RESUMO
PURPOSE: Transcriptomic profiling can shed light on the biology of small-cell bladder cancer (SCBC), nominating biomarkers, and novel therapeutic targets. EXPERIMENTAL DESIGN: Sixty-three patients with SCBC had small-cell histology confirmed and quantified by a genitourinary pathologist. Gene expression profiling was performed for 39 primary tumor samples, 1 metastatic sample, and 6 adjacent normal urothelium samples (46 total) from the same cohort. Protein levels of differentially expressed therapeutic targets, DLL3 and PDL1, and also CD56 and ASCL1, were confirmed by IHC. A SCBC PDX model was utilized to assess in vivo efficacy of DLL3-targeting antibody-drug conjugate (ADC). RESULTS: Unsupervised hierarchical clustering of 46 samples produced 4 clusters that correlated with clinical phenotypes. Patients whose tumors had the most "normal-like" pattern of gene expression had longer overall survival (OS) compared with the other 3 clusters while patients with the most "metastasis-like" pattern had the shortest OS (P = 0.047). Expression of DLL3, PDL1, ASCL1, and CD56 was confirmed by IHC in 68%, 30%, 52%, and 81% of tissue samples, respectively. In a multivariate analysis, DLL3 protein expression on >10% and CD56 expression on >30% of tumor cells were both prognostic of shorter OS (P = 0.03 each). A DLL3-targeting ADC showed durable antitumor efficacy in a SCBC PDX model. CONCLUSIONS: Gene expression patterns in SCBC are associated with distinct clinical phenotypes ranging from more indolent to aggressive disease. Overexpression of DLL3 mRNA and protein is common in SCBC and correlates with shorter OS. A DLL3-targeted ADC demonstrated in vivo efficacy superior to chemotherapy in a PDX model of SCBC.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Prognóstico , Transcriptoma/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Imunoconjugados/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteoma/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Pseudomonas aeruginosa Vfr (the virulence factor regulator) enhances P. aeruginosa virulence by positively regulating the expression of numerous virulence genes. A previous microarray analysis identified numerous genes positively regulated by Vfr in strain PAK, including the yet uncharacterized PA2782 and PA2783. RESULTS: In this study, we report the detailed characterization of PA2783 in the P. aeruginosa strain PAO1. RT-PCR analysis confirmed that PA2782-PA2783 constitute an operon. A mutation in vfr significantly reduced the expression of both genes. The predicted protein encoded by PA2783 contains a typical leader peptide at its amino terminus end as well as metalloendopeptidase and carbohydrate binding motifs at its amino terminus and carboxy terminus regions, respectively. An in-frame PA2783::phoA fusion encoded a hybrid protein that was exported to the periplasmic space of Escherichia coli and P. aeruginosa. In PAO1, the proteolytic activity of the PA2783-encoded protein was masked by other P. aeruginosa extracellular proteases but an E. coli strain carrying a PA2783 recombinant plasmid produced considerable proteolytic activity. The outer membrane fraction of an E. coli strain in which PA2783 was overexpressed contained specific endopeptidase activity. In the presence of cAMP, purified recombinant Vfr (rVfr) bound to a 98-bp fragment within the PA2782-PA2783 upstream region that carries a putative Vfr consensus sequence. Through a series of electrophoretic mobility shift assays, we localized rVfr binding to a 33-bp fragment that contains part of the Vfr consensus sequence and a 5-bp imperfect (3/5) inverted repeat at its 3' and 5' ends (TGGCG-N22-CGCTG). Deletion of either repeat eliminated Vfr binding. CONCLUSIONS: PA2782 and PA2783 constitute an operon whose transcription is positively regulated by Vfr. The expression of PA2783 throughout the growth cycle of P. aeruginosa follows a unique pattern. PA2783 codes for a secreted metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.
