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USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

ABSTRACT: 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.

SUBMITTER: Karapurkar JK

PROVIDER: S-EPMC10948558 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

Karapurkar Janardhan Keshav JK Colaco Jencia Carminha JC Suresh Bharathi B Tyagi Apoorvi A Woo Sang Hyeon SH Jo Won-Jun WJ Ko Nare N Singh Vijai V Hong Seok-Ho SH Oh Seung Jun SJ Kim Kye-Seong KS Ramakrishna Suresh S

Cellular and molecular life sciences : CMLS 20240318 1

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- ...[more]

PMID: 38498222

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Project description:BackgroundBreast cancer is the most prevalent cancer in women globally. Over-activated estrogen receptor (ER) α signaling is considered the main factor in luminal breast cancers, which can be effectively managed with selective estrogen receptor modulators (SERMs) like tamoxifen. However, approximately 30-40% of ER + breast cancer cases are recurrent after tamoxifen therapy. This implies that the treatment of breast cancer is still hindered by resistance to tamoxifen. Recent studies have suggested that post-translational modifications of ERα play a significant role in endocrine resistance. The stability of both ERα protein and its transcriptome is regulated by a balance between E3 ubiquitin ligases and deubiquitinases. According to the current knowledge, approximately 100 deubiquitinases are encoded in the human genome, but it remains unclear which deubiquitinases play a critical role in estrogen signaling and endocrine resistance. Thus, decoding the key deubiquitinases that significantly impact estrogen signaling, including the control of ERα expression and stability, is critical for the improvement of breast cancer therapeutics.MethodsWe used several ER positive breast cancer cell lines, DUB siRNA library screening, xenograft models, endocrine-resistant (ERα-Y537S) model and performed immunoblotting, real time PCR, RNA sequencing, immunofluorescence, and luciferase activity assay to investigate the function of USP36 in breast cancer progression and tamoxifen resistance.ResultsIn this study, we identify Ubiquitin-specific peptidase 36 (USP36) as a key deubiquitinase involved in ERα signaling and the advancement of breast cancer by deubiquitinases siRNA library screening. In vitro and in vivo studies showed that USP36, but not its catalytically inactive mutant (C131A), could promote breast cancer progression through ERα signaling. Conversely, silencing USP36 inhibited tumorigenesis. In models resistant to endocrine therapy, silencing USP36 destabilized the resistant form of ERα (Y537S) and restored sensitivity to tamoxifen. Molecular studies indicated that USP36 inhibited K48-linked polyubiquitination of ERα and enhanced the ERα transcriptome. It is interesting to note that our results suggest USP36 as a novel biomarker for treatment of breast cancer.ConclusionOur study revealed the possibility that inhibiting USP36 combined with tamoxifen could provide a potential therapy for breast cancer.

Project description:Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.

Project description:IntroductionCisplatin-based chemotherapy is one of the fundamental therapeutic modalities for gastric cancer (GC). Chemoresistance to cisplatin is a great clinical challenge, and its underlying mechanisms remain poorly understood. Circular RNAs (circRNAs) are involved in the pathophysiology of multiple human malignancies.MethodsHigh-throughput sequencing was performed to determine the differentially expressed profile of circRNA in GC tissues and cisplatin-resistant GC cells. Quantitative real-time polymerase chain reaction and Fluorescence in situ hybridization was utilized to confirm the dysregulation of circ_0008315 in GC tissues. To evaluate the prognostic significance of circ_0008315 in GC, we used Kaplan-Meier plot. The self-renewal ability of drug-resistant GC cell was verified through tumor sphere formation assay. GC organoids were constructed to simulate the tumor microenvironment and verified the function of circ_0008315 in cisplatin resistance of gastric cancer. In vivo evaluation was conducted using patient-derived xenograft models. Dual-luciferase reporter gene, RNA immunoprecipitation and miRNA pull-down assays were employed to investigate the molecular mechanisms of circ_0008315 in GC.ResultsWe revealed that a novel circRNA hsa_circ_0008315 was upregulated in GC and cisplatin-resistant GC cells. Elevated circ_0008315 was also observed in cisplatin-resistant GC organoid model. High circ_0008315 expression predicted unfavorable survival outcome in GC patients. Downregulation of circ_0008315 expression inhibited proliferation, mobility, and epithelial-mesenchymal transition of GC cells in vitro and in vivo. Reducing circ_0008315 expression in cisplatin-resistant GC organoid model reversed cisplatin resistance. Mechanistically, circ_0008315 modulated the stem cell properties of GC through the miR-3666/CPEB4 signaling pathway, thereby promoting cisplatin resistance and GC malignant progression. Furthermore, we developed PLGA-PEG nanoparticles targeting circ_0008315, and the nanoparticles could effectively inhibit GC proliferation and cisplatin resistance.ConclusionCirc_0008315 exacerbates GC progression and cisplatin resistance, and can be used as a prognostic predictor. Circ_0008315 may function as a promising nanotherapeutic target for GC treatment.

Dataset Information

USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

Publications

USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

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