cancers Article ALDH1A2 Is a Candidate Tumor Suppressor Gene in Ovarian Cancer Jung-A Choi 1, Hyunja Kwon 1, Hanbyoul Cho 1,* , Joon-Yong Chung 2 , Stephen M. Hewitt 2 and Jae-Hoon Kim 1 1 Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul 03722, Korea; [email protected] (J.-A.C.); [email protected] (H.K.); [email protected] (J.-H.K.) 2 Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (J.-Y.C.); [email protected] (S.M.H.) * Correspondence: [email protected]; Tel.: +82-2-2019-3430; Fax: +82-2-3462-8209 Received: 11 July 2019; Accepted: 10 October 2019; Published: 14 October 2019 Abstract: Aldehyde dehydrogenase 1 family member A2 (ALDH1A2) is a rate-limiting enzyme involved in cellular retinoic acid synthesis. However, its functional role in ovarian cancer remains elusive. Here, we found that ALDH1A2 was the most prominently downregulated gene among ALDH family members in ovarian cancer cells, according to complementary DNA microarray data. Low ALDH1A2 expression was associated with unfavorable prognosis and shorter disease-free and overall survival for ovarian cancer patients. Notably, hypermethylation of ALDH1A2 was significantly higher in ovarian cancer cell lines when compared to that in immortalized human ovarian surface epithelial cell lines. ALDH1A2 expression was restored in various ovarian cancer cell lines after treatment with the DNA methylation inhibitor 5-aza-20-deoxycytidine. Furthermore, silencing DNA methyltransferase 1 (DNMT1) or 3B (DNMT3B) restored ALDH1A2 expression in ovarian cancer cell lines. Functional studies revealed that forced ALDH1A2 expression significantly impaired the proliferation of ovarian cancer cells and their invasive activity. To the best of our knowledge, this is the first study to show that ALDH1A2 expression is regulated by the epigenetic regulation of DNMTs, and subsequently that it might act as a tumor suppressor in ovarian cancer, further suggesting that enhancing ALDH1A2-linked signaling might provide new opportunities for therapeutic intervention in ovarian cancer. Keywords: ALDH1A2; methylation; DNMT; OVARIAN cancer; invasion 1. Introduction Ovarian cancer has the highest mortality rate among all gynecological cancers because most cases are not diagnosed until the disease has reached an advanced stage [1]. Although the five-year survival rate for ovarian cancer patients has substantially improved, owing to ongoing efforts to develop an effective screening strategy [1], the lack of reliable methods for early diagnosis and the absence of specific symptoms result in diagnoses at advanced stages [1]. Furthermore, approximately 80% of women with advanced ovarian cancer show tumor progression or, more commonly, recurrence [2]. Thus, understanding the pathogenesis of ovarian cancer and the molecular mechanism of its early stage is crucial for the management of this lethal, highly metastatic disease. The aldehyde dehydrogenase 1 (ALDH1) family comprises major enzymes that produce retinoic acid (RA) via the oxidation of all-trans retinal and 9-cis-retinal, which is mainly involved in biological functions related to cell differentiation, cell cycle arrest, and eventually, apoptosis [3–5]. The ALDH1A Cancers 2019, 11, 1553; doi:10.3390/cancers11101553 www.mdpi.com/journal/cancers Cancers 2019, 11, 1553 2 of 18 subfamily consists of three members, ALDH1A1, ALDH1A2, and ALDH1A3 [6]. ALDH1A1 participates in retinal oxidation, acetaldehyde metabolism, and detoxification of cyclophosphamide, whereas the ALDH1A2 and ALDH1A3 proteins are integral to the oxidation of retinal to RA [7]. Interestingly, despite similarities in the structure and function of the isoenzymes of the ALDH1A subfamily [8], several lines of evidence suggest that these molecules might perform different functions in cancer progression [7–14]. ALDH1A1 has been implicated in conferring resistance to certain anticancer agents, such as cyclophosphamide, and was shown to play a functional role in cancer stem cells [11,12]. In contrast to that of ALDH1A1, low ALDH1A2 expression has been associated with an unfavorable prognosis in prostate cancer and head and neck squamous cell carcinoma patients [13,14]. Meanwhile, ALDH1A3 plays important roles in several metabolic and physiological processes [15], which potentially suggest different roles for ALDH1A isoforms in cancer progression. However, the functions and prognostic value of ALDH1A2 in ovarian cancer progression remain unclear. The methylation of CpG sites is an epigenetic mechanism underlying the regulation of gene expression, which usually leads to gene silencing [16]. Aberrant methylation of gene promoter regions is one of the earliest molecular alterations that occurs during carcinogenesis and a key mechanism for the inactivation of tumor suppressor genes [16–18]. Therefore, methylation has emerged as a promising marker for the early detection of cancer [19]. In fact, CpG islands in tumor suppressor genes such as CDKN2B, BRACA1, RASSF1A, MLH1, RARB2, and TIMP3 undergo aberrant methylation in ovarian cancer [20]. DNA methylation patterns have been shown to be regulated by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B [21]. DNMT1 is essential for maintaining DNA methylation patterns in proliferating cells [22], whereas DNMT3A and DNMT3B are required for de novo methylation during embryonic development [23]. However, there are no reports regarding the epigenetic regulation of ALDH1A2 by DNMTs in human ovarian cancer. Therefore, in this study, we evaluated the expression profiles of ALDH isozyme-encoding genes in ovarian cancer using a complementary DNA (cDNA) microarray. Additionally, we focused on the role of ALDH1A2 as one of the most prominently downregulated genes in ovarian cancer. 2. Results 2.1. Reduced Expression of ALDH1A2 in Ovarian Cancer To evaluate whether ALDH isozymes contribute to the progression of ovarian cancer, an Illumina microarray was used to identify differentially expressed ALDH genes in six ovarian cancer cell lines (YDOV-139, YDOV-157, YDOV-161, YDOV-13, YDOV-105, and YDOV-151) and four human ovarian surface epithelial (HOSE) cell lines (HOSE 198, 209, 211, and 213) [24]. RNA isolated from the cells was directly compared by hybridization with cDNA microarrays with 24,957 probe sets. The volcano plot shows that 24,957 genes were differentially expressed (p < 0.01 and fold change > 2) in the cancer cells compared to the normal cells (Figure1A). We found that 15 ALDH isoforms exhibited di fferential expression patterns in the ovarian cancer cell lines compared to the normal cells (Figure1A,B, Table S1). ALDH1A2, ALDH1B1, and ALDH9A1 were downregulated in the ovarian cancer cells compared to the expression in HOSE cells (Figure1A and Table S1), whereas ALDH3A1 was upregulated in the ovarian cancer cells. Among these isoforms, we further characterized the functional role of ALDH1A2 in ovarian cancer progression, because it was one of the most prominently downregulated genes, with approximately 50-fold lower expression levels in ovarian cancer cells than in normal cells (Figure1B, Table S1). These decreased expression levels were further validated by real-time PCR, and immunoblotting in ovarian cancer cell lines (SNU840, RMG1, OVCA433, OVCA429, and SKOV3) and IHOSE8695 cells (Figure1C,D). Taken together, these observations suggest that the expression of ALDH1A2 is strongly downregulated in ovarian cancer. Cancers 2019, 11, 1553 3 of 18 Cancers 2019, 11, x FOR PEER REVIEW 3 of 18 FigureFigure 1. 1. ComparativeComparative analysis analysis of of ALDH ALDH gene gene expression expression profiles profiles and ALDH1A2ALDH1A2 protein protein expression expression in inovarian ovarian cancer cancer cell cell lines. lines. (A )( VolcanoA) Volcano plot plot of the of distribution the distribution of diff erentiallyof differentially expressed expressed genes between genes betweenovarian ovarian cancer cell cancer lines cell (YDOV-139, lines (YDOV YDOV-157,‐139, YDOV YDOV-161,‐157, YDOV YDOV-13,‐161, YDOV YDOV-105,‐13, YDOV and YDOV-151)‐105, and YDOVand human‐151) and ovarian human surface ovarian epithelial surface (HOSE) epithelial cell (HOSE) lines (HOSE cell lines 198, (HOSE 209, 211, 198, and 209, 213) 211, using and a DNA213) usingmicroarray. a DNA Themicroarray. volcano The plot volcano shows that plot 24,957 shows genes that 24,957 were di genesfferentially were differentially expressed (p expressed< 0.01 and (p fold < change > 2). The log10 (p-value) of each gene is plotted against the log 2 ratio of cancer intensity 0.01 and fold change− > 2). The −log10 (p‐value) of each gene is plotted against the log 2 ratio of cancer intensityto normal to intensity.normal intensity. 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