UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma

Abstract

Introduction: Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM.

Methods: Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression.

Results: UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM.

Conclusions: UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

Keywords: DNMT1; Epigenetics; Malignant pleural mesothelioma; Prognosis; Therapeutic target; UHRF1.

Figure 1:. UHRF1 expression in MPM.

(A) qRT-PCR and immunoblot analyses demonstrating elevated UHRF1 expression in cultured MPM cells relative to normal mesothelial cells. (B) Results of IHC analysis of UHRF1 expression in MPM specimens and unmatched normal pleura. (C) qRT-PCR and immunoblot analyses demonstrating dose-dependent induction of UHRF1 expression in normal mesothelioma cells following 10-day crocidolite exposure. (D) Results of TCGA analysis demonstrating that UHRF1 expression levels correlate inversely with OS in MPM patients. (E) left panel: Results of BWH database analysis demonstrating negative impact of UHRF1 over-expression on OS of MPM patients; right panel: analysis of BWH database demonstrating impact of UHRF1 expression (high vs low) on gender specific OS of patients with epithelioid MPM. * p<0.05; ** p<0.01.

Figure 2:. Phenotypic effects of UHRF1 knock-down in MPM cells.

(A) qRT-PCR and corresponding immunoblot analyses of UHRF1 expression in parental MPM cells, and respective MPM cells transduced with either control shRNA or shRNA targeting UHRF1. (B) left panel: confocal microscopy images demonstrating that UHRF1 knock-down depletes UHRF1 as well as DNMT1 in MPM cells; right panel: confocal experiments demonstrating markedly reduced 5-mC immunoreactivity indicative of genomic hypomethylation in MPM cells relative to normal mesothelioma cells (LP9). Knock-down of UHRF1 increases 5-mC levels in MPM cells. (C) left panel: in-vitro proliferation of MES1 cells following transduction with shRNA targeting UHRF1 or control sequences; middle panel: soft agar colony formation by UHRF1 knock-down MES1 cells relative to respective controls; right panel: representative results demonstrating that knock-down of UHRF1 inhibits soft agar invasion of MES1 cells. (D) left panel: effects of UHRF1 knock-down on growth of subcutaneous MES1 xenografts in athymic nude mice; middle panel: depletion of UHRF1 significantly decreased volumes and masses of UHRF1 knock-down xenografts relative to vector control xenografts; right panel: IHC analysis of demonstrating decreased UHRF1 and Ki67 levels in MES1 UHRF1 knock-down xenografts relative to control xenografts. ** p<0.01; *** p<0.001; **** p<0.0001.

Figure 3:. Pharmacologic strategies to target UHRF1 expression in MPM.

(A) left panel and right panels: qRT-PCR and immunoblot assays demonstrating dose dependent decreases of UHRF1 and DNMT1 expression following 24 hour mithramycin (MM) exposure in-vitro, or in MPM xenografts following IP mithramycin (1 mg/kg weekly x 3 weeks). (B) Confocal images demonstrating increased 5-mC immunoreactivity in MPM cells following 24 hour mithramycin exposure. (C) Immunoblot analysis demonstrating effects of HDM2 inhibitors on UHRF1 and DNMT1 levels in MPM cells in-vitro. (D) Immunoblot analysis demonstrating decreased UHRF1 and DNMT1 levels in MPM xenografts following IP RITA treatment (2 mg/kg q Tuesday-Wednesday-Thursday x 3 weeks).

Figure 4:. Transcriptomic Effects of UHRF1 Knock-down and Mithramycin Treatment in MPM Cells.

(A) left panel: Venn diagram depicting differentially expressed genes in MES1 and MES7 following UHRF1 knock-down. Middle and right panels: top canonical pathways and top diseases and biofunctions associated with the 99 commonly regulated genes in MES1 and MES7 cells following UHRF1 knock-down. (B) left panel: Venn diagram depicting differentially expressed genes in MES1 following UHRF1 knock-down or mithramycin treatment; middle and right panels: top canonical pathways and top diseases and biofunctions associated with the 50 commonly regulated genes in MES1 cells following UHRF1 knock-down or mithramycin treatment. (C) left panel: Venn diagram depicting differentially expressed genes in MES7 following UHRF1 knock-down or mithramycin treatment; middle and right panels: top canonical pathways and top diseases and biofunctions associated with the 17 commonly regulated genes in MES7 cells following UHRF1 knock-down or mithramycin treatment. (D) left panel: Correlation of SAE1 with UHRF1 in MPM from TCGA; middle and right panels: Kaplan Meier curves demonstrating that SAE1 expression is inversely associated with OS of MPM patients in TCGA and BWH databases. (E) left panel: correlation of DEPDC1 with UHRF1 expression in MPM from TCGA-meso; middle and right panels: Kaplan Meier curves demonstrating that DEPDC1 expression is inversely associated with OS of MPM patients in TCGA and BWH databases.

Figure 5:. Potential Biomarkers of UHRF1 Expression with Clinical Relevance in MPM.

TCGA data demonstrating correlations of various potential downstream targets with UHRF1 expression (left panels) with respective Kaplan Meier curves (right panels) showing that expression of each of these targets correlates inversely with OS of MPM patients.