RESUMO
Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Adolescente , Humanos , Adulto Jovem , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Fibrolamellar carcinoma (FLC) is a rare, lethal, early-onset liver cancer with a critical need for new therapeutics. The primary driver in FLC is the fusion oncoprotein, DNAJ-PKAc, which remains challenging to target therapeutically. It is critical, therefore, to expand understanding of the FLC molecular landscape to identify druggable pathways/targets. Here, we perform the most comprehensive integrative proteo-metabolomic analysis of FLC. We also conduct nutrient manipulation, respirometry analyses, as well as key loss-of-function assays in FLC tumor tissue slices from patients. We propose a model of cellular energetics in FLC pointing to proline anabolism being mediated by ornithine aminotransferase hyperactivity and ornithine transcarbamylase hypoactivity with serine and glutamine catabolism fueling the process. We highlight FLC's potential dependency on voltage-dependent anion channel (VDAC), a mitochondrial gatekeeper for anions including pyruvate. The metabolic rewiring in FLC that we propose in our model, with an emphasis on mitochondria, can be exploited for therapeutic vulnerabilities.
RESUMO
Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.
Assuntos
Carcinoma Hepatocelular/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Fusão Oncogênica/genética , RNA Longo não Codificante/genética , Regulação para Cima , Sistemas CRISPR-Cas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , ProteômicaRESUMO
Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the chromatin regulatory landscape and the genes most critical for tumor cell survival remain unclear. Here, we use chromatin run-on sequencing to discover â¼7,000 enhancers and 141 enhancer hotspots activated in FLC relative to nonmalignant liver. Bioinformatic analyses reveal aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also define the genes most strongly associated with hotspots of FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduce cell viability and/or significantly enhance the effect of the MEK inhibitor cobimetinib. These findings highlight molecular targets for drug development, as well as drug combination approaches.
Assuntos
Carcinoma Hepatocelular/genética , Elementos Facilitadores Genéticos/genética , Adolescente , Antígeno Ca-125/genética , Carcinogênese/patologia , Proliferação de Células/genética , Cromatina/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Membrana/genética , Transportadores de Ácidos Monocarboxílicos/genética , Oncogenes/genética , Análise de Sequência de DNA/métodos , Transdução de Sinais/genéticaRESUMO
The kinase LKB1 is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of LKB1 substrates in vivo, we used CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic KRAS-driven lung adenocarcinoma. We demonstrate that members of the SIK family are critical for constraining tumor development. Histologic and gene-expression similarities between LKB1- and SIK-deficient tumors suggest that SIKs and LKB1 operate within the same axis. Furthermore, a gene-expression signature reflecting SIK deficiency is enriched in LKB1-mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key LKB1-SIK tumor-suppressive axis and underscore the need to redirect efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression. SIGNIFICANCE: Uncovering the effectors of frequently altered tumor suppressor genes is critical for understanding the fundamental driving forces of cancer growth. Our identification of the SIK family of kinases as effectors of LKB1-mediated tumor suppression will refocus future mechanistic studies and may lead to new avenues for genotype-specific therapeutic interventions.This article is highlighted in the In This Issue feature, p. 1469.
Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular Tumoral , Proliferação de Células , Edição de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
The Nkx2-1 transcription factor promotes differentiation of lung epithelial lineages and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium-binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic inactivation of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate that Selenbp1 is a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo Implications: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome. Mol Cancer Res; 16(11); 1737-49. ©2018 AACR.
Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a Selênio/genética , Fator Nuclear 1 de Tireoide/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Processos de Crescimento Celular , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Proteínas de Ligação a Selênio/biossíntese , Proteínas de Ligação a Selênio/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , TransfecçãoRESUMO
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
Assuntos
Carcinogênese/genética , Análise Mutacional de DNA/métodos , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Reparo de DNA por Recombinação/genética , Animais , Sistemas CRISPR-Cas/genética , Análise Custo-Benefício , Análise Mutacional de DNA/economia , Estudos de Viabilidade , Feminino , Genômica/economia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Masculino , Camundongos , Mutação , Neoplasias/patologia , Reprodutibilidade dos TestesRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
Carcinoma Ductal Pancreático/patologia , Perfilação da Expressão Gênica/métodos , Neoplasias Pancreáticas/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Análise de Sequência de RNA/métodos , Regulação para Cima , Animais , Carcinoma Ductal Pancreático/genética , Hipóxia Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Engenharia Genética , Humanos , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Microambiente TumoralRESUMO
Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.