RESUMEN
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/ß-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Quimioterapia Combinada , Neoplasias Pulmonares/terapia , Escape del Tumor/efectos de los fármacos , Animales , Presentación de Antígeno/efectos de los fármacos , Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Inmunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Ratones , Linfocitos T/inmunología , Transcriptoma , Microambiente TumoralRESUMEN
We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Interferón Tipo I/inmunología , Melanoma/inmunología , Melanoma/terapia , Animales , Azacitidina/farmacología , Línea Celular Tumoral , Metilasas de Modificación del ADN/antagonistas & inhibidores , Retrovirus Endógenos/genética , Femenino , Humanos , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , ARN Bicatenario/metabolismoRESUMEN
Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.
Asunto(s)
Epigénesis Genética , Terapia Genética , Células Supresoras de Origen Mieloide/fisiología , Neoplasias/terapia , Microambiente Tumoral , Animales , Azacitidina/farmacología , Benzamidas/farmacología , Diferenciación Celular , Movimiento Celular/efectos de los fármacos , Quimioterapia Adyuvante , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Ratones , Células Supresoras de Origen Mieloide/citología , Metástasis de la Neoplasia/terapia , Neoplasias/cirugía , Piridinas/farmacología , Receptores CCR2/genética , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efectos de los fármacosRESUMEN
To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.
Asunto(s)
COVID-19/patología , Animales , COVID-19/virología , Cricetinae , Modelos Animales de Enfermedad , Femenino , Pulmón/patología , Masculino , Mesocricetus , SARS-CoV-2RESUMEN
Despite recent progress, the physiological role of Hippo signaling in mammary gland development and tumorigenesis remains poorly understood. Here we show that the Hippo pathway is functionally dispensable in virgin mammary glands but specifically required during pregnancy. In contrast to many other tissues, hyperactivation of YAP in mammary epithelia does not induce hyperplasia but leads to defects in terminal differentiation. Interestingly, loss of YAP causes no obvious defects in virgin mammary glands but potently suppresses oncogene-induced mammary tumors. The selective requirement for YAP in oncogenic growth highlights the potential of YAP inhibitors as molecular targeted therapies against breast cancers.
Asunto(s)
Carcinogénesis/patología , Diferenciación Celular , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Femenino , Vía de Señalización Hippo , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones Endogámicos C57BL , Ratones Transgénicos , Oncogenes/genética , Embarazo , TiempoRESUMEN
Triple-negative breast cancer (TNBC) is treated with cytotoxic chemotherapy and is often characterized by early relapse and metastasis. To form a secondary (recurrent and/or metastatic) tumor, a breast cancer cell must evade the innate and adaptive immune systems. CD47 enables cancer cells to evade killing by macrophages, whereas CD73 and PDL1 mediate independent mechanisms of evasion of cytotoxic T lymphocytes. Here, we report that treatment of human or murine TNBC cells with carboplatin, doxorubicin, gemcitabine, or paclitaxel induces the coordinate transcriptional induction of CD47, CD73, and PDL1 mRNA and protein expression, leading to a marked increase in the percentage of CD47+CD73+PDL1+ breast cancer cells. Genetic or pharmacological inhibition of hypoxia-inducible factors (HIFs) blocked chemotherapy-induced enrichment of CD47+CD73+PDL1+ TNBC cells, which were also enriched in the absence of chemotherapy by incubation under hypoxic conditions, leading to T cell anergy or death. Treatment of mice with cytotoxic chemotherapy markedly increased the intratumoral ratio of regulatory/effector T cells, an effect that was abrogated by HIF inhibition. Our results delineate an HIF-dependent transcriptional mechanism contributing to TNBC progression and suggest that combining chemotherapy with an HIF inhibitor may prevent countertherapeutic induction of proteins that mediate evasion of innate and adaptive antitumor immunity.
Asunto(s)
5'-Nucleotidasa/metabolismo , Antineoplásicos/farmacología , Antígeno B7-H1/metabolismo , Antígeno CD47/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias de la Mama Triple Negativas/inmunología , Escape del Tumor/inmunología , 5'-Nucleotidasa/genética , Animales , Apoptosis , Antígeno B7-H1/genética , Antígeno CD47/genética , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.
Asunto(s)
Epigénesis Genética/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Interferón Tipo I/metabolismo , Neoplasias Ováricas/etiología , Neoplasias Ováricas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos , Azacitidina/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Stem-like cells in human cancer (CSCs) have multiple properties of embryonic stem cells (ESCs). In this issue, Iliopoulos et al. (2010) identify molecular mechanisms that link miRNAs to epigenetic states and drive CSCs, contributing to tumor aggressiveness.
RESUMEN
Cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers exemplify such characteristics, with BRAFV600E predominantly occurring in proximal colon cancers along with increased DNA methylation phenotype. Using mouse colon organoids, here we show that proximal and distal colon stem cells have distinct transcriptional programs that regulate stemness and differentiation. We identify that the homeobox transcription factor, CDX2, which is silenced by DNA methylation in proximal colon cancers, is a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program concurrently is tumor suppressive, and Cdx2 loss sufficiently creates permissive state for BRAFV600E-driven transformation. Human proximal colon cancers with CDX2 downregulation showed similar transcriptional program as in mouse proximal organoids with Cdx2 loss. Developmental transcription factors, such as CDX2, are thus critical in maintaining tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor development.
Asunto(s)
Neoplasias del Colon , Proteínas Proto-Oncogénicas B-raf , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas B-raf/genética , Factor de Transcripción CDX2/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteínas de Unión al ADN , Factores de Transcripción/genética , Proteínas de Homeodominio/genéticaRESUMEN
BACKGROUND: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure. RESULTS: We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis. CONCLUSIONS: Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.
Asunto(s)
Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal , Fumar/efectos adversos , Animales , Mama/metabolismo , Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre/metabolismoRESUMEN
Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinß (C/EBPß) in a variety of tumor cell lines. C/EBPß activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPß inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPß knock-down. Therefore, C/EBPß mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Linfoma de Burkitt , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Pirazinas/farmacología , Elementos de Respuesta , Activación Viral/efectos de los fármacos , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 6/metabolismo , Bortezomib , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/virología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Factores de Transcripción del Factor Regulador X , Tapsigargina/farmacología , Transactivadores/biosíntesis , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteína 1 de Unión a la X-BoxRESUMEN
NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.
RESUMEN
NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.
Asunto(s)
Carcinoma , Proteínas Nucleares , Animales , Humanos , Ratones , Carcinoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Genes Supresores de Tumor , Histonas/metabolismo , Recurrencia Local de Neoplasia/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The gene hypermethylated in cancer 1 (HIC1) is epigenetically inactivated, but not mutated, in cancer. Here we show that cooperative loss of Hic1 with p53, but not INK4a, yields distinct tumor phenotypes in mice. Germline deletion of one allele of each gene on the opposite chromosome yields breast and ovarian carcinomas and metastatic osteosarcomas with epigenetic inactivation of the wild-type Hic1 allele. Germline deletion of the two genes on the same chromosome results in earlier appearance and increased prevalence and aggressiveness of osteosarcomas with genetic deletion of both wild-type genes. In human osteosarcomas, hypermethylation of HIC1 is frequent only in tumors with p53 mutations. Our results indicate the importance of genes altered only through epigenetic mechanisms in cancer progression in conjunction with genetically modified tumor suppressor genes.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Epigénesis Genética/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Deleción Cromosómica , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Genes Supresores de Tumor , Heterocigoto , Humanos , Inmunohistoquímica , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Noqueados , Mutación/genética , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Osteosarcoma/genética , Osteosarcoma/patología , Fenotipo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: The transcription factor, CCAAT enhancer binding protein-ß (C/EBPß), is expressed as several distinct protein isoforms (LAP1, LAP2 and LIP) that have opposing actions in cellular proliferation and differentiation. Increases in the ratio of LIP/LAP are associated with aggressive, metastatic breast cancer; however, little is known regarding the molecular mechanisms that regulate LIP expression or the biological actions of an increase in the LIP/LAP ratio. Metastasis is highly dependent upon the suppression of anoikis and the role of C/EBPß and LIP in this anchorage-independent, survival process is currently not known in mammary epithelial cells. IGF-1R signaling is important for the survival of breast cancer cells and crosstalk between IGF-1R and EGFR signaling pathways have been implicated in the development of more aggressive disease. We therefore evaluated in mammary epithelial cells whether IGF-1R signaling regulates the LIP/LAP ratio, analyzed the potential interplay between EGFR and IGF-1R signaling and addressed the biological significance of increased LIP expression in cellular survival and suppression of anoikis. RESULTS: Our data provide the first evidence that IGF-1R signaling regulates LIP expression in an EGFR independent manner to increase the LIP/LAP ratio in mammary epithelial cells. Although crosstalk between IGF-1R signaling and EGFR signaling is detectable in MCF10A cells, this crosstalk is not required for the IGF-1 mediated regulation of LIP expression. Rather, the critical regulator of IGF-1 induced LIP expression appears to be EGFR-independent, Akt activity. Our data also demonstrate that increases in LIP expression promote cell survival via suppression of anoikis. Likewise, knockdown of total C/EBPß leads to increased cell death and suggest that C/EBPß expression is important for survival and resistance to anoikis. IGF-1 treatment can partially rescue vector control cells from anoikis; however, cells with reduced C/EBPß expression do not survive anoikis. CONCLUSIONS: Taken together, our data demonstrate that IGF-1R signaling regulates LIP expression in an EGFR independent manner to increase the LIP/LAP ratio in mammary epithelial cells. C/EBPß expression and elevations in LIP play an important role in regulating cellular survival via suppression of anoikis, in an IGF-1R mediated context or in a manner independent of IGF-1R signaling.
Asunto(s)
Anoicis , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Receptor IGF Tipo 1/fisiología , Transducción de Señal , Neoplasias de la Mama , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Supervivencia Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Expresión Génica , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/farmacologíaRESUMEN
BACKGROUND: The flank is commonly used for primary xenografts in mice, but it is rare for these tumors to metastasize. Tail vein injection creates a pattern of metastases, but is artificial. We hypothesized that the liver is a convenient alternative xenograft site and that metastases would gradually proceed spontaneously. MATERIALS AND METHODS: Using 15 NOD.CB17-Prkdc(scid)/NcrCrl (NOD/SCID) mice, 10,000 A549 cells were xenografted into the liver while a second group of five mice were xenografted in the flank with 100,000 A549 cells as a control. Mice were euthanized and grossly dissected at 7 wk. A third group of seven mice received liver xenografts with A549 and a mouse each week was euthanized for 7 wk and evaluated. The liver, lung, and spleen were examined histologically. RESULTS: At 7 wk, 15/15 liver xenografted mice had gross primary tumor in the liver. Histologic review confirmed multiple microscopic foci of metastatic disease in all mice (15/15) throughout the lungs, mediastinal nodes, and spleen. The control group had primary tumor in the flank (4/5), but none had histologic evidence of metastases. Serially euthanized liver xenografted mice revealed evidence of a gradual spontaneous metastatic model system with the first histologic findings of micrometastases appearing in the lungs by wk 5, which became wide spread by wk 7. Splenic and mediastinal lymph node metastases developed in wk 6 and 7. CONCLUSIONS: Liver xenografting of A549 cells into NOD/SCID mice is a reliable way of developing widespread micrometastases. This model allows the study of a gradually developing solid tumor with subsequent metastatic spread.