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The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors.

ABSTRACT: Y-box-binding protein 1 (YB-1) is a multifunctional cellular factor overexpressed in tumors resistant to chemotherapy. An intrinsically disordered structure together with a high positive charge peculiar to YB-1 allows this protein to function in almost all cellular events related to nucleic acids including RNA, DNA and poly(ADP-ribose) (PAR). In the present study we show that YB-1 acts as a potent poly(ADP-ribose) polymerase 1 (PARP1) cofactor that can reduce the efficiency of PARP1 inhibitors. Similarly to that of histones or polyamines, stimulatory effect of YB-1 on the activity of PARP1 was significantly higher than the activator potential of Mg2+ and was independent of the presence of EDTA. The C-terminal domain of YB-1 proved to be indispensable for PARP1 stimulation. We also found that functional interactions of YB-1 and PARP1 can be mediated and regulated by poly(ADP-ribose).

SUBMITTER: Alemasova EE

PROVIDER: S-EPMC5955111 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors.

Alemasova Elizaveta E EE Naumenko Konstantin N KN Kurgina Tatyana A TA Anarbaev Rashid O RO Lavrik Olga I OI

Oncotarget 20180504 34

Y-box-binding protein 1 (YB-1) is a multifunctional cellular factor overexpressed in tumors resistant to chemotherapy. An intrinsically disordered structure together with a high positive charge peculiar to YB-1 allows this protein to function in almost all cellular events related to nucleic acids including RNA, DNA and poly(ADP-ribose) (PAR). In the present study we show that YB-1 acts as a potent poly(ADP-ribose) polymerase 1 (PARP1) cofactor that can reduce the efficiency of PARP1 inhibitors. ...[more]

PMID: 29805738

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Project description:BackgroundCyclic-dependent kinase (CDK) 4/6 kinases, as the critical drivers of the cell cycle, are involved in the tumor progression of various malignancies. Pharmacologic inhibitors of CDK4/6 have shown significant clinical prospects in treating hormone receptor-positive and human epidermal growth factor receptor-negative (HR + /HER2-) breast cancer (BC) patients. However, acquired resistance to CDK4/6 inhibitors (CDK4/6i), as a common issue, has developed rapidly. It is of great significance that the identification of novel therapeutic targets facilitates overcoming the CDK4/6i resistance. PARP1, an amplified gene for CDK4/6i-resistant patients, was found to be significantly upregulated during the construction of CDK4/6i-resistant strains. Whether PARP1 drives CDK4/6i resistance in breast cancer is worth further study.MethodPARP1 and p-YB-1 protein levels in breast cancer cells and tissues were quantified using Western blot (WB) analysis, immunohistochemical staining (IHC) and immunofluorescence (IF) assays. Bioinformatics analyses of Gene Expression Profiling Interactive Analysis (GEPIA), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets were applied to explore the relationship between YB-1/PARP1 protein levels and CDK4/6i IC50. Cell Counting Kit-8 (CCK-8) and crystal violet staining assays were performed to evaluate cell proliferation rates and drug killing effects. Flow cytometry assays were conducted to assess apoptosis rates and the G1/S ratio in the cell cycle. An EdU proliferation assay was used to detect the DNA replication ratio after treatment with PARP1 and YB-1 inhibitors. A ChIP assay was performed to assess the interaction of the transcription factor YB-1 and associated DNA regions. A double fluorescein reporter gene assay was designed to assess the influence of WT/S102A/S102E YB-1 on the promoter region of PARP1. Subcutaneous implantation models were applied for in vivo tumor growth evaluations.ResultsHere, we reported that PARP1 was amplified in breast cancer cells and CDK4/6i-resistant patients, and knockdown or inhibition of PARP1 reversed drug resistance in cell experiments and animal models. In addition, upregulation of transcription factor YB-1 also occurred in CDK4/6i-resistant breast cancer, and YB-1 inhibition can regulate PARP1 expression. p-YB-1 and PARP1 were upregulated when treated with CDK4/6i based on the WB and IF results, and elevated PARP1 and p-YB-1 were almost simultaneously observed during the construction of MCF7AR-resistant strains. Inhibition of YB-1 or PAPR1 can cause decreased DNA replication, G1/S cycle arrest, and increased apoptosis. We initially confirmed that YB-1 can bind to the promoter region of PARP1 through a ChIP assay. Furthermore, we found that YB-1 phosphorylated at S102 was crucial for PARP1 transcription according to the double fluorescein reporter gene assay. The combination therapy of YB-1 inhibitors and CDK4/6i exerted a synergistic antitumor effect in vitro and in vivo. The clinical data suggested that HR + /HER2- patients with low expression of p-YB-1/PARP1 may be sensitive to CDK4/6i in breast cancer.ConclusionThese findings indicated that a ''YB-1/PARP1'' loop conferred resistance to CDK4/6 inhibitors. Furthermore, interrupting the loop can enhance tumor killing in the xenograft tumor model, which provides a promising strategy against drug resistance in breast cancer.

Project description:BackgroundEnhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.MethodsWe investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.ResultsTrabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.ConclusionsPARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

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The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors.

Publications

The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors.

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