Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers

Notaro et al. BMC Cancer (2016) 16:589 DOI 10.1186/s12885-016-2637-y RESEARCH ARTICLE Open Access Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers Sara Notaro1,2, Daniel Reimer1, Heidi Fiegl1, Gabriel Schmid1, Annamarie Wiedemair1, Julia Rössler1, Christian Marth1 and Alain Gustave Zeimet1* Abstract Background: In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer. Methods: Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique. Results: No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012). Conclusion: No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a reliable indicator of improved platinum responsiveness reflecting a transient better one-year follow up outcome in highly FOLR1 expressing type I cancers. An independent prognostic role of global DNA hypomethylation was demonstrated in type I tumours. Keywords: Ovarian cancer, Folate receptor 1, FOLR1, FOLR1 promoter methylation, Global DNA hypomethylation, Type I, Type II, Platinum sensitivity * Correspondence: 1 Department of Obstetrics and Gynecology, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Notaro et al. BMC Cancer (2016) 16:589 Background Ovarian carcinomas account for the highest mortality among all gynecological cancers in the world [1]. Despite aggressive treatment including highly offensive surgical cytoreduction and multiagent chemotherapy, the prognosis of patients with advanced ovarian cancer remains unacceptably poor. Folic acid is an essential component in DNA synthesis, replication and repair, protein synthesis and methylation reactions. This is especially true for rapidly dividing cells [2]. Three different mechanisms exist for cellular folate uptake: one via the membrane-associated Folate Receptor (FOLR) and the physiologically more important mechanism via the Reduced Folate Carrier (RFC). More recently, a third, the proton-coupled folate transporter, responsible exclusively for the intestinal uptake of folate, was identified [3, 4]. FOLR internalizes folates by means of receptor mediated endocytosis and RFC uses a bidirectional anionexchange mechanism to transport folates into cytoplasm [5, 6]. Human FOLR is encoded by a family of genes whose homologous products are the FOLR types -α, -β and –γ. Principally FOLR isoforms α and β are capable of transporting folate into cells, but generally the ubiquitously expressed RFC is exclusively used for this purpose by adult tissues [7]. In fact, most normal tissues virtually lack FOLR, and its physiologic importance appears to be confined to situations where the availability of folate is limited [8]. In contrast, the ability of FOLR1 (encodes for folate receptor α) to bind folate has been demonstrated in malignant tissue [9, 10]. Under such pathological circumstances FOLR1 may be overexpressed to increase folate uptake in order to cope with the augmented turnover of nucleic acid synthesis and reparation during accelerated cellular growth [11, 12]. FOLR1 was found to be strongly expressed in renal, pancreatic, endometrial carcinomas, squamous cervical cancer and ovarian cancer [13]. Furthermore, FOLR1 is reported to be expressed in the majority of non-mucinous epithelial ovarian cancers at levels 10- to 100-fold higher than its normal expression in the kidney and on lung and breast epithelial cells [14]. Recently, it was shown that FOLR1 can be exploited for specific delivery of drugs linked to folic acid into ovarian cancer cells. Proof of principle was provided with folatedesacetyl vinblastine monohydrazide (Vintafolide®) in the clinical PRECEDENT trial in ovarian cancer. Moreover, companion diagnostics with radio-labelled folate (Etarfolatide®) -based tumour imaging has been found to predict response to Vintafolide® [15]. Furthermore, folic acid is crucially involved in DNA methylation via metabolic cycling of methionine towards homocysteine. Epigenetic modifications of DNA through CpG site methylation are recognized to play a fundamental Page 2 of 13 role in tumorigenesis. Two distinct DNA methylation abnormalities are observed in cancer. The first is a global genome-wide reduction of DNA methylation (global DNA hypomethylation) and the second is hypo- or hypermethylation within the CpG islands within specific gene promoters. Promoter hypomethylation is believed to induce proto-oncogene activation, and global DNA hypomethylation is strongly related to chromosomal instability. Regional hypermet (...truncated)