High PRMT5 levels, maintained by KEAP1 inhibition, drive chemoresistance in high-grade serous ovarian cancer
Harun Ozturk, Fidan Seker-Polat, Neda Abbaszadeh, Yasemin Kingham, Sandra Orsulic, Mazhar Adli
Harun Ozturk, Fidan Seker-Polat, Neda Abbaszadeh, Yasemin Kingham, Sandra Orsulic, Mazhar Adli
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Research Article Cell biology Genetics Oncology

High PRMT5 levels, maintained by KEAP1 inhibition, drive chemoresistance in high-grade serous ovarian cancer

Abstract

Protein arginine methyl transferases (PRMTs) are generally upregulated in cancers. However, the mechanisms leading to this upregulation and its biological consequences are poorly understood. Here, we identify PRMT5, the main symmetric arginine methyltransferase, as a critical driver of chemoresistance in high-grade serous ovarian cancer (HGSOC). PRMT5 levels and its enzymatic activity are induced in a platinum-resistant (Pt-resistant) state at the protein level. To reveal potential regulators of high PRMT5 protein levels, we optimized intracellular immunostaining conditions and performed unbiased CRISPR screening. We identified Kelch-like ECH-associated protein 1 (KEAP1) as a top-scoring negative regulator of PRMT5. Our mechanistic studies show that KEAP1 directly interacted with PRMT5, leading to its ubiquitin-dependent degradation under normal physiological conditions. At the genomic level, ChIP studies showed that elevated PRMT5 directly interacted with the promoters of stress response genes and positively regulated their transcription. Combined PRMT5 inhibition with Pt resulted in synergistic cellular cytotoxicity in vitro and reduced tumor growth in vivo in Pt-resistant patient-derived xenograft tumors. Overall, the findings from this study identify PRMT5 as a critical therapeutic target in Pt-resistant HGSOC cells and reveal the molecular mechanisms that lead to high PRMT5 levels in Pt-treated and chemo-resistant tumors.

Authors

Harun Ozturk, Fidan Seker-Polat, Neda Abbaszadeh, Yasemin Kingham, Sandra Orsulic, Mazhar Adli

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Figure 2

PRMT5 protein levels are further induced in chemotherapy-treated and chemotherapy-resistant HGSOC tumors.

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PRMT5 protein levels are further induced in chemotherapy-treated and che...
(A) Schematic displays TMAs used for PRMT5 staining. Figure was created with BioRender.com. (B) Representative IHC images show QuPath annotation of tumor stroma and epithelial cells (left), and a violin plot shows PRMT5 staining intensity in stroma versus tumor epithelial cells (right). P value was determined by Wilcoxon rank-sum test. Original magnification: ×5. (C) Representative IHC images show PRMT5 staining in primary and recurrent tumors. Original magnification: ×5. (D) The density plot displays H-scores for PRMT5 staining in primary tumors (left), and the dot plot shows PRMT5 levels (H-score) between primary and recurrent tumors for the primary tumors with low PRMT5 staining (right). P values were quantified by 2-tailed, paired Student’s t test. (E) Western blots show PRMT5 and SDMA levels in chemo-naive and -resistant isogenic cell line pairs. Noncontiguous lanes on different blots have been separated by thin dashed lines. (F) Western blots show PRMT5 expression after different doses of Dox induction (72 hours) in chemo-naive OVCAR4 cells. Carboplatin-resistant OVCAR4 cells were used to determine Dox levels for overexpression. (G) Line plots show the relative apoptosis rate of OVCAR4 cells treated with the indicated doses of Dox. Cells were subjected to 40 μM carboplatin, and apoptosis (caspase 3/7 activity) was monitored by the IncuCyte live-cell imaging platform for 72 hours. Data are shown as the mean ± SEM (n = 3). P values were quantified by 1-way ANOVA with Dunnett’s multiple-comparison test. (H) Western blots show PRMT5 levels in OVCAR4 cells expressing sgRNAs targeting PRMT5 promoter. (I) Line plots show the relative apoptosis rates of OVCAR4 cells expressing the indicated sgRNAs. Cells were treated with 20 μM carboplatin, and apoptosis was monitored over 72 hours using the IncuCyte live-cell imaging platform. Data are shown as the mean ± SEM (n = 3). P values were determined by 1-way ANOVA with Dunnett’s multiple-comparison test.