Epigenetic Modifier UHRF1 May Be a Potential Target in Malignant Pleural Mesothelioma

Exposure to multiple environmental contaminants has been linked to an increased risk of several cancers. A case in point is asbestos exposure and the associated mesothelioma risk, which was first conclusively reported in a study from South Africa in 1960.1Wagner J.C. Sleggs C.A. Marchand P. Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province.Br J Ind Med. 1960; 17: 260-271PubMed Google Scholar Despite bans and the reduced use of asbestos in many jurisdictions, it is still widely used in several industrialized and developing countries.2Chen T. Sun X.M. Wu L. High time for complete ban on asbestos use in developing countries.JAMA Oncol. 2019; 5: 779-780Crossref PubMed Scopus (9) Google Scholar Recent data from Global Cancer Incidence, Mortality and Prevalence indicate just over 30,000 cases and in excess of 25,000 deaths because of malignant pleural mesothelioma (MPM)3Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries [published correction appears in CA Cancer J Clin. 2020;70:313].CA Cancer J Clin. 2018; 68: 394-424Crossref PubMed Scopus (31864) Google Scholar; however, many believe this to be an underestimate of both the incidence and mortality of this lethal disease. Given the significant latency between exposure and the development of overt disease, cases of MPM are expected to increase for some time when combined with the so-called “third wave” exposure.4Carbone M. Adusumilli P.S. Alexander Jr., H.R. et al.Mesothelioma: scientific clues for prevention, diagnosis, and therapy [published correction appears in CA Cancer J Clin. 2020;70:313–314].CA Cancer J Clin. 2019; 69: 402-429Crossref PubMed Scopus (111) Google Scholar Most people diagnosed with MPM tend to present with late-stage refractory disease, in which standard first-line therapy, consisting of cisplatin-pemetrexed, has largely remained unchanged since 2004.5Vogelzang N.J. Rusthoven J.J. Symanowski J. et al.Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma.J Clin Oncol. 2003; 21: 2636-2644Crossref PubMed Scopus (2267) Google Scholar Clinical trials have reported the added benefit of bevacizumab,6Dowell J.E. Dunphy F.R. Taub R.N. et al.A multicenter phase II study of cisplatin, pemetrexed, and bevacizumab in patients with advanced malignant mesothelioma.Lung Cancer. 2012; 77: 567-571Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar and more recently, the advent of immunotherapy has initiated many clinical trials in the mesothelioma space, with results eagerly awaited.7de Gooijer C.J. Borm F.J. Scherpereel A. Baas P. Immunotherapy in malignant pleural mesothelioma.Front Oncol. 2020; 10: 187Crossref PubMed Scopus (24) Google Scholar The combination of durvalumab, cisplatin, and pemetrexed has exhibited promising results in the DREAM (Durvalumab with first-line chemotherapy in previously untreated malignant pleural mesothelioma) trial,8Nowak A.K. Lesterhuis W.J. Kok P.S. et al.Durvalumab with first-line chemotherapy in previously untreated malignant pleural mesothelioma (DREAM): a multicentre, single-arm, phase 2 trial with a safety run-in.Lancet Oncol. 2020; 21: 1213-1223Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar and data presented at the virtual presidential symposium at the World Conference on Lung Cancer revealed positive results for first-line nivolumab plus ipilimumab versus chemotherapy in nonepithelioid mesothelioma.9Baas P. Scherpereel A. Nowak A. et al.ID:2908 first-line nivolumab + ipilimumab vs chemotherapy in unresectable malignant pleural mesothelioma: CheckMate 743.J Thorac Oncol. 2020; 15: E42Abstract Full Text Full Text PDF PubMed Google Scholar Further clinical trials are evaluating immunotherapy combined with epigenetic modifiers, given their known immunomodulatory effects.10Aspeslagh S. Morel D. Soria J.C. Postel-Vinay S. Epigenetic modifiers as new immunomodulatory therapies in solid tumours.Ann Oncol. 2018; 29: 812-824Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar This may also hold promise in MPM, as epigenetic changes are frequently identified in this disease.11McLoughlin K.C. Kaufman A.S. Schrump D.S. Targeting the epigenome in malignant pleural mesothelioma.Transl Lung Cancer Res. 2017; 6: 350-365Crossref PubMed Scopus (21) Google Scholar,12Ferrari L. Carugno M. Mensi C. Pesatori A.C. Circulating epigenetic biomarkers in malignant pleural mesothelioma: state of the art and critical evaluation.Front Oncol. 2020; 10: 445Crossref PubMed Scopus (8) Google Scholar One such epigenetic modifier is UHRF1 (also known as ICBP90 and Np95), which is a multidomain protein that principally serves to recruit DNA methyltransferase (DNMT) 1 to newly synthesized DNA. Increased levels of UHRF1 are evident in several tumor types, leading to global DNA methylation and histone modifications, primarily in tumor suppressor genes and oncogenes, thus contributing to carcinogenesis.13Ashraf W. Ibrahim A. Alhosin M. et al.The epigenetic integrator UHRF1: on the road to become a universal biomarker for cancer.Oncotarget. 2017; 8: 51946-51962Crossref PubMed Scopus (46) Google Scholar However, little is known regarding this marker in MPM, which this recent study by Reardon et al.14Reardon E.S. Shukla V. Xi S. et al.UHRF1 is a novel druggable epigenetic target in malignant pleural mesothelioma.J Thorac Oncol. 2021; 16: 89-103Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar has sought to address. Initially identified as altered through Affymetrix microarray data, higher levels of UHRF1 mRNA and protein were confirmed in a broad panel of MPM cell lines (n = 9) compared with normal mesothelial cells (n = 2) by Reardon et al.14Reardon E.S. Shukla V. Xi S. et al.UHRF1 is a novel druggable epigenetic target in malignant pleural mesothelioma.J Thorac Oncol. 2021; 16: 89-103Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar. Microarray analysis also revealed increased expression of DNMTs and components of the PRC2. In this study, UHRF1 protein and mRNA levels did not always correspond, which may result, in part, because of the regulation of UHRF1 expression by miRNAs, as reported in other cancer studies.15Choudhry H. Zamzami M.A. Omran Z. et al.Targeting microRNA/UHRF1 pathways as a novel strategy for cancer therapy.Oncol Lett. 2018; 15: 3-10PubMed Google Scholar In addition, in a small cohort of unmatched MPM (n = 49) and normal mesothelial (n = 11) patient tissue samples, UHRF1 was detected in all MPM tissues and not in normal samples. This result is not unexpected, given the substantial literature in other cancers illustrating the same trends.13Ashraf W. Ibrahim A. Alhosin M. et al.The epigenetic integrator UHRF1: on the road to become a universal biomarker for cancer.Oncotarget. 2017; 8: 51946-51962Crossref PubMed Scopus (46) Google Scholar Next, the group determined the impact of asbestos exposure on UHRF1 levels in a normal mesothelial cell line (LP9). After 10 days of exposure to crocidolite fibers, UHRF1 expression levels were significantly elevated compared with untreated controls. The asbestos burden has previously been significantly associated with epigenetic changes in MPM.16Christensen B.C. Houseman E.A. Godleski J.J. et al.Epigenetic profiles distinguish pleural mesothelioma from normal pleura and predict lung asbestos burden and clinical outcome.Cancer Res. 2009; 69: 227-234Crossref PubMed Scopus (120) Google Scholar Analysis of The Cancer Genome Atlas (TCGA) MPM cohort revealed that UHRF1 levels correlated inversely with overall survival (OS), which was validated in an additional data set of 155 people with MPM (Brigham and Women’s Hospital database). The median OS in those with tumors showing high UHRF1 expression was 8.7 months, compared with those with low expression, in which the OS was 22.2 months (hazard ratio [HR] = 3.61, 95% confidence interval [CI]: 2.48–5.28, p < 0.001). This mirrors what has been identified in other cancer types, with higher expression of UHFR1 associated with poor outcomes and shorter survival times.13Ashraf W. Ibrahim A. Alhosin M. et al.The epigenetic integrator UHRF1: on the road to become a universal biomarker for cancer.Oncotarget. 2017; 8: 51946-51962Crossref PubMed Scopus (46) Google Scholar The authors next sought to determine if an association between sex and UHRF1 levels existed, given that sex is an element related to survival in MPM.17Taioli E. Wolf A.S. Camacho-Rivera M. Flores R.M. Women with malignant pleural mesothelioma have a threefold better survival rate than men.Ann Thorac Surg. 2014; 98: 1020-1024Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar When stratified according to sex, the same patterns of high expression and poor OS were observed (epithelioid histologic diagnosis). The median OS for women—high versus low expression—were 14.0 versus 40.7 months (HR = 4.7, 95% CI: 2.5–8.3, p < 0.001). However, men with high expression of UHRF1 had shorter OS (7.5 mo, HR = 7.5, 95% CI: 4.4–12.9, p < 0.001) than women, and those with low expression had a reduced OS compared with women (15.5 mo, HR = 2.4, 95% CI: 1.5–4.2, p < 0.001). To assess phenotypic changes in vitro, UHRF1 levels were subjected to stable knockdown using a short hairpin RNA strategy in three MPM cell lines (MES1, MES7, and H2542). As with other cancer studies, this knockdown resulted in depleted DNMT1 and increased 5-methylcytosine levels (knockdown versus controls). Reduced levels of UHRF1 resulted in decreases in both proliferation levels and soft agar clonogenicity. In cells with UHRF1 knockdown, this resulted in significantly decreased invasion (in vitro) and tumor growth in athymic nude mice (compared with controls). These data are similar to data observed in other cancer studies, and not unexpected, given the link between UHFR1 levels and cell cycle regulation and apoptosis. Given the lack of specific UHRF1 targeted agents in the clinical sphere, the authors next sought to devise a targeting strategy for UHRF1 in MPM. Previous work by the same group has found that the repurposed chemotherapy agent mithramycin depleted SP1 and activated p53 to promote senescence and apoptosis in MPM.18Rao M. Atay S.M. Shukla V. et al.Mithramycin depletes specificity protein 1 and activates p53 to mediate senescence and apoptosis of malignant pleural mesothelioma cells.Clin Cancer Res. 2016; 22: 1197-1210Crossref PubMed Scopus (22) Google Scholar Mithramycin is a clinically approved DNA-binding antitumor antibiotic and has previously been reported to interact with core histones19Banerjee A. Sanyal S. Kulkarni K.K. et al.Anticancer drug mithramycin interacts with core histones: an additional mode of action of the DNA groove binder.FEBS Open Bio. 2014; 4: 987-995Crossref PubMed Scopus (14) Google Scholar and impact DNA methylation patterns.20Lin R.K. Hsu C.H. Wang Y.C. Mithramycin A inhibits DNA methyltransferase and metastasis potential of lung cancer cells.Anticancer Drugs. 2007; 18: 1157-1164Crossref PubMed Scopus (50) Google Scholar Numerous recognition elements for SP1 (activator) and p53 (repressor) have been identified within the UHRF1 promoter, and Reardon et al.,14Reardon E.S. Shukla V. Xi S. et al.UHRF1 is a novel druggable epigenetic target in malignant pleural mesothelioma.J Thorac Oncol. 2021; 16: 89-103Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar in this additional study, have illustrated that treatment with mithramycin reduced DNMT1 levels in MPM cells. In a subcutaneous MES1 xenograft in athymic nude mice, treatment with mithramycin reduced UHRF1 expression; however, the effect on DNMT1 was less remarkable, indicating that other pathways may be in play. Given that UHRF1 has a clear regulatory function in relation to p53, p53 levels were increased in MPM cell lines (MES1, MES7, and H2542) using two treatments: the Reactivating p53 and Inducing Tumor Apoptosis and RG7388 (inhibits HDM2-mediated ubiquitination of p53). Both treatments decreased the levels of UHRF1 in wild-type p53 cell lines (MES1 and MES7); however, only Reactivating p53 and Inducing Tumor Apoptosis was effective in the p53-mutated cell line, H2542. Concomitant increases in p53, and decreased DNMT1 were associated with depleted UHRF1 in MPM cells. Similar trends were observed in vivo, with HDM2 inhibitors decreasing UHRF1 and DNMT1 in an MPM xenograft model. To further assess the transcriptomic effects of UHRF1 inhibition in MPM cells, UHRF1 was knocked down in two MPM cell lines (MES1 and MES7) using the small interfering RNA strategy. Overall, 99 genes were differentially expressed in both cell lines and followed the same pattern of expression between the cells (55 down, 44 up). A total of 83 genes were cancer-associated and were involved in cancer-linked signaling pathways such as p38 MAPK signaling. The same cell lines were then treated with 25 nM mithramycin for 24 hours. Comparing small interfering UHRF1 and mithramycin treated cells, there was an overlap of 50 genes in MES1 and only 17 in MES7. Little overlap was evident in top conical pathways between the two cell lines; however, common themes regarding diseases and biofunctions were evident. Further investigation of the 50 (MES1) and 17 (MES7) gene set was undertaken using the TCGA and Brigham and Women’s Hospital data sets. In MES1, 11 of 50 genes had a positive correlation with UHRF1 (i.e., SAE1), with all 11 genes associated with increased levels of UHRF1 and decreased OS survival in the TCGA data set. In MES7, only one of the 17 genes, DEPDC1, revealed a positive correlation with the expression of UHRF1 in TCGA and was associated with significantly reduced OS in people with MPM. Previous studies have found that targeting the epigenetic machinery is fraught with difficulty, and unfortunately, the preclinical promise is often not translated to clinical success; however, newer studies such as tazemetostat (EZH2 inhibitor) in BAP-mutated MPM are exhibiting promise.21Zauderer M.G. Szlosarek P.W. Le Moulec S. et al.Safety and efficacy of tazemetostat, an enhancer of zeste-homolog 2 inhibitor, in patients with relapsed or refractory malignant mesothelioma.J Clin Oncol. 2020; 38 (9058–9058)Google Scholar This work by Reardon et al.,14Reardon E.S. Shukla V. Xi S. et al.UHRF1 is a novel druggable epigenetic target in malignant pleural mesothelioma.J Thorac Oncol. 2021; 16: 89-103Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar has identified a number of UHRF1-responsive targets and exhibited value in pursuing indirect and direct targeting of UHRF1 in MPM. Dual targeting with other epigenetic therapies and mithramycin may provide a potential vulnerability to exploit in the future, with further studies on this critical epigenetic modifier warranted. UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural MesotheliomaJournal of Thoracic OncologyVol. 16Issue 1PreviewUbiquitin-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. Full-Text PDF