RESUMEN
BACKGROUND: Evolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear. METHODS: To ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 "metastasis-specific" mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition. RESULTS: The ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable. CONCLUSIONS: Our results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Mutación , Secuenciación del ExomaRESUMEN
Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. In vivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Supervivencia Celular/genética , Recurrencia Local de Neoplasia/genética , Heparitina Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Galactosiltransferasas/genéticaRESUMEN
BACKGROUND: Breast cancer mortality is principally due to recurrent disease that becomes resistant to therapy. We recently identified copy number (CN) gain of the putative membrane progesterone receptor PAQR8 as one of four focal CN alterations that preferentially occurred in recurrent metastatic tumors compared to primary tumors in breast cancer patients. Whether PAQR8 plays a functional role in cancer is unknown. Notably, PAQR8 CN gain in recurrent tumors was mutually exclusive with activating ESR1 mutations in patients treated with anti-estrogen therapies and occurred in > 50% of both patients treated with anti-estrogen therapies and those treated with chemotherapy or anti-Her2 agents. METHODS: We used orthotopic mouse models to determine whether PAQR8 overexpression or deletion alters breast cancer dormancy or recurrence following therapy. In vitro studies, including assays for colony formation, cell viability, and relative cell fitness, were employed to identify effects of PAQR8 in the context of therapy. Cell survival and proliferation were quantified by immunofluorescence staining for markers of apoptosis and proliferation. Sphingolipids were quantified by liquid chromatography-high resolution mass spectrometry. RESULTS: We show that PAQR8 is necessary and sufficient for efficient mammary tumor recurrence in mice, spontaneously upregulated and CN gained in recurrent tumors that arise following therapy in multiple mouse models, and associated with poor survival following recurrence as well as poor overall survival in breast cancer patients. PAQR8 promoted resistance to therapy by enhancing tumor cell survival following estrogen receptor pathway inhibition by fulvestrant or estrogen deprivation, Her2 pathway blockade by lapatinib or Her2 downregulation, and treatment with chemotherapeutic agents. Pro-survival effects of PAQR8 were mediated by a Gi protein-dependent reduction in cAMP levels, did not require progesterone, and involved a PAQR8-dependent decrease in ceramide levels and increase in sphingosine-1-phosphate levels, suggesting that PAQR8 may possess ceramidase activity. CONCLUSIONS: Our data provide in vivo evidence that PAQR8 plays a functional role in cancer, implicate PAQR8, cAMP, and ceramide metabolism in breast cancer recurrence, and identify a novel mechanism that may commonly contribute to the acquisition of treatment resistance in breast cancer patients.
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Resistencia a Antineoplásicos , Recurrencia Local de Neoplasia , Animales , Ratones , Resistencia a Antineoplásicos/genética , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Lapatinib , Fulvestrant , Receptor ErbB-2/metabolismo , Estrógenos , Receptores de ProgesteronaRESUMEN
BACKGROUND: Breast cancer mortality is principally due to tumor recurrence, which can occur following extended periods of clinical remission that may last decades. While clinical latency has been postulated to reflect the ability of residual tumor cells to persist in a dormant state, this hypothesis remains unproven since little is known about the biology of these cells. Consequently, defining the properties of residual tumor cells is an essential goal with important clinical implications for preventing recurrence and improving cancer outcomes. METHODS: To identify conserved features of residual tumor cells, we modeled minimal residual disease using inducible transgenic mouse models for HER2/neu and Wnt1-driven tumorigenesis that recapitulate cardinal features of human breast cancer progression, as well as human breast cancer cell xenografts subjected to targeted therapy. Fluorescence-activated cell sorting was used to isolate tumor cells from primary tumors, residual lesions following oncogene blockade, and recurrent tumors to analyze gene expression signatures and evaluate tumor-initiating cell properties. RESULTS: We demonstrate that residual tumor cells surviving oncogenic pathway inhibition at both local and distant sites exist in a state of cellular dormancy, despite adequate vascularization and the absence of adaptive immunity, and retain the ability to re-enter the cell cycle and give rise to recurrent tumors after extended latency periods. Compared to primary or recurrent tumor cells, dormant residual tumor cells possess unique features that are conserved across mouse models for human breast cancer driven by different oncogenes, and express a gene signature that is strongly associated with recurrence-free survival in breast cancer patients and similar to that of tumor cells in which dormancy is induced by the microenvironment. Although residual tumor cells in both the HER2/neu and Wnt1 models are enriched for phenotypic features associated with tumor-initiating cells, limiting dilution experiments revealed that residual tumor cells are not enriched for cells capable of giving rise to primary tumors, but are enriched for cells capable of giving rise to recurrent tumors, suggesting that tumor-initiating populations underlying primary tumorigenesis may be distinct from those that give rise to recurrence following therapy. CONCLUSIONS: Residual cancer cells surviving targeted therapy reside in a well-vascularized, desmoplastic microenvironment at both local and distant sites. These cells exist in a state of cellular dormancy that bears little resemblance to primary or recurrent tumor cells, but shares similarities with cells in which dormancy is induced by microenvironmental cues. Our observations suggest that dormancy may be a conserved response to targeted therapy independent of the oncogenic pathway inhibited or properties of the primary tumor, that the mechanisms underlying dormancy at local and distant sites may be related, and that the dormant state represents a potential therapeutic target for preventing cancer recurrence.
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Terapia Molecular Dirigida , Neoplasia Residual/patología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida/efectos adversos , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Neoplasia Residual/irrigación sanguínea , Neoplasia Residual/etiología , Neoplasia Residual/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neovascularización Patológica/patología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Proteína Wnt1/antagonistas & inhibidores , Proteína Wnt1/genéticaRESUMEN
Nearly all breast cancer deaths result from metastatic disease. Despite this, the genomic events that drive metastatic recurrence are poorly understood. We performed whole-exome and shallow whole-genome sequencing to identify genes and pathways preferentially mutated or copy-number altered in metastases compared with the paired primary tumors from which they arose. Seven genes were preferentially mutated in metastases - MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1 - 5 of which are not significantly mutated in any type of human primary cancer. Four regions were preferentially copy-number altered: loss of STK11 and CDKN2A/B, as well as gain of PTK6 and the membrane-bound progesterone receptor, PAQR8. PAQR8 gain was mutually exclusive with mutations in the nuclear estrogen and progesterone receptors, suggesting a role in treatment resistance. Several pathways were preferentially mutated or altered in metastases, including mTOR, CDK/RB, cAMP/PKA, WNT, HKMT, and focal adhesion. Immunohistochemical analyses revealed that metastases preferentially inactivate pRB, upregulate the mTORC1 and WNT signaling pathways, and exhibit nuclear localization of activated PKA. Our findings identify multiple therapeutic targets in metastatic recurrence that are not significantly mutated in primary cancers, implicate membrane progesterone signaling and nuclear PKA in metastatic recurrence, and provide genomic bases for the efficacy of mTORC1, CDK4/6, and PARP inhibitors in metastatic breast cancer.
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Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica , Mutación , Proteínas de Neoplasias , Vía de Señalización Wnt , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genéticaRESUMEN
BACKGROUND: Obesity is associated with an increased risk of breast cancer recurrence and cancer death. Recurrent cancers arise from the pool of residual tumor cells, or minimal residual disease (MRD), that survives primary treatment and persists in the host. Whether the association of obesity with recurrence risk is causal is unknown, and the impact of obesity on MRD and breast cancer recurrence has not been reported in humans or in animal models. METHODS: Doxycycline-inducible primary mammary tumors were generated in intact MMTV-rtTA;TetO-HER2/neu (MTB/TAN) mice or orthotopic recipients fed a high-fat diet (HFD; 60% kcal from fat) or a control low-fat diet (LFD; 10% kcal from fat). Following oncogene downregulation and tumor regression, mice were followed for clinical recurrence. Body weight was measured twice weekly and used to segregate HFD mice into obese (i.e., responders) and lean (i.e., nonresponders) study arms, and obesity was correlated with body fat percentage, glucose tolerance (measured using intraperitoneal glucose tolerance tests), serum biomarkers (measured by enzyme-linked immunosorbent assay), and tissue transcriptomics (assessed by RNA sequencing). MRD was quantified by droplet digital PCR. RESULTS: HFD-Obese mice weighed significantly more than HFD-Lean and LFD control mice (p < 0.001) and had increased body fat percentage (p < 0.001). Obese mice exhibited fasting hyperglycemia, hyperinsulinemia, and impaired glucose tolerance, as well as decreased serum levels of adiponectin and increased levels of leptin, resistin, and insulin-like growth factor 1. Tumor recurrence was accelerated in HFD-Obese mice compared with HFD-Lean and LFD control mice (median relapse-free survival 53.0 days vs. 87.0 days vs. 80.0 days, log-rank p < 0.001; HFD-Obese compared with HFD-Lean HR 2.52, 95% CI 1.52-4.16; HFD-Obese compared with LFD HR 2.27, 95% CI 1.42-3.63). HFD-Obese mice harbored a significantly greater number of residual tumor cells than HFD-Lean and LFD mice (12,550 ± 991 vs. 7339 ± 2182 vs. 4793 ± 1618 cells, p < 0.001). CONCLUSION: These studies provide a genetically engineered mouse model for study of the association of diet-induced obesity with breast cancer recurrence. They demonstrate that this model recapitulates physiological changes characteristic of obese patients, establish that the association between obesity and recurrence risk is causal in nature, and suggest that obesity is associated with the increased survival and persistence of residual tumor cells.
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Neoplasias de la Mama/mortalidad , Neoplasias Mamarias Experimentales/patología , Recurrencia Local de Neoplasia/patología , Obesidad/patología , Animales , Índice de Masa Corporal , Peso Corporal , Neoplasias de la Mama/patología , Línea Celular Tumoral/trasplante , Conjuntos de Datos como Asunto , Dieta Alta en Grasa/efectos adversos , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/mortalidad , Ratones Obesos , Ratones Transgénicos , Recurrencia Local de Neoplasia/mortalidad , Neoplasia Residual , Obesidad/etiología , Receptor ErbB-2/genética , Análisis de SupervivenciaRESUMEN
Breast cancer mortality is principally due to recurrent tumors that arise from a reservoir of residual tumor cells that survive therapy. Remarkably, breast cancers can recur after extended periods of clinical remission, implying that at least some residual tumor cells pass through a dormant phase prior to relapse. Nevertheless, the mechanisms that contribute to breast cancer recurrence are poorly understood. Using a mouse model of recurrent mammary tumorigenesis in combination with bioinformatics analyses of breast cancer patients, we have identified a role for Notch signaling in mammary tumor dormancy and recurrence. Specifically, we found that Notch signaling is acutely upregulated in tumor cells following HER2/neu pathway inhibition, that Notch signaling remains activated in a subset of dormant residual tumor cells that persist following HER2/neu downregulation, that activation of Notch signaling accelerates tumor recurrence, and that inhibition of Notch signaling by either genetic or pharmacological approaches impairs recurrence in mice. Consistent with these findings, meta-analysis of microarray data from over 4,000 breast cancer patients revealed that elevated Notch pathway activity is independently associated with an increased rate of recurrence. Together, these results implicate Notch signaling in tumor recurrence from dormant residual tumor cells and provide evidence that dormancy is a targetable stage of breast cancer progression.
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Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/metabolismo , Receptor ErbB-2 , Receptores Notch/metabolismo , Transducción de Señal , Anciano , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Metaanálisis como Asunto , Ratones , Ratones Desnudos , Ratones Transgénicos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Notch/genética , Células Tumorales CultivadasRESUMEN
Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTC) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that ceramide kinase (Cerk) is required for mammary tumor recurrence following HER2/neu pathway inhibition and is spontaneously upregulated during tumor recurrence in multiple genetically engineered mouse models for breast cancer. We find that Cerk is rapidly upregulated in tumor cells following HER2/neu downregulation or treatment with Adriamycin and that Cerk is required for tumor cell survival following HER2/neu downregulation. Consistent with our observations in mouse models, analysis of gene expression profiles from more than 2,200 patients revealed that elevated CERK expression is associated with an increased risk of recurrence in women with breast cancer. In addition, although CERK expression is associated with aggressive subtypes of breast cancer, including those that are estrogen receptor-negative, HER2(+), basal-like, or high grade, its association with poor clinical outcome is independent of these clinicopathologic variables. Together, our findings identify a functional role for Cerk in breast cancer recurrence and suggest the clinical utility of agents targeted against this prosurvival pathway.
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Neoplasias de la Mama/genética , Neoplasias Mamarias Animales/genética , Recurrencia Local de Neoplasia/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Neoplasias de la Mama/patología , Supervivencia Celular/genética , Femenino , Humanos , Neoplasias Mamarias Animales/patología , Ratones , Recurrencia Local de Neoplasia/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptor ErbB-2RESUMEN
UNLABELLED: Breast cancer mortality is principally due to tumor recurrence; however, the molecular mechanisms underlying this process are poorly understood. We now demonstrate that the suppressor of cytokine signaling protein SPSB1 is spontaneously upregulated during mammary tumor recurrence and is both necessary and sufficient to promote tumor recurrence in genetically engineered mouse models. The recurrence-promoting effects of SPSB1 result from its ability to protect cells from apoptosis induced by HER2/neu pathway inhibition or chemotherapy. This, in turn, is attributable to SPSB1 potentiation of c-MET signaling, such that preexisting SPSB1-overexpressing tumor cells are selected for following HER2/neu downregulation. Consistent with this, SPSB1 expression is positively correlated with c-MET activity in human breast cancers and with an increased risk of relapse in patients with breast cancer in a manner that is dependent upon c-MET activity. Our findings define a novel pathway that contributes to breast cancer recurrence and provide the first evidence implicating SPSB proteins in cancer. SIGNIFICANCE: The principal cause of death from breast cancer is recurrence. This study identifies SPSB1 as a critical mediator of breast cancer recurrence, suggests activation of the SPSB1-c-MET pathway as an important mechanism of therapeutic resistance in breast cancers, and emphasizes that pharmacologic targets for recurrence may be unique to this stage of tumor progression.
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Neoplasias de la Mama/genética , Recurrencia Local de Neoplasia/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
MOTIVATION: Combinatorial therapies play increasingly important roles in combating complex diseases. Owing to the huge cost associated with experimental methods in identifying optimal drug combinations, computational approaches can provide a guide to limit the search space and reduce cost. However, few computational approaches have been developed for this purpose, and thus there is a great need of new algorithms for drug combination prediction. RESULTS: Here we proposed to formulate the optimal combinatorial therapy problem into two complementary mathematical algorithms, Balanced Target Set Cover (BTSC) and Minimum Off-Target Set Cover (MOTSC). Given a disease gene set, BTSC seeks a balanced solution that maximizes the coverage on the disease genes and minimizes the off-target hits at the same time. MOTSC seeks a full coverage on the disease gene set while minimizing the off-target set. Through simulation, both BTSC and MOTSC demonstrated a much faster running time over exhaustive search with the same accuracy. When applied to real disease gene sets, our algorithms not only identified known drug combinations, but also predicted novel drug combinations that are worth further testing. In addition, we developed a web-based tool to allow users to iteratively search for optimal drug combinations given a user-defined gene set. AVAILABILITY: Our tool is freely available for noncommercial use at http://www.drug.liuzlab.org/. CONTACT: zhandong.liu@bcm.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Programación Lineal , Algoritmos , Bases de Datos Factuales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Combinación de Medicamentos , Redes Reguladoras de Genes , Humanos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/genética , Diseño de SoftwareRESUMEN
Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.
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Proteínas Reguladoras de la Apoptosis/fisiología , Neoplasias de la Mama/etiología , Recurrencia Local de Neoplasia/etiología , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Miosinas Cardíacas/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Regulación hacia Abajo , Femenino , Humanos , Ratones , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Receptor ErbB-2/análisis , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Trithorax and polycomb group proteins antagonistically regulate the transcription of many genes, and cancer can result from the disruption of this regulation. Deregulation of trithorax function occurs through chromosomal translocations involving the trithorax gene MLL, leading to the expression of MLL fusion proteins and acute leukemia. It is poorly understood how MLL fusion proteins block differentiation, a hallmark of leukemogenesis. We analyzed the effect of acute depletion of menin, a close partner of MLL that is critical for MLL and MLL-AF9 recruitment to target genes, on MLL-AF9 leukemia cell differentiation using an in vivo model. We performed cDNA microarray analysis of menin-regulated genes from primary leukemia cells to determine menin-regulated pathways involved in suppressing MLL-AF9 leukemia cell differentiation. We found that menin binds the promoter of the polycomb gene Ezh2, and promotes its expression. EZH2 interacts with the differentiation-promoting transcription factor C/EBPα and represses C/EBPα target genes. Menin depletion reduces MLL binding to the Ezh2 locus, EZH2 expression, and EZH2 binding and repressive H3K27 methylation at C/EBPα target genes, thereby inducing the expression of pro-differentiation C/EBPα targets. In conclusion, our results show that in contrast to its classical role antagonizing trithorax function, the polycomb group protein EZH2 collaborates with trithorax-associated menin to block MLL-AF9 leukemia cell differentiation, uncovering a novel mechanism for suppression of C/EBPα and leukemia cell differentiation, through menin-mediated upregulation of EZH2.
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Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Leucemia/genética , Leucemia/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Activación TranscripcionalRESUMEN
MicroRNAs typically function at the level of posttranscriptional gene silencing within the cytoplasm; however, increasing evidence suggests that they may also function in nuclear, Argonaut-containing complexes, to directly repress target gene transcription. We have investigated the role of microRNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/ß, PERK, and ATF6, whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the proapoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter, where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 downregulation. Our data suggest a model in which PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment.
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Regulación de la Expresión Génica , MicroARNs/genética , Factor de Transcripción CHOP/genética , eIF-2 Quinasa/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Estrés del Retículo Endoplásmico/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Metilación , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Células 3T3 NIH , Fosforilación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tapsigargina/farmacología , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. The oxygen-sensitive hypoxia inducible factor (HIF) transcriptional regulators HIF-1alpha and HIF-2alpha are overexpressed in many human NSCLCs, and constitutive HIF-2alpha activity can promote murine lung tumor progression, suggesting that HIF proteins may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted Hif-1alpha or Hif-2alpha in an established Kras(G12D)-driven murine NSCLC model. Deletion of Hif-1alpha had no obvious effect on tumor growth, whereas Hif-2alpha deletion resulted in an unexpected increase in tumor burden that correlated with reduced expression of the candidate tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 as a direct HIF-2alpha target gene and demonstrate that HIF-2alpha regulates Scgb3a1 expression and tumor formation in human Kras(G12D)-driven NSCLC cells. AKT pathway activity, reported to be repressed by Scgb3a1, was enhanced in HIF-2alpha-deficient human NSCLC cells and xenografts. Finally, a direct correlation between HIF-2alpha and SCGB3a1 expression was observed in approximately 70% of human NSCLC samples analyzed. These data suggest that, whereas HIF-2alpha overexpression can contribute to NSCLC progression, therapeutic inhibition of HIF-2alpha below a critical threshold may paradoxically promote tumor growth by reducing expression of tumor suppressor genes, including Scgb3a1.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma de Pulmón de Células no Pequeñas/etiología , Eliminación de Gen , Proteínas Proto-Oncogénicas/fisiología , Proteínas ras/fisiología , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Genes Supresores de Tumor , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas p21(ras) , Trasplante Heterólogo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The Extracellular signal Regulated Kinase (ERK) pathway is one of the most well-studied signaling pathways in cell cycle regulation. Disruption in the normal functioning of this pathway is linked to many forms of cancer. In a previous study [D.K. Pant, A. Ghosh, Automated oncogene detection in complex protein networks, with applications to the MAPK signal transduction pathway, Biophys. Chem. 113 (2005) 275-288.], we developed a novel approach to predict single point mutations that are likely to cause cellular transformation in signaling transduction networks. We have extended this method to study disparate pair mutation in enzyme/protein interactions and in expression levels in signal transduction pathway and have applied it to the MAPK signaling pathway to study how synergistic or cooperative mutation within signaling networks acts in unison to cause malignant transformation. The method provides a quantitative ranking of the modifier pair of ERK activation. It is seen that the highest ranking single point mutations comprise the highest ranking pair mutations. We validate some of our results with experimental literature on multiple mutations. A second order sensitivity analysis scheme is additionally used to determine the effect of correlations among mutations at different sites in the pathways.
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Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oncogenes/fisiología , Transducción de Señal/fisiología , Biología de Sistemas/métodos , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Cinética , Mutación , Neoplasias/metabolismoRESUMEN
Activation of the extracellular signal-regulated kinases (ERK1/2; p42/p44 mitogen-activated protein kinase (MAPK)) is one of the most extensively studied signaling pathways not least because it occurs downstream of oncogenic RAS. Here, we take advantage of the wealth of experimental data available on the canonical RAS/RAF/MEK/ERK pathway of Bhalla et al. to test the utility of a newly developed nonlinear analysis algorithm designed to predict likelihood of cellular transformation. By using ERK phosphorylation as an "output signal", the method analyzes experimentally determined kinetic data and predicts putative oncogenes and tumor suppressor gene products impacting the RAS/MAPK module using a purely theoretical approach. This analysis identified several modifiers of ERK/MAPK activation described previously. In addition, several novel enzymes are identified which are not previously described to affect ERK/MAPK phosphorylation. Importantly, the nonlinear analysis enables a ranking of modifiers of MAPK activation predicting their relative importance in RAS-dependent oncogenesis. The results are compared with a linearized analysis based on sensitivity analysis about the steady state or metabolic control analysis (MCA). The results are favorable, pointing to the utility of first-order sensitivity analysis and MCA in the analysis of complex signaling networks for oncogenes.