RESUMO
Cisplatin is a chemotherapy drug that causes a plethora of DNA lesions and inhibits DNA transcription and replication, resulting in the induction of apoptosis in cancer cells. However, over time, patients develop resistance to cisplatin due to repeated treatment and thus the treatment efficacy is limited. Therefore, identifying an alternative therapeutic strategy combining cisplatin treatment along with targeting factors that drive cisplatin resistance is needed. CRISPR/Cas9 system-based genome-wide screening for the deubiquitinating enzyme (DUB) subfamily identified USP28 as a potential DUB that governs cisplatin resistance. USP28 regulates the protein level of microtubule-associated serine/threonine kinase 1 (MAST1), a common kinase whose expression is elevated in several cisplatin-resistant cancer cells. The expression level and protein turnover of MAST1 is a major factor driving cisplatin resistance in many cancer types. Here we report that the USP28 interacts and extends the half-life of MAST1 protein by its deubiquitinating activity. The expression pattern of USP28 and MAST1 showed a positive correlation across a panel of tested cancer cell lines and human clinical tissues. Additionally, CRISPR/Cas9-mediated gene knockout of USP28 in A549 and NCI-H1299 cells blocked MAST1-driven cisplatin resistance, resulting in suppressed cell proliferation, colony formation ability, migration and invasion in vitro. Finally, loss of USP28 destabilized MAST1 protein and attenuated tumor growth by sensitizing cells to cisplatin treatment in mouse xenograft model. We envision that targeting the USP28-MAST1 axis along with cisplatin treatment might be an alternative therapeutic strategy to overcome cisplatin resistance in cancer patients.
Assuntos
Cisplatino , Neoplasias , Animais , Humanos , Camundongos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas Associadas aos Microtúbulos , Microtúbulos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina TiolesteraseRESUMO
Cell division cycle 25A (Cdc25A) is a dual-specificity phosphatase that is overexpressed in several cancer cells and promotes tumorigenesis. In normal cells, Cdc25A expression is regulated tightly, but the changes in expression patterns in cancer cells that lead to tumorigenesis are unknown. In this study, we showed that ubiquitin-specific protease 29 (USP29) stabilized Cdc25A protein expression in cancer cell lines by protecting it from ubiquitin-mediated proteasomal degradation. The presence of USP29 effectively blocked polyubiquitination of Cdc25A and extended its half-life. CRISPR-Cas9-mediated knockdown of USP29 in HeLa cells resulted in cell cycle arrest at the G0/G1 phase. We also showed that USP29 knockdown hampered Cdc25A-mediated cell proliferation, migration, and invasion of cancer cells in vitro. Moreover, NSG nude mice transplanted with USP29-depleted cells significantly reduced the size of the tumors, whereas the reconstitution of Cdc25A in USP29-depleted cells significantly increased the tumor size. Altogether, our results implied that USP29 promoted cell cycle progression and oncogenic transformation by regulating protein turnover of Cdc25A.
Assuntos
Carcinogênese/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteases Específicas de Ubiquitina/metabolismo , Fosfatases cdc25/metabolismo , Animais , Apoptose , Sistemas CRISPR-Cas , Carcinogênese/genética , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Oncogenes , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Fosfatases cdc25/genéticaRESUMO
Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the degradation pathway of the amino acids tyrosine and phenylalanine in mammals that catalyzes the hydrolysis of 4-fumarylacetoacetate into acetoacetate and fumarate. Mutations of the FAH gene are associated with hereditary tyrosinemia type I (HT1), resulting in reduced protein stability, misfolding, accelerated degradation and deficiency in functional proteins. Identifying E3 ligases, which are necessary for FAH protein stability and degradation, is essential. In this study, we demonstrated that the FAH protein level is elevated in liver cancer tissues compared to that in normal tissues. Further, we showed that the FAH protein undergoes 26S proteasomal degradation and its protein turnover is regulated by the anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 E3 ubiquitin ligase complex. APC/CCdh1 acts as a negative stabilizer of FAH protein by promoting FAH polyubiquitination and decreases the half-life of FAH protein. Thus, we envision that Cdh1 might be a key factor in the maintenance of FAH protein level to regulate FAH-mediated physiological functions.
