and oncologic outcomes of patients who underwent RH between 2011 and 2019 were retrospectively collected. The difficulty level of each operation was assessed using the IWATE criteria, and outcomes were compared at each level. Univariate linear regression was performed to characterize the relationship between IWATE criteria and perioperative outcomes (OR time, EBL, and LOS), and a multivariable model was also developed to address potential confounding by patient characteristics (age, sex, BMI, prior abdominal surgery, ASA class, and simultaneous non-hepatectomy operation). RESULTS: Two hundred and twenty-five RH were performed. Median IWATE criteria for RH were 6 (IQR 5-9), with low, intermediate, advanced, and expert resections accounting for 23% (n = 51), 34% (n = 77), 32% (n = 72), and 11% (n = 25) of resections, respectively. The majority of resections were parenchymal-sparing approaches, including anatomic segmentectomies and non-anatomic partial resections. 30-day complication rate was 14%, conversion to open surgery occurred in 9 patients (4%), and there were no deaths within 30 days postoperatively. In the univariate linear regression analysis, IWATE criteria were positively associated with OR time, EBL, and LOS. In the multivariable model, IWATE criteria were independently associated with greater OR time, EBL, and LOS. Two-year overall survival for hepatocellular carcinoma and intrahepatic cholangiocarcinoma was 94% and 50%, respectively. CONCLUSION: In conclusion, the IWATE criteria are associated with surgical outcomes after RH. This series highlights the utility of RH for difficult hepatic resections, particularly parenchymal-sparing resections in the posterosuperior sector, extending the indication of minimally invasive hepatectomy in experienced hands and potentially offering select patients an alternative to open hepatectomy or other less definitive liver-directed treatment options.
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Neoplasias dos Ductos Biliares , Laparoscopia , Neoplasias Hepáticas , Procedimentos Cirúrgicos Robóticos , Neoplasias dos Ductos Biliares/cirurgia , Ductos Biliares Intra-Hepáticos/patologia , Hepatectomia/efeitos adversos , Humanos , Laparoscopia/efeitos adversos , Tempo de Internação , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/efeitos adversosRESUMO
Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell-like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).
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Carcinoma Hepatocelular/patologia , Histonas/fisiologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Proliferação de Células , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Fosforilação , Fator de Transcrição RelA/metabolismoRESUMO
PURPOSE: To assess the postprocedure findings of irreversible electroporation (IRE) in patients with hepatocellular carcinoma (HCC) at magnetic resonance (MR) imaging. MATERIALS AND METHODS: This retrospective study was Institutional Review Board approved, and informed consent was waived. Twenty patients with HCC were treated with IRE over a 2.5-year period. The median patient age was 62 years, and 75% of patients had cirrhosis with a Child-Pugh score of A. The median tumor diameter was 2.0 cm (range, 1.0-3.3 cm). Contrast material-enhanced multiphase MR imaging was performed on postprocedure days 1 and 30 and every 90 days thereafter. Ablation zone sizes and signal intensities were compared between each time point for both T1- and T2-weighted images. Trends in signal intensity and tumor dimensions over time were quantified by using generalized linear models. RESULTS: MR imaging appearances of treated tumors include a zone of peripheral enhancement with centripetal filling on delayed contrast-enhanced images. Compared with postprocedure day 1, every 90 days there is a decrease of 28.9% (mean, axis) in the size of the enhancing ablation zone. Over time, there is a trend toward decreasing signal intensity in the peripheral ablation zone on both T2-weighted (P = .01) and contrast-enhanced T1-weighted (P < .08) images. Conversely, the tumor itself typically has increased signal intensity on the same sequences. CONCLUSION: IRE of HCC results in a large region of enhancement on immediate postprocedure MR images that, over time, involutes and is associated with decreasing signal intensity of the peripheral ablation zone. This phenomenon may represent resolution of the reversible penumbra.
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Carcinoma Hepatocelular/patologia , Eletroporação/métodos , Neoplasias Hepáticas/patologia , Imageamento por Ressonância Magnética/métodos , Idoso , Meios de Contraste , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND: Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. METHODS: The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. RESULTS: The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. CONCLUSION: FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.
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8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Carcinoma Hepatocelular/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Neoplasias Hepáticas/enzimologia , Mutação , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Carcinoma Hepatocelular/genética , Domínio Catalítico , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/genética , Metástase Linfática , Recidiva Local de NeoplasiaRESUMO
Fibrolamellar hepatocellular carcinoma (FL-HCC) has historically been classified as a rare subtype of HCC. However, unlike "classic" HCC, it occurs in children and young adults without underlying liver disease. The recent discovery of a deletion mutation in all FL-HCCs represented a major advancement in understanding the pathogenesis of this disease. This deletion results in the fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PKA, PRKACA), and overexpression of PRKACA and enhanced cAMP-dependent PKA activity. This review summarizes recent advancements in FL-HCC pathogenesis and characteristics of the HSP40-PKA C protein.
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Sequência de Bases , Carcinoma Hepatocelular/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Proteínas de Fusão Oncogênica/genética , Deleção de Sequência , Adolescente , Adulto , Carcinoma Hepatocelular/classificação , Carcinoma Hepatocelular/patologia , Criança , Pré-Escolar , Feminino , Humanos , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/patologia , MasculinoRESUMO
Fibrolamellar hepatocellular carcinoma, or fibrolamellar carcinoma, is a rare form of primary liver cancer that afflicts healthy young men and women without underlying liver disease. There are currently no effective treatments for fibrolamellar carcinoma other than resection or transplantation. In this study, we sought evidence of mechanistic target of rapamycin complex 1 (mTORC1) activation in fibrolamellar carcinoma, based on anecdotal reports of tumor response to rapamycin analogs. Using a tissue microarray of 89 primary liver tumors, including a subset of 10 fibrolamellar carcinomas, we assessed the expression of phosphorylated S6 ribosomal protein (P-S6), a downstream target of mTORC1, along with fibroblast growth factor receptor 1 (FGFR1). These results were extended and confirmed using an additional 13 fibrolamellar carcinomas, whose medical records were reviewed. In contrast to weak staining in normal livers, all fibrolamellar carcinomas on the tissue microarray showed strong immunostaining for FGFR1 and P-S6, whereas only 13% of non-fibrolamellar hepatocellular carcinomas had concurrent activation of FGFR1 and mTORC1 signaling (P<0.05). When individual samples were stratified according to staining intensity (scale 0-4), the average score in fibrolamellar carcinomas was 2.46 for FGFR1 and 3.77 for P-S6, compared with 0 and 0, respectively, in non-tumor liver. Immunoblot analyses of fibrolamellar carcinomas revealed high mTORC1 activities relative to AKT activities accompanied by reduced TSC2 expression, which was not observed in non-fibrolamellar hepatocellular carcinomas. Our findings provide evidence for mTORC1 activation and FGFR1 overexpression in human fibrolamellar carcinoma, and support the use of FGFR1 inhibitors and rapamycin analogs in the treatment of patients with unresectable fibrolamellar carcinoma.
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Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Complexos Multiproteicos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adolescente , Adulto , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Análise Serial de Tecidos , Adulto JovemRESUMO
BACKGROUND & AIMS: Phosphatidylinositide 3-kinase (PI3K) is deregulated in many human tumor types, including primary liver malignancies. The kinase v-akt murine thymoma viral oncogene homolog 1 (Akt) and mammalian target of rapamycin complex (mTORC1) are effectors of PI3K that promote cell growth and survival, but their individual roles in tumorigenesis are not well defined. METHODS: In livers of albumin (Alb)-Cre mice, we selectively deleted tuberous sclerosis (Tsc)1, a negative regulator of Ras homolog enriched in brain and mTORC1, along with Phosphatase and tensin homolog (Pten), a negative regulator of PI3K. Tumor tissues were characterized by histologic and biochemical analyses. RESULTS: The Tsc1fl/fl;AlbCre, Ptenfl/fl;AlbCre, and Tsc1fl/fl;Ptenfl/fl;AlbCre mice developed liver tumors that differed in size, number, and histologic features. Livers of Tsc1fl/fl;AlbCre mice did not develop steatosis; tumors arose later than in the other strains of mice and were predominantly hepatocellular carcinomas. Livers of the Ptenfl/fl;AlbCre mice developed steatosis and most of the tumors that formed were intrahepatic cholangiocarcinomas. Livers of Tsc1fl/fl;Ptenfl/fl;AlbCre formed large numbers of tumors, of mixed histologies, with the earliest onset of any strain, indicating that loss of Tsc1 and Pten have synergistic effects on tumorigenesis. In these mice, the combination of rapamycin and MK2206 was more effective in reducing liver cell proliferation and inducing cell death than either reagent alone. Tumor differentiation correlated with Akt and mTORC1 activities; the ratio of Akt:mTORC1 activity was high throughout the course of intrahepatic cholangiocarcinomas development and low during hepatocellular carcinoma development. Compared with surrounding nontumor liver tissue, tumors from all 3 strains had increased activities of Akt, mTORC1, and mitogen-activated protein kinase and overexpressed fibroblast growth factor receptor 1. Inhibition of fibroblast growth factor receptor 1 in Tsc1-null mice suppressed Akt and mitogen-activated protein kinase activities in tumor cells. CONCLUSIONS: Based on analyses of knockout mice, mTORC1 and Akt have different yet synergistic effects during the development of liver tumors in mice.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas Experimentais/genética , Complexos Multiproteicos/genética , Mutação , Proteínas Proto-Oncogênicas c-akt/genética , RNA Neoplásico/genética , Serina-Treonina Quinases TOR/genética , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais CultivadasRESUMO
Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.
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Present cancer disease models - typically based on cell cultures and animal models that lack the human tumor microenvironment (TME) - are extremely poor predictors of human disease outcomes. Microscale cancer models that combine the micromanipulation of tissues and fluids offer the exciting possibility of miniaturizing the drug testing workflow, enabling inexpensive, more efficient tests of high clinical biomimicry that maximize the use of scarce human tissue and minimize animal testing. Critically, these microscale models allow for precisely addressing the impact of the structural features of the heterogeneous TME to properly target and understand the contributions of these unique zones to therapeutic response. We have recently developed a precision slicing method that yields large numbers of cuboidal micro-tissues ("cuboids", â¼ (400 µm) 3 ) from a single tumor biopsy. Here we evaluate cuboids from syngeneic mouse tumor models and human tumors, which contain native immune cells, as models for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as their cellular architecture (immune cells and vasculature), cytokine secretion, proteomics profiles, and their response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong functional responses such as cytokine and drug responses. Overall, our results suggest that cuboids make an excellent model for applications that require the TME, such as immunotherapy drug evaluations, including for clinical trials and personalized oncology approaches.
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The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
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Carcinoma Hepatocelular , Humanos , Sobrevivência Celular , Carcinoma Hepatocelular/tratamento farmacológico , Apoptose , Proteínas de Choque Térmico HSP70 , Proteínas Proto-Oncogênicas c-bcl-2 , Chaperonas MolecularesRESUMO
Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter coordinates metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial metabolic pathways has remained unexplored. Here, we investigate the metabolic consequences of uniporter loss- and gain-of-function, and identify a key transcriptional regulator that mediates these effects. Using gene expression profiling and proteomic, we find that loss of uniporter function increases the expression of proteins in the branched-chain amino acid (BCAA) catabolism pathway. Activity is further augmented through phosphorylation of the enzyme that catalyzes this pathway's committed step. Conversely, in the liver cancer fibrolamellar carcinoma (FLC)-which we demonstrate to have high mitochondrial calcium levels- expression of BCAA catabolism enzymes is suppressed. We also observe uniporter-dependent suppression of the transcription factor KLF15, a master regulator of liver metabolic gene expression, including those involved in BCAA catabolism. Notably, loss of uniporter activity upregulates KLF15, along with its transcriptional target ornithine transcarbamylase (OTC), a component of the urea cycle, suggesting that uniporter hyperactivation may contribute to the hyperammonemia observed in FLC patients. Collectively, we establish that FLC has increased mitochondrial calcium levels, and identify an important role for mitochondrial calcium signaling in metabolic adaptation through the transcriptional regulation of metabolism.
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Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.
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Proteínas Adaptadoras de Transdução de Sinal , Colangiocarcinoma , Dasatinibe , Isocitrato Desidrogenase , Mutação , Quinases da Família src , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Dasatinibe/farmacologia , Isocitrato Desidrogenase/metabolismo , Isocitrato Desidrogenase/genética , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Quinases da Família src/antagonistas & inibidores , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismoRESUMO
Tuberin is a negative regulator of mTOR pathway. To investigate the function of tuberin during liver regeneration, we performed 70% hepatectomy on wild-type and Tsc2+/- mice. We found the tuberin phosphorylation correlated with mTOR activation during early liver regeneration in wild-type mice. However, liver regeneration in the Tsc2+/- mice was not enhanced. Instead, the Tsc2+/- livers failed to accumulate lipid bodies, and this was accompanied by increased mortality. These findings suggest that tuberin plays a critical role in liver energy balance by regulating hepatocellular lipid accumulation during early liver regeneration. These effects may influence the role of mTORC1 on cell growth and proliferation.
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Heterozigoto , Metabolismo dos Lipídeos , Regeneração Hepática , Fígado/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Peso Corporal , Ativação Enzimática , Hepatectomia , Fígado/patologia , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiênciaRESUMO
In the era of personalized oncology, there have been accelerated efforts to develop clinically relevant platforms to test drug sensitivities of individual cancers. An ideal assay will serve as a diagnostic companion to inform the oncologist of the various treatments that are sensitive and insensitive, thus improving outcome while minimizing unnecessary toxicities and costs. To date, no such platform exists for clinical use, but promising approaches are on the horizon that take advantage of improved techniques in creating human cancer models that encompass the entire tumor microenvironment, alongside technologies for assessing and analyzing tumor response. This review summarizes a number of current strategies that make use of intact human cancer tissues as organotypic cultures in drug sensitivity testing.
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The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12 DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12 DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
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Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.
Assuntos
Carcinoma Hepatocelular , Proteínas Quinases Dependentes de AMP Cíclico , Humanos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Carcinoma Hepatocelular/genética , Transdução de Sinais , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Radiofrequency ablation (RFA) use among patients with hepatocellular carcinoma (HCC) has increased dramatically over the last decade, but assessments outside specialized centers are lacking. This population-based study was intended to evaluate the safety and effectiveness of RFA when used to treat HCC. METHODS: A cohort study of HCC patients (diagnosed 2002-2005) was performed using linked Surveillance, Epidemiology, and End Results (SEER)-Medicare data. Early (≤90-day) mortality and readmission as well as survival among patients undergoing RFA, resection, or no treatment were compared using multivariate and propensity score adjusted Poisson and Cox regression models. RESULTS: Of 2631 patients (mean age 76.1±6.1 years, 65.9% male), 16% underwent RFA (49.6%) or resection (50.4%). Early mortality (13.6 vs. 18.7%, P=.16) and readmission (34.5 vs. 32.1%, P=.60) rates were similar among RFA and resection patients. The 1-year survival after RFA and resection was similar (72.2 vs. 79.7%, P=.18), but beyond 3 years there was a survival benefit among patients undergoing resection (39.2 vs. 58.0%, P<.001). Patients treated with RFA as a sole therapeutic intervention in the 1st year had a similar hazard of death compared with untreated patients (hazard ratio [HR] 0.84, 95% confidence interval [95% CI] 0.54-1.33). CONCLUSIONS: In the general community, patients treated with RFA have a similar risk of early adverse events compared with those treated with resection with no clear survival benefit when used as a sole intervention. Although RFA has been described as a safe and effective treatment for HCC at specialized centers, this experience may not extrapolate to the general community and requires further evaluation.
Assuntos
Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/cirurgia , Ablação por Cateter/estatística & dados numéricos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/cirurgia , Medicare/estatística & dados numéricos , Readmissão do Paciente/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Ablação por Cateter/efeitos adversos , Estudos de Coortes , Feminino , Humanos , Masculino , Análise Multivariada , Análise de Regressão , Medição de Risco , Taxa de Sobrevida , Estados Unidos/epidemiologiaRESUMO
Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion:227Th-octapa-αGPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.