RESUMEN
ID proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. ID proteins inhibit basic-HLH transcription factors often blocking differentiation and sustaining proliferation. A small-molecule, AGX51, targets ID proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impairs cell growth and viability that results from an increase in reactive oxygen species (ROS) production upon ID degradation. In mouse models, AGX51 treatment suppresses breast cancer colonization in the lung, regresses the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduces tumor burden in sporadic colorectal neoplasia. Furthermore, in cells and mice, we fail to observe acquired resistance to AGX51 likely the result of the inability to mutate the binding pocket without loss of ID function and efficient degradation of the ID proteins. Thus, AGX51 is a first-in-class compound that antagonizes ID proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.
RESUMEN
BACKGROUND: As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses. METHODS: The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing. RESULTS: Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement. CONCLUSION: Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.
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Perfilación de la Expresión Génica/métodos , Luminiscencia , Mediciones Luminiscentes/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Ováricas/genética , Biomarcadores de Tumor , Línea Celular Tumoral , Sondas de ADN , Interpretación Estadística de Datos , Femenino , Expresión Génica , Perfilación de la Expresión Génica/instrumentación , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Células HCT116 , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neovascularización Patológica/metabolismoRESUMEN
Polymeric nanocarriers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAM) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b+ TAMs costained with pan-macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development. Mol Cancer Ther; 16(12); 2701-10. ©2017 AACR.
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Neoplasias de la Mama/genética , Macrófagos/metabolismo , Metacrilatos/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , PolímerosRESUMEN
The frequent loss of chromosome 17 in epithelial ovarian carcinomas (EOC), particularly high-grade serous carcinomas (HGSC), has been attributed to the disruption of TP53 (at 17p13.1) and other chromosome 17 genes suspected to play a role in tumour suppressor pathways. In a transcriptome analysis of HGSC, we showed underexpression of a number of chromosome 17 genes, which included FKBP10 (at 17q21.1) and collagen I α 1 (COL1A1; at 17q21.33). FKBP10 codes for the immunophilin FKBP65 and is suspected to act as a chaperone for COL1A1. We have investigated FKBP10 (gene) and FKBP65 (protein) expression in HGSC samples and EOC cell lines that differ in their tumourigenic potential. COL1A1 expression was also investigated given the purported function of FKBP65. RT-PCR analysis verified underexpression of FKBP10 and COL1A1 in HGSCs (n=14) and six tumourigenic EOC cell lines, relative to normal ovarian surface epithelial cells and a non-tumourigenic EOC cell line. Immunohistochemistry analyses of 196 HGSC samples using tissue microarrays revealed variable staining intensities in the epithelial tumour component where only 7.8% and 1.0% of samples stained intensely for FKBP65 and COL1A1, respectively. Variable staining intensities were also observed for the stromal component where 23.6% and 24.1% stained intensely for FKBP65 and COL1A1, respectively. There was no significant correlation of staining intensity of either protein with disease stage. Staining of FKBP65 was clearly visible in normal epithelial cells of the ovarian surface and fallopian tube. There was a significant correlation between absence of FKBP65 staining in the epithelial cell component of the tumour and prolonged overall survival (p<0.001). Our results suggest that underexpression of FKBP65 protein is characteristic of HGSCs and that this expression profile may be linked to molecular pathways associated with an unfavourable outcome in cancer patients.
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Cromosomas Humanos Par 17/genética , Colágeno Tipo I/biosíntesis , Cistadenocarcinoma Seroso , Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Proteínas de Unión a Tacrolimus/biosíntesis , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Deleción Cromosómica , Cadena alfa 1 del Colágeno Tipo I , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/mortalidad , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Síndrome de Smith-Magenis , Sobrevida , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.
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Genes Supresores de Tumor , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ovario/patología , Factores de Transcripción/genética , Animales , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular , Células Clonales , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Ovario/metabolismo , Fenotipo , Factores de Transcripción/análisisRESUMEN
It has been proposed that the frequent loss of heterozygosity (LOH) of an entire chromosome 17 contig in epithelial ovarian cancers (EOC) is the consequence of the inactivation of multiple tumour suppressor genes on this chromosome. We report the characterization of a 453 Kb 17q25 locus shown previously to exhibit a high frequency of LOH in EOC samples. LOH analysis further defined the minimal region of deletion to a 65 Kb interval flanked by D17S2239 and D17S2244, which contains RHBDF2, CYGB and PRCD as tumour suppressor gene candidates. Tissue specific expression excluded PRCD as a candidate. RHBDF2 was expressed at low levels in the majority of benign and low malignant potential (LMP) tumours, and in a subset of malignant ovarian tumour samples, as compared with primary cultures of normal ovarian surface epithelial cell (NOSE) samples. CYGB was expressed at low levels in the majority of LMP and malignant samples compared with benign and NOSE samples. In contrast to CYGB expression, RHBDF2 was expressed at low or undetectable levels in EOC cell lines exhibiting tumourigenic characteristics and up-regulated in a genetically modified EOC cell line rendered non-tumourigenic. DNA sequence analysis identified variants but no apparent deleterious mutations in either gene. Methylation-specific PCR analysis suggested that promoter methylation of CYGB but not RHBDF2 occurred in 6 of 31 malignant samples. The results combined suggest that RHBDF2 and CYGB may play distinctive roles in ovarian cancer and could be added to the growing roster of chromosome 17 genes implicated in this disease.
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Cromosomas Humanos Par 17/genética , Genes Supresores de Tumor , Globinas/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Secuencia de Aminoácidos , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Secuencia Conservada , Citoglobina , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Globinas/metabolismo , Humanos , Pérdida de Heterocigocidad , Datos de Secuencia Molecular , Mutación Missense , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.