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1.
Genes Dev ; 33(11-12): 641-655, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048544

RESUMO

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of ß-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.


Assuntos
Carcinogênese , Carcinoma Ductal Pancreático/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/fisiologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carcinoma in Situ/patologia , Carcinoma in Situ/fisiopatologia , Carcinoma Ductal Pancreático/patologia , Transdiferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Metaplasia , Camundongos , Camundongos Transgênicos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo
2.
Genes Dev ; 29(2): 171-83, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25593307

RESUMO

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of ß-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.


Assuntos
Carcinoma Ductal Pancreático/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica/genética , Neoplasias Pancreáticas/enzimologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco , beta Catenina/metabolismo
3.
Int J Mol Sci ; 23(1)2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35008431

RESUMO

The substantial biological heterogeneity of metastatic prostate cancer has hindered the development of personalized therapeutic approaches. Therefore, it is difficult to predict the course of metastatic hormone-sensitive prostate cancer (mHSPC), with some men remaining on first-line androgen deprivation therapy (ADT) for several years while others progress more rapidly. Improving our ability to risk-stratify patients would allow for the optimization of systemic therapies and support the development of stratified prospective clinical trials focused on patients likely to have the greatest potential benefit. Here, we applied a liquid biopsy approach to identify clinically relevant, blood-based prognostic biomarkers in patients with mHSPC. Gene expression indicating the presence of CTCs was greater in CHAARTED high-volume (HV) patients (52% CTChigh) than in low-volume (LV) patients (23% CTChigh; * p = 0.03). HV disease (p = 0.005, q = 0.033) and CTC presence at baseline prior to treatment initiation (p = 0.008, q = 0.033) were found to be independently associated with the risk of nonresponse at 7 months. The pooled gene expression from CTCs of pre-ADT samples found AR, DSG2, KLK3, MDK, and PCA3 as genes predictive of nonresponse. These observations support the utility of liquid biomarker approaches to identify patients with poor initial response. This approach could facilitate more precise treatment intensification in the highest risk patients.


Assuntos
Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/métodos , Células Neoplásicas Circulantes/química , Neoplasias da Próstata/genética , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Antígenos de Neoplasias/genética , Desmogleína 2/genética , Humanos , Calicreínas/genética , Masculino , Midkina/genética , Reação em Cadeia da Polimerase Multiplex , Medicina de Precisão , Prognóstico , Estudos Prospectivos , Antígeno Prostático Específico/genética , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética
4.
J Biol Chem ; 290(45): 27146-27157, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26381412

RESUMO

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348-588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Tolerância a Radiação/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos da radiação , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica/efeitos da radiação , Domínios e Motivos de Interação entre Proteínas , Tolerância a Radiação/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitinação
5.
Oncology (Williston Park) ; 30(5): 377-85, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27188668

RESUMO

Metastatic prostate cancer remains a highly lethal disease with no curative therapeutic options. A significant subset of patients with prostate cancer harbor either germline or somatic mutations in DNA repair enzyme genes such as BRCA1, BRCA2, or ATM. Emerging data suggest that drugs that target poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) enzymes may represent a novel and effective means of treating tumors with these DNA repair defects, including prostate cancers. Here we will review the molecular mechanism of action of PARP inhibitors and discuss how they target tumor cells with faulty DNA repair functions and transcriptional controls. We will review emerging data for the utility of PARP inhibition in the management of metastatic prostate cancer. Finally, we will place PARP inhibitors within the framework of precision medicine-based care of patients with prostate cancer.


Assuntos
Antineoplásicos/uso terapêutico , Desenho de Fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Predisposição Genética para Doença , Humanos , Masculino , Terapia de Alvo Molecular , Mutação , Metástase Neoplásica , Seleção de Pacientes , Fenótipo , Inibidores de Poli(ADP-Ribose) Polimerases/efeitos adversos , Medicina de Precisão , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos
6.
Neoplasia ; 57: 101036, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39173508

RESUMO

Assessing the molecular profiles of bladder cancer (BC) from patients with locally advanced or metastatic disease provides valuable insights, such as identification of invasive markers, to guide personalized treatment. Currently, most molecular profiling of BC is based on highly invasive biopsy or transurethral tumor resection. Liquid biopsy takes advantage of less-invasive procedures to longitudinally profile disease. Circulating tumor cells (CTCs) isolated from blood are one of the key analytes of liquid biopsy. In this study, we developed a protein and mRNA co-analysis workflow for BC CTCs utilizing the graphene oxide (GO) microfluidic chip. The GO chip was conjugated with antibodies against both EpCAM and EGFR to isolate CTCs from 1 mL of blood drawn from BC patients. Following CTC capture, protein and mRNA were analyzed using immunofluorescent staining and ion-torrent-based whole transcriptome sequencing, respectively. Elevated CTC counts were significantly associated with patient disease status at the time of blood draw. We found a count greater than 2.5 CTCs per mL was associated with shorter overall survival. The invasive markers EGFR, HER2, CD31, and ADAM15 were detected in CTC subpopulations. Whole transcriptome sequencing showed distinct RNA expression profiles from patients with or without tumor burden at the time of blood draw. In patients with advanced metastatic disease, we found significant upregulation of metastasis-related and chemotherapy-resistant genes. This methodology demonstrates the capability of GO chip-based assays to identify tumor-related RNA signatures, highlighting the prognostic potential of CTCs in metastatic BC patients.


Assuntos
Biomarcadores Tumorais , Microfluídica , Células Neoplásicas Circulantes , Neoplasias da Bexiga Urinária , Humanos , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/sangue , Feminino , Microfluídica/métodos , Masculino , Pessoa de Meia-Idade , Idoso , Metástase Neoplásica , Biópsia Líquida/métodos , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Prognóstico
7.
Clin Cancer Res ; 30(15): 3200-3210, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38787530

RESUMO

PURPOSE: CDK12 inactivation in metastatic castration-resistant prostate cancer (mCRPC) may predict immunotherapy responses. This phase 2 trial evaluated the efficacy of immune checkpoint inhibitor (ICI) therapy in patients with CDK12-altered mCRPC. PATIENTS AND METHODS: Eligible patients had mCRPC with deleterious CDK12 alterations and any prior therapies except ICI. Cohort A received ipilimumab (1 mg/kg) with nivolumab (3 mg/kg) every 3 weeks for up to four cycles, followed by nivolumab 480 mg every 4 weeks. Cohort C received nivolumab alone 480 mg every 4 weeks. Patients with CDK12-altered nonprostate tumors were enrolled in cohort B and not reported. The primary endpoint was a 50% reduction in PSA (PSA50). Key secondary endpoints included PSA progression-free survival, overall survival, objective response rate, and safety. RESULTS: PSA was evaluable in 23 patients in cohort A and 14 in cohort C. Median lines of prior therapy were two in cohorts A and C, including any prior novel hormonal agent (74% and 79%) and chemotherapy (57% and 36%). The PSA50 rate was 9% [95% confidence interval (CI), 1%-28%] in cohort A with two responders; neither had microsatellite instability or a tumor mutational burden >10 mutations/megabase. No PSA50 responses occurred in cohort C. Median PSA progression-free survival was 7.0 months (95% CI, 3.6-11.4) in cohort A and 4.5 months (95% CI, 3.4-13.8) in cohort C. Median overall survival was 9.0 months (95% CI, 6.2-12.3) in cohort A and 13.8 months (95% CI, 3.6-not reached) in cohort C. CONCLUSIONS: There was minimal activity with ICI therapy in patients with CDK12-altered mCRPC.


Assuntos
Quinases Ciclina-Dependentes , Inibidores de Checkpoint Imunológico , Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/mortalidade , Idoso , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/efeitos adversos , Pessoa de Meia-Idade , Quinases Ciclina-Dependentes/antagonistas & inibidores , Idoso de 80 Anos ou mais , Mutação , Nivolumabe/uso terapêutico , Nivolumabe/administração & dosagem , Ipilimumab/uso terapêutico , Ipilimumab/administração & dosagem , Ipilimumab/efeitos adversos , Metástase Neoplásica , Antígeno Prostático Específico/sangue , Biomarcadores Tumorais , Intervalo Livre de Progressão , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos
8.
Eur Urol ; 2024 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-39155193

RESUMO

BACKGROUND AND OBJECTIVE: Predicting response to therapy for each patient's tumor is critical to improving long-term outcomes for muscle-invasive bladder cancer. This study aims to establish ex vivo bladder cancer patient-derived organoid (PDO) models that are representative of patients' tumors and determine the potential efficacy of standard of care and curated experimental therapies. METHODS: Tumor material was collected prospectively from consented bladder cancer patients to generate short-term PDO models, which were screened against a panel of clinically relevant drugs in ex vivo three-dimensional culture. Multiomic profiling was utilized to validate the PDO models, establish the molecular characteristics of each tumor, and identify potential biomarkers of drug response. Gene expression (GEX) patterns between paired primary tissue and PDO samples were assessed using Spearman's rank correlation coefficients. Molecular correlates of therapy response were identified using Pearson correlation coefficients and Kruskal-Wallis tests with Dunn's post hoc pairwise comparison testing. KEY FINDINGS AND LIMITATIONS: A total of 106 tumors were collected from 97 patients, with 65 samples yielding sufficient material for complete multiomic molecular characterization and PDO screening with six to 32 drugs/combinations. Short-term PDOs faithfully represent the tumor molecular characteristics, maintain diverse cell types, and avoid shifts in GEX-based subtyping that accompany long-term PDO cultures. Utilizing an integrative approach, novel correlations between ex vivo drug responses and genomic alterations, GEX, and protein expression were identified, including a multiomic signature of gemcitabine response. The positive predictive value of ex vivo drug responses and the novel multiomic gemcitabine response signature need to be validated in future studies. CONCLUSIONS AND CLINICAL IMPLICATIONS: Short-term PDO cultures retain the molecular characteristics of tumor tissue and avoid shifts in expression-based subtyping that have plagued long-term cultures. Integration of multiomic profiling and ex vivo drug screening data identifies potential predictive biomarkers, including a novel signature of gemcitabine response. PATIENT SUMMARY: Better models are needed to predict patient response to therapy in bladder cancer. We developed a platform that uses short-term culture to best mimic each patient's tumor and assess potential sensitivity to therapeutics.

9.
Mol Cancer Res ; 20(10): 1574-1588, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-35852380

RESUMO

Splicing of the hTERT gene to produce the full-length (FL) transcript is necessary for telomerase enzyme activity and telomere-dependent cellular immortality in the majority of human tumors, including non-small cell lung cancer (NSCLC) cells. The molecular machinery to splice hTERT to the FL isoform remains mostly unknown. Previously, we reported that an intron 8 cis-element termed "direct repeat 8" (DR8) promotes FL hTERT splicing, telomerase, and telomere length maintenance when bound by NOVA1 and PTBP1 in NSCLC cells. However, some NSCLC cells and patient tumor samples lack NOVA1 expression. This leaves a gap in knowledge about the splicing factors and cis-elements that promote telomerase in the NOVA1-negative context. We report that DR8 regulates FL hTERT splicing in the NOVA1-negative and -positive lung cancer contexts. We identified splicing factor 3b subunit 4 (SF3B4) as an RNA trans-factor whose expression is increased in lung adenocarcinoma (LUAD) tumors compared with adjacent normal tissue and predicts poor LUAD patient survival. In contrast to normal lung epithelial cells, which continued to grow with partial reductions of SF3B4 protein, SF3B4 knockdown reduced hTERT splicing, telomerase activity, telomere length, and cell growth in lung cancer cells. SF3B4 was also demonstrated to bind the DR8 region of hTERT pre-mRNA in both NOVA1-negative and -positive NSCLC cells. These findings provide evidence that DR8 is a critical binding hub for trans-factors to regulate FL hTERT splicing in NSCLC cells. These studies help define mechanisms of gene regulation important to the generation of telomerase activity during carcinogenesis. IMPLICATIONS: Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Telomerase , Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Íntrons , Neoplasias Pulmonares/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo
10.
Tomography ; 8(2): 644-656, 2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35314631

RESUMO

This observer study investigates the effect of computerized artificial intelligence (AI)-based decision support system (CDSS-T) on physicians' diagnostic accuracy in assessing bladder cancer treatment response. The performance of 17 observers was evaluated when assessing bladder cancer treatment response without and with CDSS-T using pre- and post-chemotherapy CTU scans in 123 patients having 157 pre- and post-treatment cancer pairs. The impact of cancer case difficulty, observers' clinical experience, institution affiliation, specialty, and the assessment times on the observers' diagnostic performance with and without using CDSS-T were analyzed. It was found that the average performance of the 17 observers was significantly improved (p = 0.002) when aided by the CDSS-T. The cancer case difficulty, institution affiliation, specialty, and the assessment times influenced the observers' performance without CDSS-T. The AI-based decision support system has the potential to improve the diagnostic accuracy in assessing bladder cancer treatment response and result in more consistent performance among all physicians.


Assuntos
Sistemas de Apoio a Decisões Clínicas , Neoplasias da Bexiga Urinária , Inteligência Artificial , Humanos , Tomografia Computadorizada por Raios X , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/terapia , Urografia
11.
Clin Cancer Res ; 27(11): 3017-3027, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33727260

RESUMO

PURPOSE: Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, blocks proliferation in a RB and cyclin D-dependent manner in preclinical prostate cancer models. We hypothesized that cotargeting androgen receptor and cell cycle with palbociclib would improve outcomes in patients with metastatic hormone-sensitive prostate cancer (mHSPC). PATIENTS AND METHODS: A total of 60 patients with RB-intact mHSPC were randomized (1:2) to Arm 1: androgen deprivation (AD) or Arm 2: AD + palbociclib. Primary endpoint was PSA response rate (RR) after 28 weeks of therapy. Secondary endpoints included safety, PSA, and clinical progression-free survival (PFS), as well as PSA and radiographic RR. Tumors underwent exome sequencing when available. Circulating tumor cells (CTC) were enumerated at various timepoints. RESULTS: A total of 72 patients with mHSPC underwent metastatic disease biopsy and 64 had adequate tissue for RB assessment. A total of 62 of 64 (97%) retained RB expression. A total of 60 patients initiated therapy (Arm 1: 20; Arm 2: 40). Neutropenia was the most common grade 3/4 adverse event in Arm 2. Eighty percent of patients (Arm 1: 16/20, Arm 2: 32/40; P = 0.87) met primary PSA endpoint ≤4 ng/mL at 28 weeks. PSA undetectable rate at 28 weeks was 50% and 43% in Arms 1 and 2, respectively (P = 0.5). Radiographic RR was 89% in both arms. Twelve-month biochemical PFS was 69% and 74% in Arms 1 and 2, respectively (P = 0.72). TP53 and PIK3 pathway mutations, 8q gains, and pretreatment CTCs were associated with reduced PSA PFS. CONCLUSIONS: Palbociclib did not impact outcome in RB-intact mHSPC. Pretreatment CTC, TP53 and PIK3 pathway mutations, and 8q gain were associated with poor outcome.


Assuntos
Antagonistas de Androgênios/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Piperazinas/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Piridinas/administração & dosagem , Proteína do Retinoblastoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/secundário , Intervalo Livre de Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Transdução de Sinais/genética , Neoplasias de Tecidos Moles/secundário , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo
12.
EBioMedicine ; 51: 102561, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31927310

RESUMO

BACKGROUND: Half of muscle-invasive bladder cancer patients will relapse with metastatic disease and molecular tests to predict relapse are needed. TP63 has been proposed as a prognostic biomarker in bladder cancer, but reports associating it with clinical outcomes are conflicting. Since TP63 is expressed as multiple isoforms, we hypothesized that these conflicting associations with clinical outcome may be explained by distinct opposing effects of differential TP63 isoform expression. METHODS: Using RNA-Seq data from The Cancer Genome Atlas (TCGA), TP63 isoform-level expression was quantified and associated with clinical covariates (e.g. survival, stage) across 8,519 patients from 29 diseases. A comprehensive catalog of TP63 isoforms was assembled using gene annotation databases and de novo discovery in bladder cancer patients. Quantifications and un-annotated TP63 isoforms were validated using quantitative RT-PCR and a separate bladder cancer cohort. FINDINGS: DNp63 isoform expression was associated with improved bladder cancer patient survival in patients with a luminal subtype (HR = 0.89, CI 0.80-0.99, Cox p = 0.034). Conversely, TAp63 isoform expression was associated with reduced bladder cancer patient survival in patients with a basal subtype (HR = 2.35, CI 1.64-3.37, Cox p < 0.0001). These associations were observed in multiple TCGA disease cohorts and correlated with epidermal differentiation (DNp63) and immune-related (TAp63) gene signatures. INTERPRETATION: These results comprehensively define TP63 isoform expression in human cancer and suggest that TP63 isoforms are involved in distinct transcriptional programs with opposing effects on clinical outcome.


Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Humanos , Modelos de Riscos Proporcionais , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Resultado do Tratamento , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/classificação
13.
Oncotarget ; 11(44): 3921-3932, 2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33216841

RESUMO

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

14.
Tomography ; 6(2): 194-202, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32548296

RESUMO

We evaluated the intraobserver variability of physicians aided by a computerized decision-support system for treatment response assessment (CDSS-T) to identify patients who show complete response to neoadjuvant chemotherapy for bladder cancer, and the effects of the intraobserver variability on physicians' assessment accuracy. A CDSS-T tool was developed that uses a combination of deep learning neural network and radiomic features from computed tomography (CT) scans to detect bladder cancers that have fully responded to neoadjuvant treatment. Pre- and postchemotherapy CT scans of 157 bladder cancers from 123 patients were collected. In a multireader, multicase observer study, physician-observers estimated the likelihood of pathologic T0 disease by viewing paired pre/posttreatment CT scans placed side by side on an in-house-developed graphical user interface. Five abdominal radiologists, 4 diagnostic radiology residents, 2 oncologists, and 1 urologist participated as observers. They first provided an estimate without CDSS-T and then with CDSS-T. A subset of cases was evaluated twice to study the intraobserver variability and its effects on observer consistency. The mean areas under the curves for assessment of pathologic T0 disease were 0.85 for CDSS-T alone, 0.76 for physicians without CDSS-T and improved to 0.80 for physicians with CDSS-T (P = .001) in the original evaluation, and 0.78 for physicians without CDSS-T and improved to 0.81 for physicians with CDSS-T (P = .010) in the repeated evaluation. The intraobserver variability was significantly reduced with CDSS-T (P < .0001). The CDSS-T can significantly reduce physicians' variability and improve their accuracy for identifying complete response of muscle-invasive bladder cancer to neoadjuvant chemotherapy.


Assuntos
Sistemas de Apoio a Decisões Clínicas , Neoplasias da Bexiga Urinária , Humanos , Variações Dependentes do Observador , Médicos , Tomografia Computadorizada por Raios X , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/tratamento farmacológico
15.
Genetics ; 180(4): 1809-19, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18832348

RESUMO

Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70-Yku80 (Ku), Dnl4-Lif1-Nej1 (DNA ligase IV), and Mre11-Rad50-Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2-Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2-Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80-Dnl4 and Xrs2-Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs-Lif1 and Yku80-Dnl4 interactions are important for formation of a productive ligase-DSB intermediate.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Trends Biochem Sci ; 28(2): 62-6, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12575991

RESUMO

The repair of DNA double-strand breaks by non-homologous end-joining (NHEJ) has long been thought to be restricted to eukaryotes. However, recent papers document the existence of operons encoding functional NHEJ complexes in some bacteria. These findings provide new evolutionary insights into the core biochemistry of this repair pathway, and suggest that one function driving the selection of NHEJ in bacteria, and perhaps eukaryotes, relates to prolonged periods of mitotic exit.


Assuntos
Bactérias/genética , Cromossomos Bacterianos/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bactérias/metabolismo , DNA Ligases/genética , Humanos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
17.
Nat Protoc ; 14(3): 738-755, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30683938

RESUMO

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. This technique consists of a small laparotomy and direct implantation of human cancer cells into the bladder lumen. Unlike other protocols, it does not require debriding of the urothelial lining, injection into the bladder wall, specialized imaging equipment, bladder catheterization or costly surgical equipment. With minimal practice, the procedure can be executed in <10 min. Tumors develop with a high take rate, and most cell lines exhibit local invasion within 4 weeks of implantation.


Assuntos
Progressão da Doença , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica
18.
Oncogene ; 38(18): 3340-3354, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30643195

RESUMO

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Invasividade Neoplásica/patologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasia de Células Basais/metabolismo , Neoplasia de Células Basais/patologia , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patologia , Transcrição Gênica/fisiologia
19.
Mol Cell Biol ; 25(24): 10782-90, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16314503

RESUMO

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antígenos Nucleares/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Análise Mutacional de DNA , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Autoantígeno Ku , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Telômero/metabolismo , Técnicas do Sistema de Duplo-Híbrido
20.
J Vis Exp ; (139)2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30272657

RESUMO

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.


Assuntos
Imageamento Tridimensional/métodos , Microambiente Tumoral/imunologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/terapia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Humanos , Invasividade Neoplásica , Neoplasias da Bexiga Urinária/patologia
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