RESUMO
Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.
Assuntos
Benzamidas , Resistencia a Medicamentos Antineoplásicos , Mutação , Nitrilas , Feniltioidantoína , Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Masculino , Nitrilas/uso terapêutico , Benzamidas/uso terapêutico , Humanos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
The DEK oncoprotein regulates cellular chromatin function via a number of protein-protein interactions. However, the biological relevance of its unique pseudo-SAP/SAP-box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis-driven mutations failed to yield DNA-binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re-expression of a DEK-DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK-DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Mutação com Perda de Função , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Recombinantes/toxicidade , Viés , Núcleo Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/toxicidade , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Humanos , Mutagênese , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/toxicidade , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testes de Toxicidade/métodosRESUMO
In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
