RESUMO
Prostate cancer (PCa) remains the most diagnosed non-skin cancer amongst the American male population. Treatment for localized prostate cancer consists of androgen deprivation therapies (ADTs), which typically inhibit androgen production and the androgen receptor (AR). Though initially effective, a subset of patients will develop resistance to ADTs and the tumors will transition to castration-resistant prostate cancer (CRPC). Second generation hormonal therapies such as abiraterone acetate and enzalutamide are typically given to men with CRPC. However, these treatments are not curative and typically prolong survival only by a few months. Several resistance mechanisms contribute to this lack of efficacy such as the emergence of AR mutations, AR amplification, lineage plasticity, AR splice variants (AR-Vs) and increased kinase signaling. Having identified SRC kinase as a key tyrosine kinase enriched in CRPC patient tumors from our previous work, we evaluated whether inhibition of SRC kinase synergizes with enzalutamide or chemotherapy in several prostate cancer cell lines expressing variable AR isoforms. We observed robust synergy between the SRC kinase inhibitor, saracatinib, and enzalutamide, in the AR-FL+/AR-V+ CRPC cell lines, LNCaP95 and 22Rv1. We also observed that saracatinib significantly decreases AR Y534 phosphorylation, a key SRC kinase substrate residue, on AR-FL and AR-Vs, along with the AR regulome, supporting key mechanisms of synergy with enzalutamide. Lastly, we also found that the saracatinib-enzalutamide combination reduced DNA replication compared to the saracatinib-docetaxel combination, resulting in marked increased apoptosis. By elucidating this combination strategy, we provide pre-clinical data that suggests combining SRC kinase inhibitors with enzalutamide in select patients that express both AR-FL and AR-Vs.
RESUMO
Prostate cancer (PCa) remains the most diagnosed non-skin cancer amongst the American male population. Treatment for localized prostate cancer consists of androgen deprivation therapies (ADTs), which typically inhibit androgen production and the androgen receptor (AR). Though initially effective, a subset of patients will develop resistance to ADTs and the tumors will transition to castration-resistant prostate cancer (CRPC). Second generation hormonal therapies such as abiraterone acetate and enzalutamide are typically given to men with CRPC. However, these treatments are not curative and typically prolong survival only by a few months. Several resistance mechanisms contribute to this lack of efficacy such as the emergence of AR mutations, AR amplification, lineage plasticity, AR splice variants (AR-Vs) and increased kinase signaling. Having identified SRC kinase as a key tyrosine kinase enriched in CRPC patient tumors from our previous work, we evaluated whether inhibition of SRC kinase synergizes with enzalutamide or chemotherapy in several prostate cancer cell lines expressing variable AR isoforms. We observed robust synergy between the SRC kinase inhibitor, saracatinib, and enzalutamide, in the AR-FL+/AR-V+ CRPC cell lines, LNCaP95 and 22Rv1. We also observed that saracatinib significantly decreases AR Y 534 phosphorylation, a key SRC kinase substrate residue, on AR-FL and AR-Vs, along with the AR regulome, supporting key mechanisms of synergy with enzalutamide. Lastly, we also found that the saracatinib-enzalutamide combination reduced DNA replication compared to the saracatinib-docetaxel combination, resulting in marked increased apoptosis. By elucidating this combination strategy, we provide pre-clinical data that suggests combining SRC kinase inhibitors with enzalutamide in select patients that express both AR-FL and AR-Vs.
RESUMO
Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by â¼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Precursores Enzimáticos , Engenharia de Proteínas , gama-Glutamil Hidrolase , Domínio Catalítico , Desenho de Fármacos/métodos , Precursores Enzimáticos/química , Precursores Enzimáticos/farmacologia , Humanos , Células PC-3 , Engenharia de Proteínas/métodos , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/farmacologiaRESUMO
The increased treatment of metastatic castration-resistant prostate cancer (mCRPC) with second-generation antiandrogen therapies (ADT) has coincided with a greater incidence of lethal, aggressive variant prostate cancer (AVPC) tumors that have lost dependence on androgen receptor (AR) signaling. These AR-independent tumors may also transdifferentiate to express neuroendocrine lineage markers and are termed neuroendocrine prostate cancer (NEPC). Recent evidence suggests kinase signaling may be an important driver of NEPC. To identify targetable kinases in NEPC, we performed global phosphoproteomics comparing several AR-independent to AR-dependent prostate cancer cell lines and identified multiple altered signaling pathways, including enrichment of RET kinase activity in the AR-independent cell lines. Clinical NEPC patient samples and NEPC patient-derived xenografts displayed upregulated RET transcript and RET pathway activity. Genetic knockdown or pharmacologic inhibition of RET kinase in multiple mouse and human models of NEPC dramatically reduced tumor growth and decreased cell viability. Our results suggest that targeting RET in NEPC tumors with high RET expression could be an effective treatment option. Currently, there are limited treatment options for patients with aggressive neuroendocrine prostate cancer and none are curative. IMPLICATIONS: Identification of aberrantly expressed RET kinase as a driver of tumor growth in multiple models of NEPC provides a significant rationale for testing the clinical application of RET inhibitors in patients with AVPC.
Assuntos
Carcinoma Neuroendócrino/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Proteômica/métodos , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Camundongos , Células PC-3 , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor suppressor p53 is frequently mutated in human cancer. Mutant p53 often promotes tumor progression through gain-of-function (GOF) mechanisms. However, the mechanisms underlying mutant p53 GOF are not well understood. In this study, we found that mutant p53 activates small GTPase Rac1 as a critical mechanism for mutant p53 GOF to promote tumor progression. Mechanistically, mutant p53 interacts with Rac1 and inhibits its interaction with SUMO-specific protease 1 (SENP1), which in turn inhibits SENP1-mediated de-SUMOylation of Rac1 to activate Rac1. Targeting Rac1 signaling by RNAi, expression of the dominant-negative Rac1 (Rac1 DN), or the specific Rac1 inhibitor NSC23766 greatly inhibits mutant p53 GOF in promoting tumor growth and metastasis. Furthermore, mutant p53 expression is associated with enhanced Rac1 activity in clinical tumor samples. These results uncover a new mechanism for Rac1 activation in tumors and, most importantly, reveal that activation of Rac1 is an unidentified and critical mechanism for mutant p53 GOF in tumorigenesis, which could be targeted for therapy in tumors containing mutant p53.
Assuntos
Carcinogênese/genética , Mutação , Sumoilação , Proteína Supressora de Tumor p53/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Progressão da Doença , Humanos , Metástase Neoplásica , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Phosphoproteomic analysis of tumor samples has the potential to uncover significant insights into kinase signaling networks present in late stage prostate cancer that are complementary to genomic and transcriptomic approaches. Phosphoproteomics could potentially aid drug development in clinical trial design as well as provide utility for oncologists in the personalized therapeutic management of individual cancers through identifying novel biomarkers and druggable targets. Rapid advancement of targeted mass spectrometry platforms is underway to integrate phosphoproteomic technology with genomic assays to soon translate this information into the cancer clinic.
RESUMO
Integration of phosphoproteomics with traditional genomics and transcriptomics provides a more comprehensive overview of the signaling networks in advanced prostate cancer for immediate preclinical and future clinical use. Our recent publication introduces computational approaches for integrating the phosphoproteome, specifically with the intent of identifying important kinase signaling networks in advanced-stage prostate cancer.