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1.
Biomaterials ; 220: 119402, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31400612

RESUMO

Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.

2.
Mol Oncol ; 13(4): 738-756, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30537101

RESUMO

Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.

3.
J Cell Sci ; 131(15)2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30002137

RESUMO

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis. Here, we report that expression of the activating mutations FGFR2N550K and FGFR2Y376C in an endometrial cancer cell model induce Golgi fragmentation, and loss of polarity and directional migration. In mutant FGFR2-expressing cells, this was associated with an inability to polarise intracellular pools of FGFR2 towards the front of migrating cells. Such polarization defects were exacerbated in three-dimensional culture, where FGFR2 mutant cells were unable to form well-organised acini, instead undergoing exogenous ligand-independent invasion. Our findings uncover collective cell polarity and invasion as common targets of disease-associated FGFR2 mutations that lead to poor outcome in endometrial cancer patients.

4.
Mod Pathol ; 31(12): 1851-1861, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29955143

RESUMO

The TransPORTEC consortium previouslclassified high-risk endometrial cancer including poor-risk histologies such as clear cells, into four molecular subtypes "POLE mutated," "microsatellite unstable," "TP53 mutated," and "no specific molecular profile." We evaluated whether DNA damage response biomarkers could further refine this high-risk tumors classification, in particular the heterogeneous "no specific molecular profile" and "TP53 mutated" subsets recently qualified as poor prognosis in high-risk endometrial cancer. DNA damage response biomarkers including proteins involved in DNA damage (δ-H2AX), homologous recombination (RAD51), regulators of error-prone Non Homologous End-Joining (DNA-pk, FANCD2), and PARP-1 were evaluated in 116 high-risk tumors by immunohistochemistry. CD8 and PD-1 expression by immunochemistry and mutation analyses were performed previously. Survival outcome were calculated using Kaplan-Meier and Log-rank test. None of the DNA damage response biomarkers alone were prognostic. However markers were informative within molecular subsets. Among the "no specific molecular profile" subset, δ-H2AX+ was significantly predictive of poor disease free survival (Hazard Ratio = 2.56; p = 0.026), and among "TP53 mutated," a DNA-pk+/FANCD2- profile (favouring error-prone Non Homologous End-Joining) predicted worst disease free survival (Hazard Ratio = 4.95; p = 0.009) resulting in five distinct prognostic subgroups from best to worst prognosis: group1 "POLE mutated/Microsatellite unstable" > group2 "no specific molecular profile with no DNA damage" > group3 "TP53 mutated/Non Homologous End-Joining negative" > group4 "no specific molecular profile with high DNA damage" > group5 "TP53 mutated/Non Homologous End-Joining positive"; p = 0.0002). Actionable targets were also different among subsets. Group3 had significantly higher infiltration of PD-1+ immune cells (p = 0.003), segregating with group1. Group2 had frequent PI3K pathway mutations and ER positivity. While group5, with the worst prognosis, had high DNA damage and PARP-1 expression providing a rationale for PARP inhibition. Our findings have refined the TransPORTEC prognostic classification of high-risk endometrial cancer into five distinct subgroups by integrating DNA damage response biomarkers and identified molecular subtype-specific therapeutic strategies.

5.
DNA Repair (Amst) ; 54: 30-39, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28448822

RESUMO

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.


Assuntos
Reparo do DNA , Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/química , Humanos , Proteínas Mitocondriais/química , Fosforilação
6.
Oncoimmunology ; 6(2): e1264565, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28344870

RESUMO

High-risk endometrial cancer (EC) is an aggressive disease for which new therapeutic options are needed. Aims of this study were to validate the enhanced immune response in highly mutated ECs and to explore immune profiles in other EC subgroups. We evaluated immune infiltration in 116 high-risk ECs from the TransPORTEC consortium, previously classified into four molecular subtypes: (i) ultramutated POLE exonuclease domain-mutant ECs (POLE-mutant); (ii) hypermutated microsatellite unstable (MSI); (iii) p53-mutant; and (iv) no specific molecular profile (NSMP). Within The Cancer Genome Atlas (TCGA) EC cohort, significantly higher numbers of predicted neoantigens were demonstrated in POLE-mutant and MSI tumors compared with NSMP and p53-mutants. This was reflected by enhanced immune expression and infiltration in POLE-mutant and MSI tumors in both the TCGA cohort (mRNA expression) and the TransPORTEC cohort (immunohistochemistry) with high infiltration of CD8+ (90% and 69%), PD-1+ (73% and 69%) and PD-L1+ immune cells (100% and 71%). Notably, a subset of p53-mutant and NSMP cancers was characterized by signs of an antitumor immune response (43% and 31% of tumors with high infiltration of CD8+ cells, respectively), despite a low number of predicted neoantigens. In conclusion, the presence of enhanced immune infiltration, particularly high numbers of PD-1 and PD-L1 positive cells, in highly mutated, neoantigen-rich POLE-mutant and MSI endometrial tumors suggests sensitivity to immune checkpoint inhibitors.

7.
Gynecol Oncol ; 145(2): 366-373, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28314589

RESUMO

PURPOSE: Activating FGFR2 mutations have been identified in ~10% of endometrioid endometrial cancers (ECs). We have previously reported that mutations in FGFR2 are associated with shorter disease free survival (DFS) in stage I/II EC patients. Here we sought to validate the prognostic importance of FGFR2 mutations in a large, multi-institutional patient cohort. METHODS: Tumors were collected as part of the GOG 210 clinical trial "Molecular Staging of Endometrial Cancer" where samples underwent rigorous pathological review and had more than three years of detailed clinical follow-up. DNA was extracted and four exons encompassing the FGFR2 mutation hotspots were amplified and sequenced. RESULTS: Mutations were identified in 144 of the 973 endometrioid ECs, of which 125 were classified as known activating mutations and were included in the statistical analyses. Consistent with FGFR2 having an association with more aggressive disease, FGFR2 mutations were more common in patients initially diagnosed with stage III/IV EC (29/170;17%) versus stage I/II EC (96/803; 12%; p=0.07, Chi-square test). Additionally, incidence of progression (progressed, recurred or died from disease) was significantly more prevalent (32/125, 26%) among patients with FGFR2 mutation versus wild type (120/848, 14%; p<0.001, Chi-square test). Using Cox regression analysis adjusting for known prognostic factors, patients with FGFR2 mutation had significantly (p<0.025) shorter progression-free survival (PFS; HR 1.903; 95% CI 1.177-3.076) and endometrial cancer specific survival (ECS; HR 2.013; 95% CI 1.096-3.696). CONCLUSION: In summary, our findings suggest that clinical trials testing the efficacy of FGFR inhibitors in the adjuvant setting to prevent recurrence and death are warranted.


Assuntos
Carcinoma Endometrioide/genética , Neoplasias do Endométrio/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Idoso , Carcinoma Endometrioide/patologia , Estudos de Coortes , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Neoplasias do Endométrio/patologia , Éxons , Feminino , Humanos , Estimativa de Kaplan-Meier , Estadiamento de Neoplasias
8.
Mol Cancer Ther ; 16(4): 637-648, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28119489

RESUMO

Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Endometrial cancers display hyperactivation of the MAPK and PI3K pathways, the result of somatic aberrations in genes such as FGFR2, KRAS, PTEN, PIK3CA, and PIK3R1 The FGFR2 and PI3K pathways, have emerged as potential therapeutic targets in endometrial cancer. Activation of the PI3K pathway is seen in more than 90% of FGFR2mutant endometrial cancers. This study aimed to examine the efficacy of the pan-FGFR inhibitor BGJ398 with pan-PI3K inhibitors (GDC-0941, BKM120) and the p110α-selective inhibitor BYL719. We assessed synergy in three FGFR2mutant endometrial cancer cell lines (AN3CA, JHUEM2, and MFE296), and the combination of BGJ398 and GDC-0941 or BYL719 showed strong synergy. A significant increase in cell death and decrease in long-term survival was seen when PI3K inhibitors were combined with BGJ398. Importantly, these effects were seen at low concentrations correlating to only partial inhibition of AKT. The combination of BGJ398 and GDC-0941 showed tumor regressions in vivo, whereas each drug alone only showed moderate tumor growth inhibition. BYL719 alone resulted in increased tumor growth of AN3CA xenografts but in combination with BGJ398 resulted in tumor regression in both AN3CA- and JHUEM2-derived xenografts. These data provide evidence that subtherapeutic doses of PI3K inhibitors enhance the efficacy of anti-FGFR therapies, and a combination therapy may represent a superior therapeutic treatment in patients with FGFR2mutant endometrial cancer. Mol Cancer Ther; 16(4); 637-48. ©2017 AACR.


Assuntos
Aminopiridinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias do Endométrio/tratamento farmacológico , Indazóis/administração & dosagem , Morfolinas/administração & dosagem , Compostos de Fenilureia/administração & dosagem , Pirimidinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Sulfonamidas/administração & dosagem , Tiazóis/administração & dosagem , Aminopiridinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias do Endométrio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Camundongos , Morfolinas/farmacologia , Mutação , Compostos de Fenilureia/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Sulfonamidas/farmacologia , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Oncotarget ; 8(3): 3881-3894, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28002804

RESUMO

PTEN loss and constitutive activation of the class I phosphoinositide 3-kinase (PI3K) pathway are key drivers of endometrial tumorigenesis. In some cancer types, PTEN-deficient tumors are reliant on class I PI3K p110ß (encoded by PIK3CB) activity but little is known about this contribution in endometrial tumorigenesis. In this study, we find that p110ß is overexpressed in a panel of 7 endometrial cancer cell lines compared to non-transformed cells. Furthermore, in 234 clinically annotated patient samples, PIK3CB mRNA levels increase significantly in the early phase of tumorigenesis from precursors to low grade primary malignant lesions whereas PIK3CA levels are higher in non-endometrioid compared to endometrioid primary tumors. While high levels of either PIK3CA or PIK3CB associate with poor prognosis, only elevated PIK3CB mRNA levels correlate with a high cell cycle signature score in clinical samples. In cancer cell lines, p110α inhibition reduces cell viability by inducing cell death in PIK3CA mutant cells while p110ß inhibition delayed proliferation in PTEN-deficient cells, but not in WT cells. Taken together, our findings suggest that PIK3CB/p110ß contributes to some of the pleiotropic functions of PI3K in endometrial cancer, particularly in the early steps by contributing to cell proliferation.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias do Endométrio/patologia , Regulação para Cima , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Morfolinas/farmacologia , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida
10.
Am J Hum Genet ; 98(6): 1159-1169, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27259051

RESUMO

A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.


Assuntos
Cromossomos Humanos Par 14/genética , Neoplasias do Endométrio/genética , Fosfatidilinositol 3-Quinases/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Uterinas/genética , Fator de Transcrição YY1/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Risco , Transdução de Sinais , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Fator de Transcrição YY1/genética
11.
Oncotarget ; 7(31): 49677-49687, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27331623

RESUMO

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the 'cell cycle' pathway and the 'melanosome'. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and the melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , MicroRNAs/metabolismo , Neoplasias Cutâneas/metabolismo , Algoritmos , Biotina/química , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Genes Supressores de Tumor , Humanos , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/metabolismo , Transdução de Sinais
12.
EBioMedicine ; 2(7): 671-80, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26288839

RESUMO

The overall 5-year survival for melanoma is 91%. However, if distant metastasis occurs (stage IV), cure rates are < 15%. Hence, melanoma detection in earlier stages (stages I-III) maximises the chances of patient survival. We measured the expression of a panel of 17 microRNAs (miRNAs) (MELmiR-17) in melanoma tissues (stage III; n = 76 and IV; n = 10) and serum samples (collected from controls with no melanoma, n = 130; and patients with melanoma (stages I/II, n = 86; III, n = 50; and IV, n = 119)) obtained from biobanks in Australia and Germany. In melanoma tissues, members of the 'MELmiR-17' panel were found to be predictors of stage, recurrence, and survival. Additionally, in a minimally-invasive blood test, a seven-miRNA panel (MELmiR-7) detected the presence of melanoma (relative to controls) with high sensitivity (93%) and specificity (≥ 82%) when ≥ 4 miRNAs were expressed. Moreover, the 'MELmiR-7' panel characterised overall survival of melanoma patients better than both serum LDH and S100B (delta log likelihood = 11, p < 0.001). This panel was found to be superior to currently used serological markers for melanoma progression, recurrence, and survival; and would be ideally suited to monitor tumour progression in patients diagnosed with early metastatic disease (stages IIIa-c/IV M1a-b) to detect relapse following surgical or adjuvant treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/sangue , Melanoma/patologia , MicroRNAs/sangue , MicroRNAs/genética , Adulto , Estudos de Coortes , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Sensibilidade e Especificidade , Análise de Sobrevida , Adulto Jovem
13.
Oncotarget ; 6(19): 17753-63, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25980496

RESUMO

To identify 'melanoma-specific' microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA 'pull-down' experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Melanoma/genética , MicroRNAs/genética , Neurofibromina 1/biossíntese , Proteínas Proto-Oncogênicas B-raf/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Indóis/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Mutagênese Sítio-Dirigida , Neurofibromina 1/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Sulfonamidas/farmacologia , Transfecção , Vemurafenib
14.
Cancer Discov ; 5(4): 355-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25847957

RESUMO

In this issue of Cancer Discovery, Hagel and colleagues report the design and the in vitro and in vivo activity of a novel, irreversible, paralog-specific kinase inhibitor of FGFR4, BLU9931. This compound binds covalently to a cysteine residue in the hinge region of FGFR4 but not in FGFR1-3. BLU9931 induces tumor shrinkage in hepatocellular carcinoma models that express a functioning ligand/receptor complex consisting of FGF19/FGFR4/KLB and adds to a growing list of anti-FGFR4 agents.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Humanos
15.
Mod Pathol ; 28(6): 836-44, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25720322

RESUMO

This study aimed to investigate whether molecular analysis can be used to refine risk assessment, direct adjuvant therapy, and identify actionable alterations in high-risk endometrial cancer. TransPORTEC, an international consortium related to the PORTEC3 trial, was established for translational research in high-risk endometrial cancer. In this explorative study, routine molecular analyses were used to detect prognostic subgroups: p53 immunohistochemistry, microsatellite instability and POLE proofreading mutation. Furthermore, DNA was analyzed for hotspot mutations in 13 additional genes (BRAF, CDKNA2, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, and PTEN) and protein expression of ER, PR, PTEN, and ARID1a was analyzed. Rates of distant metastasis, recurrence-free, and overall survival were calculated using the Kaplan-Meier method and log-rank test. In total, samples of 116 high-risk endometrial cancer patients were included: 86 endometrioid; 12 serous; and 18 clear cell. For endometrioid, serous, and clear cell cancers, 5-year recurrence-free survival rates were 68%, 27%, and 50% (P=0.014) and distant metastasis rates 23%, 64%, and 50% (P=0.001), respectively. Four prognostic subgroups were identified: (1) a group of p53-mutant tumors; (2) microsatellite instable tumors; (3) POLE proofreading-mutant tumors; and (4) a group with no specific molecular profile (NSMP). In group 3 (POLE-mutant; n=14) and group 2 (microsatellite instable; n=19) patients, no distant metastasis occurred, compared with 50% distant metastasis rate in group 1 (p53-mutant; n=36) and 39% in group 4 (NSMP; P<0.001). Five-year recurrence-free survival was 93% and 95% for group 3 (POLE-mutant) and group 2 (microsatellite instable) vs 42% (group 1, p53-mutant) and 52% (group 4, NSMP; P<0.001). Targetable FBXW7 and FGFR2 mutations (6%), alterations in the PI3K-AKT pathway (60%) and hormone receptor positivity (45%) were frequently found. In conclusion, molecular analysis of high-risk endometrial cancer identifies four distinct prognostic subgroups, with potential therapeutic implications. High frequencies of targetable alterations were identified and may serve as targets for individualized treatment.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias do Endométrio/classificação , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Fatores de Risco , Análise Serial de Tecidos
16.
Gynecol Oncol ; 135(1): 38-43, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25019571

RESUMO

PURPOSE: Brivanib, an oral, multi-targeted tyrosine kinase inhibitor with activity against vascular endothelial growth factor (VEGF) and fibroblast growth factor receptor (FGFR) was investigated as a single agent in a phase II trial to assess the activity and tolerability in recurrent or persistent endometrial cancer (EMC). PATIENTS AND METHODS: Eligible patients had persistent or recurrent EMC after receiving one to two prior cytotoxic regimens, measurable disease, and performance status of ≤2. Treatment consisted of brivanib 800 mg orally every day until disease progression or prohibitive toxicity. Primary endpoints were progression-free survival (PFS) at six months and objective tumor response. Expression of multiple angiogenic proteins and FGFR2 mutation status was assessed. RESULTS: Forty-five patients were enrolled. Forty-three patients were eligible and evaluable. Median age was 64 years. Twenty-four patients (55.8%) received prior radiation. Median number of cycles was two (range 1-24). No GI perforations but one rectal fistula were seen. Nine patients had grade 3 hypertension, with one experiencing grade 4 confusion. Eight patients (18.6%; 90% CI 9.6%-31.7%) had responses (one CR and seven PRs), and 13 patients (30.2%; 90% CI 18.9%-43.9%) were PFS at six months. Median PFS and overall survival (OS) were 3.3 and 10.7 months, respectively. When modeled jointly, VEGF and angiopoietin-2 expression may diametrically predict PFS. Estrogen receptor-α (ER) expression was positively correlated with OS. CONCLUSION: Brivanib is reasonably well tolerated and worthy of further investigation based on PFS at six months in recurrent or persistent EMC.


Assuntos
Alanina/análogos & derivados , Neoplasias do Endométrio/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Triazinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Alanina/uso terapêutico , Intervalo Livre de Doença , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Neoplasia ; 15(8): 975-88, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23908597

RESUMO

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.


Assuntos
Benzimidazóis/farmacologia , Imidazóis/farmacologia , Mutação , Piridazinas/farmacologia , Pirimidinas/farmacologia , Quinolonas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Eletroforese em Gel de Poliacrilamida , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo
18.
Anticancer Drugs ; 24(2): 181-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23059386

RESUMO

Mebendazole (MBZ) was identified as a promising therapeutic on the basis of its ability to induce apoptosis in melanoma cell lines through a B-cell lymphoma 2 (BCL2)-dependent mechanism. We now show that in a human xenograft melanoma model, oral MBZ is as effective as the current standard of care temozolomide in reducing tumor growth. Inhibition of melanoma growth in vivo is accompanied by phosphorylation of BCL2 and decreased levels of X-linked inhibitor of apoptosis (XIAP). Reduced expression of XIAP on treatment with MBZ is partially mediated by its proteasomal degradation. Furthermore, exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating XIAP's inhibition on apoptosis. XIAP expression on exposure to MBZ is indicative of sensitivity to MBZ as MBZ-resistant cells do not show reduced levels of XIAP after treatment. Resistance to MBZ can be reversed partially by siRNA knockdown of cellular levels of XIAP. Our data indicate that MBZ is a promising antimelanoma agent on the basis of its effects on key antiapoptotic proteins.


Assuntos
Mebendazol/farmacologia , Melanoma/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Temozolomida , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Cancer Discov ; 2(12): 1081-3, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23230185

RESUMO

In this issue of Cancer Discovery, Guagnano and colleagues use a large and diverse annotated collection of cancer cell lines, the Cancer Cell Line Encyclopedia, to correlate whole-genome expression and genomic alteration datasets with cell line sensitivity data to the novel pan-fibroblast growth factor receptor (FGFR) inhibitor NVP-BGJ398. Their findings underscore not only the preclinical use of such cell line panels in identifying predictive biomarkers, but also the emergence of the FGFRs as valid therapeutic targets, across an increasingly broad range of malignancies.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Humanos
20.
Int J Gynecol Cancer ; 22(9): 1517-26, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23060048

RESUMO

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.


Assuntos
Carcinoma Endometrioide/patologia , Doxorrubicina/farmacologia , Neoplasias do Endométrio/patologia , Paclitaxel/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/genética , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Feminino , Humanos , Mutação/fisiologia , Paclitaxel/administração & dosagem , Pirimidinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Células Tumorais Cultivadas
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