RESUMEN
Semaphorin 3B (SEMA3B) is a secreted axonal guidance molecule that is expressed during development and throughout adulthood. Recently, SEMA3B has emerged as a tumor suppressor in non-neuronal cells. Here, we show that SEMA3B is a direct target of GATA3 transcriptional activity. GATA3 is a key transcription factor that regulates genes involved in mammary luminal cell differentiation and tumor suppression. We show that GATA3 relies on SEMA3B for suppression of tumor growth. Loss of SEMA3B renders GATA3 inactive and promotes aggressive breast cancer development. Overexpression of SEMA3B in cells lacking GATA3 induces a GATA3-like phenotype and higher levels of SEMA3B are associated with better cancer patient prognosis. Moreover, SEMA3B interferes with activation of LIM kinases (LIMK1 and LIMK2) to abrogate breast cancer progression. Our data provide new insights into the role of SEMA3B in mammary gland and provides a new branch of GATA3 signaling that is pivotal for inhibition of breast cancer progression and metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Factor de Transcripción GATA3/metabolismo , Glicoproteínas de Membrana/metabolismo , Semaforinas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Quinasas Lim/metabolismo , Glicoproteínas de Membrana/genética , Ratones Desnudos , Metástasis de la Neoplasia , Pronóstico , Semaforinas/genética , Transducción de SeñalRESUMEN
Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/fisiología , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Factor de Transcripción E2F1/fisiología , Transición Epitelial-Mesenquimal , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Activación TranscripcionalRESUMEN
Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.
Asunto(s)
Neoplasias Pulmonares/terapia , Neoplasias Experimentales/inducido químicamente , Proteínas Proto-Oncogénicas p21(ras)/genética , Vacunas de ADN/administración & dosificación , Animales , Doxiciclina , Vectores Genéticos/administración & dosificación , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/inmunología , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida , Mutación , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Plásmidos/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Vacunas de ADN/farmacologíaRESUMEN
Histone deacetylase (HDAC) inhibitors are used in treating certain human malignancies. Our laboratories demonstrated their capability in enhancing antitumor effect of DNA vaccine driven by an RNA polymerase II (RNA pol II) promoter. However, it is unknown whether HDAC inhibitors enhance the therapeutic short hairpin RNA (shRNA) expressed by an RNA polymerase III (RNA pol III) promoter. We investigated whether HDAC inhibitors augmented antitumor effect of indoleamine 2,3 dioxygenase (IDO) shRNA. HDAC inhibitor OSU-HDAC42 and suberoylanilide hydroxamic acid enhanced RNA pol III-driven U6 and H1 promoter activity in three different cell types in vitro: 293, NIH3T3 and dendritic cell line DC2.4. Subcutaneous injection of OSU-HDAC42 enhanced U6 and H1 promoter activity on abdominal skin of mice in vivo. Combination of IDO shRNA and OSU-HDAC42 increased antitumor effect of IDO shRNA in MBT-2 murine bladder tumor model. IDO shRNA induced tumor-infiltrating CD8⺠and CD4⺠T cells, whereas OSU-HDAC42 treatment induced tumor-infiltrating CD4⺠T cells. Combination of OSU-HDAC42 and IDO shRNA further induced tumor-infiltrating natural killer cells and enhanced interferon-γ in lymphocytes, but suppressed interleukin (IL)-4 expression of lymphocytes. In addition, OSU-HDAC42 treatment did not alter mRNA expression of IL-12 and tumor necrosis factor-α. In conclusion, HDAC inhibitor OSU-HDAC42 may serve as adjuvant of the therapeutic shRNA expressed by an RNA pol III promoter.
Asunto(s)
Neoplasias/tratamiento farmacológico , Fenilbutiratos/administración & dosificación , ARN Polimerasa III/genética , Vacunas de ADN/administración & dosificación , Animales , Células Dendríticas/efectos de los fármacos , Sinergismo Farmacológico , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genéticaRESUMEN
The cytomegalovirus (CMV) promoter is considered to be one of the strongest promoters for driving the in vivo expression of genes encoded by DNA vaccines. However, the efficacy of DNA vaccines has so far been disappointing (particularly in humans), and this might be explained in part by histone deacetylase (HDAC)-mediated chromatin condensation. Hence, we sought to investigate whether increasing the expression of DNA vaccine antigens with the HDAC inhibitor OSU-HDAC42 would enhance the efficacy of DNA vaccines in vivo. A luciferase assay was used to determine the effects of OSU-HDAC42 on CMV promoter-driven DNA plasmids in vitro and in vivo. Three HDAC inhibitors were able to activate expression from the CMV promoter in NIH3T3 cells and MBT-2 bladder cancer cells. The expression of luciferase was significantly enhanced by co-administration of pCMV-luciferase and OSU-HDAC42 in mice. To explore whether OSU-HDAC42 could enhance the specific antitumor activity of a neu DNA vaccine driven by the CMV promoter, we evaluated therapeutic effects and immune responses in a mouse tumor natively overexpressing HER2/neu. Mice receiving OSU-HDAC42 in combination with the CMV-promoter neu DNA vaccine exhibited stronger antitumor effects than mice given the DNA vaccine only. In addition, a correlation between the antitumor effects and the specific cellular immune responses was observed in the mice receiving the DNA vaccine and OSU-HDAC42.
Asunto(s)
Citomegalovirus/genética , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias/terapia , Fenilbutiratos/farmacología , Fenilbutiratos/uso terapéutico , Vacunas de ADN/genética , Vacunas de ADN/uso terapéutico , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
The biggest challenge to gene therapy is how to efficiently deliver the desired therapeutic gene into a sufficient number of recipient cells to achieve significant clinical efficacy. Here, we identified a partially purified extract from rat muscle probably containing myoblast specific fusion factor(s) (MSF), which significantly enhanced fusion of donor myoblast with host muscle fibers. Once incorporated, the introduced genetic construct could instruct the machinery of the hybrid cells to express the desired protein(s). Rat satellite cells containing a plasmid carrying a marker bone morphogenetic protein-4 (BMP-4) coding sequence were used as foreign gene delivery vehicle. BrdU labeling of the MSF-pretreated satellite cells allowed tracing the fate of the genetically modified satellite cells in the host muscles. Immunohistochemistry using anti-BMP-4 antibody demonstrated the translation of the introduced gene construct. It was demonstrated that in the presence of MSF, numerous BrdU positive nuclei and the expression of BMP-4 polypeptides could be observed in host hybrid fibers, while in the control group using rat serum to replace MSF containing fraction, only a few BrdU positive signals were detected. The expression of osteocalcin and the elevated alkaline phosphatase activity detected in the hybrid fibers indicated the proper folding, secretion and, post-translational modification of the expressed foreign protein. This strategy of enhanced myoblast-mediated gene transfer would break the major barrier in current practice of normal or engineered myoblast transplantation in the management of genetic muscle diseases or systemic genetic disorders.