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BACKGROUND: The mutational status of ovarian cancer cell line IGROV-1 is inconsistent across the literature, suggestive of multiple clonal populations of the cell line. IGROV-1 has previously been categorised as an inappropriate model for high-grade serous ovarian cancer. METHODS: IGROV-1 cells were obtained from the Netherlands Cancer Institute (IGROV-1-NKI) and the MD Anderson Cancer Centre (IGROV-1-MDA). Cell lines were STR fingerprinted and had their chromosomal copy number analysed and BRCA1/2 genes sequenced. Mutation status of ovarian cancer-related genes were extracted from the literature. RESULTS: The IGROV-1-NKI cell line has a tetraploid chromosomal profile. In contrast, the IGROV-1-MDA cell line has pseudo-normal chromosomes. The IGROV-1-NKI and IGROV-MDA are both STR matches (80.7% and 84.6%) to the original IGROV-1 cells isolated in 1985. However, IGROV-1-NKI and IGROV-1-MDA are not an STR match to each other (78.1%) indicating genetic drift. The BRCA1 and BRCA2 gene sequences are 100% identical between IGROV-1-MDA and IGROV-1-NKI, including a BRCA1 heterozygous deleterious mutation. The IGROV-1-MDA cells are more resistant to cisplatin and olaparib than IGROV-1-NKI. IGROV-1 has a mutational profile consistent with both Type I (PTEN, PIK3CA and ARID1A) and Type II ovarian cancer (BRCA1, TP53) and is likely to be a Type II high-grade serous carcinoma of the SET (Solid, pseudo-Endometroid and Transitional cell carcinoma-like morphology) subtype. CONCLUSIONS: Routine testing of chromosomal copy number as well as the mutational status of ovarian cancer related genes should become the new standard alongside STR fingerprinting to ensure that ovarian cancer cell lines are appropriate models.
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Mutação , Neoplasias Ovarianas , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Humanos , Linhagem Celular Tumoral , Mutação/genética , Variações do Número de Cópias de DNA/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Dosagem de GenesRESUMO
BACKGROUND: Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance. METHODS: We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins. RESULTS: The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11-0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl-2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells. CONCLUSION: This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.
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Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proliferação de Células , Quinase 4 Dependente de CiclinaRESUMO
To prevent the development of endocrine-resistant breast cancer, additional targeted therapies are increasingly being trialled in combination with endocrine therapy. The molecular mechanisms facilitating cancer cell survival during endocrine treatment remain unknown but could help direct selection of additional targeted therapies. We present a novel proteomic timecourse dataset, profiling potential drug targets in a population of MCF7 cells during 1 year of tamoxifen treatment. Reverse phase protein arrays profiled >70 proteins across 30 timepoints. A biphasic response to tamoxifen was evident, which coincided with changes in growth rate. Tamoxifen strongly impeded cell growth for the first 160 days, followed by gradual growth recovery and eventual resistance development. The growth-impeded phase was distinguished by the phosphorylation of Stat3 (y705) and Src (y527). Tumour tissue from patients treated with neo-adjuvant endocrine therapy (<4 months) also displayed increased Stat3 and Src signalling. Inhibitors of Stat3 (napabucasin) and Src (dasatinib), were effective at killing tamoxifen-treated MCF7 and T47D cells. Sensitivity to both drugs was significantly enhanced once tamoxifen had induced the growth-impeded phase. This novel proteomic resource identifies key mechanisms enabling cell survival during tamoxifen treatment. It provides valuable insight into potential drug combinations and timing that may prevent the development of endocrine resistance.
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Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Proteômica , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Células MCF-7 , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. METHODS: The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. RESULTS: Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. CONCLUSION: A clinical trial may be justified to further investigate the utility of IAP inhibitors.
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Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Apoptose , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
BACKGROUND: Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear. METHODS: Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies. RESULTS: Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM-1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05). CONCLUSIONS: PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.
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Neoplasias Gástricas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Lapatinib , Fosfatidilinositol 3-Quinases , Receptor ErbB-2/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Trastuzumab/farmacologia , Trastuzumab/uso terapêuticoRESUMO
BACKGROUND: Activation of the phosphoinositide-3 kinase (PI3K) pathway is a resistance mechanism to anti-human epidermal growth factor receptor 2 (HER2) therapy. This phase Ib trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an intravenous (IV) pan-class I PI3K inhibitor, combined with trastuzumab. METHODS: Patients with advanced HER2-positive breast cancer and disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with copanlisib (45 or 60 mg) IV on days 1, 8 and 15 of a 28-day cycle with a fixed dose of trastuzumab 2 mg/kg weekly. RESULTS: Twelve patients were enrolled. The MTD was determined as copanlisib 60 mg plus trastuzumab 2 mg/kg weekly. The most common adverse events of any grade occurring in more than two patients were hyperglycaemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Stable disease was confirmed at 16 weeks in six participants (50%). PIK3CA mutations were detected in archival tumour of six participants (50%). PIK3CA hotspot mutations, were detectable in pre- and on-treatment plasma of all participants. Pre- and post-treatment tumour biopsies for two patients identified temporal genomic heterogeneity, somatic mutations in the TRRAP gene, which encodes a PI3K-like protein kinase, and emergent somatic mutations related to protein kinase signalling. CONCLUSION: Copanlisib and trastuzumab can be safely administered with fair overall tolerability. Preliminary evidence of tumour stability was observed in patients with heavily pre-treated, metastatic HER2 positive breast cancer. Several potential biomarkers were identified for further study in the current phase 2 clinical trial. NCT: 02705859.
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Objective: The therapeutic benefits of poly(ADP-ribose) polymerase inhibitors highlight the need to evaluate BRCA1/2 defects in tubal/ovarian cancer (OC). We sought to determine the pattern and disease characteristics associated with tumor BRCA1/2 mutations and BRCA1 methylation in women with OC. Methods: We obtained 111 OC specimens from 2 university hospitals and assessed BRCA1/2 mutations and BRCA1 methylation in tumor DNA. The frequency and pattern of BRCA1/2 defects were examined. Associations between patient/disease characteristics and BRCA1/2 defects were ascertained (Fisher's exact test). Platinum-free interval (PFI), progression-free survival (PFS), and overall survival (OS) based on the underlying BRCA1/2 defect were determined (Kaplan-Meier analysis [log-rank test]). Results: We observed a BRCA1/2 dysfunction rate of 40% (28/70) in high-grade serous tubal/ovarian cancer (HGSC), including 14.3% BRCA1 methylation (n=10), 7.1% BRCA1 mutation (n=5), and 18.6% BRCA2 mutation (n=13). Defects in BRCA1/2 genes were associated with stage III/IV HGSC (BRCA1 methylation: P=0.005 [stage III/IV] and P=0.004 [HGSC]; BRCA1/2 mutation: P=0.03 [stage III/IV] and P<0.001 [HGSC]). Patients with BRCA1/2-mutated cancers showed improved OS (hazard ratio [HR], 0.65; 95% confidence interval [CI], 0.43-0.99; P=0.045) and a trend toward improved PFI (HR, 0.48; 95% CI, 0.22-1.06; P=0.07) and PFS (HR, 0.72; 95% CI, 0.51-1.03; P=0.07). No survival differences were observed between BRCA1-methylated and BRCA1/2 wild-type non-BRCA1-methylated cancers. Conclusion: We observed a high tumor BRCA1/2 dysfunction rate in HGSC with a unique predominance of BRCA2 over BRCA1 mutations. While BRCA1/2 mutations conferred survival benefits in OC, no such association was observed with BRCA1 methylation.
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BACKGROUND: An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS: The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS: Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS: Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.
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Antineoplásicos , Neoplasias Colorretais , Antineoplásicos/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/genética , Humanos , Masculino , Mutação/genética , Oncogenes/genética , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.
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Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína BRCA1/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Metilação de DNA/genética , Análise Mutacional de DNA , Proteína p300 Associada a E1A/genética , Feminino , Humanos , Neoplasias Ovarianas/genética , Poli(ADP-Ribose) Polimerase-1/genética , Regiões Promotoras Genéticas/genética , Temozolomida/farmacologiaRESUMO
We designed and synthesised a series of novel chalcones, incorporating the heterocyclic framework of 2,2-dimethyl-2,3-dihydro-4(1H)-quinolinone, which was prepared via Sonogashira coupling of a substituted orthoaniline under aqueous conditions using Pd catalysis followed by acid-mediated cyclisation. The compounds were screened against the NCI-N87 and DLD-1 cancer cell lines, with most compounds showing low micromolar cytotoxic activity.
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Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Mutação , Neoplasias Ovarianas/genética , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Perda de Heterozigosidade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
HNF1A-maturity onset diabetes of the young (HNF1A-MODY) is caused by mutations in Hnf1a gene encoding the transcription factor hepatocyte nuclear factor 1alpha (HNF1A). An increased rate of apoptosis has been associated with the decrease in beta-cell mass that is a hallmark of HNF1A-MODY and other forms of diabetes. In a cellular model of HNF1A-MODY, we have recently shown that signalling through mammalian target of rapamycin (mTOR) is decreased by the overexpression of a dominant-negative mutant of HNF1A (DN-HNF1A). mTOR is a protein kinase which has important roles in cell metabolism and growth, but also in cell survival, where it has been shown to be both protective and detrimental. Here, we show that pharmacological inhibition of mTOR activity with rapamycin protected INS-1 cells against DN-HNF1A-induced apoptosis. Rapamycin also prevented DN-HNF1A-induced activation of AMP-activated protein kinase (AMPK), an intracellular energy sensor which we have previously shown to mediate DN-HNF1A-induced apoptosis. Conversely, activation of mTOR with leucine potentiated DN-HNF1A-induced apoptosis. Gene silencing of raptor (regulatory associated protein of mTOR), a subunit of mTOR complex 1 (mTORC1), also conferred protection on INS-1 cells against DN-HNF1A-induced apoptosis, confirming that mTORC1 mediates the protective effect. The potential relevance of this effect with regards to the clinical use of rapamycin as an immunosuppressant in diabetics post-transplantation is discussed.
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Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Diabetes Mellitus Tipo 1/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Dominantes , Fator 1-alfa Nuclear de Hepatócito/genética , Células Secretoras de Insulina/citologia , Insulinoma/genética , Insulinoma/patologia , Leucina/farmacologia , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A-maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.
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Proteína Morfogenética Óssea 3/farmacologia , Regulação para Baixo/efeitos dos fármacos , Mutação da Fase de Leitura/efeitos dos fármacos , Fator 1-alfa Nuclear de Hepatócito/genética , Insulina/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RatosRESUMO
Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation.
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Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Metabolismo Energético/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Hipoglicemiantes/farmacologia , Insulinoma/genética , Insulinoma/metabolismo , Insulinoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeos/farmacologiaRESUMO
OBJECTIVE: In diabetes, ß-cell mass is not static but in a constant process of cell death and renewal. Inactivating mutations in transcription factor 1 (tcf-1)/hepatocyte nuclear factor1a (hnf1a) result in decreased ß-cell mass and HNF1A-maturity onset diabetes of the young (HNF1A-MODY). Here, we investigated the effect of a dominant-negative HNF1A mutant (DN-HNF1A) induced apoptosis on the regenerative capacity of INS-1 cells. RESEARCH DESIGN AND METHODS: DN-HNF1A was expressed in INS-1 cells using a reverse tetracycline-dependent transactivator system. Gene(s)/protein(s) involved in ß-cell regeneration were investigated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Pancreatic stone protein/regenerating protein (PSP/reg) serum levels in human subjects were detected by enzyme-linked immunosorbent assay. RESULTS: We detected a prominent induction of PSP/reg at the gene and protein level during DN-HNF1A-induced apoptosis. Elevated PSP/reg levels were also detected in islets of transgenic HNF1A-MODY mice and in the serum of HNF1A-MODY patients. The induction of PSP/reg was glucose dependent and mediated by caspase activation during apoptosis. Interestingly, the supernatant from DN-HNF1A-expressing cells, but not DN-HNF1A-expressing cells treated with zVAD.fmk, was sufficient to induce PSP/reg gene expression and increase cell proliferation in naïve, untreated INS-1 cells. Further experiments demonstrated that annexin-V-positive microparticles originating from apoptosing INS-1 cells mediated the induction of PSP/reg. Treatment with recombinant PSP/reg reversed the phenotype of DN-HNF1A-induced cells by stimulating cell proliferation and increasing insulin gene expression. CONCLUSIONS: Our results suggest that apoptosing INS-1 cells shed microparticles that may stimulate PSP/reg induction in neighboring cells, a mechanism that may facilitate the recovery of ß-cell mass in HNF1A-MODY.