www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 31 BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism Zhi-Qiang Wang1,2, Adnen Faddaoui1,2, Magdalena Bachvarova2, Marie Plante2,3, Jean Gregoire2,3, Marie-Claude Renaud2,3, Alexandra Sebastianelli2,3, Chantal Guillemette4,5, Stéphane Gobeil1,4, Elizabeth Macdonald6, Barbara Vanderhyden6, Dimcho Bachvarov1,2 1Department of Molecular Medicine, Laval University, Québec PQ, Canada 2Centre de recherche du CHU de Québec, L’Hôtel-Dieu de Québec, Québec PQ, Canada 3Department of Obstetrics and Gynecology, Laval University, Québec PQ, Canada 4Centre de recherche du CHU de Québec, CHUL, Québec PQ, Canada 5Faculty of Pharmacy, Laval University, Québec PQ, Canada 6Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Correspondence to: Dimcho Bachvarov, e-mail: [email protected] Keywords: BCAT1, ovarian cancer, cancer metabolism, metastasis, DNA hypomethylation Received: May 07, 2015 Accepted: August 28, 2015 Published: September 10, 2015 ABSTRACT Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target. INTRODUCTION strategies against this deadly disease, it is essential to understand its molecular pathology. Epithelial ovarian cancer (EOC) accounts for 4% of Recently, the importance of epigenetic perturbation all cancers in women and is the leading cause of death of gene regulation in cancer [3], including EOC [4], has from gynecologic malignancies [1]. Despite treatment begun to be more fully appreciated. The most studied improvements, long-term survival rates for patients with epigenetic alteration is DNA methylation, the addition advanced disease remain disappointing [2]. The molecular of a methyl moiety to the cytosine-5 position within basis of EOC initiation and progression is still poorly the context of a CpG dinucleotide, mediated by DNA understood. To establish novel therapeutic and diagnostic methyltransferases [3]. Similar to other malignancies, www.impactjournals.com/oncotarget 31522 Oncotarget aberrant DNA methylation occurs in EOC and samples, using tissue microarrays (TMAs). Our TMAs contributes to ovarian tumorigenesis and mechanisms of included triplicate cores of 117 serous EOC tumors, chemoresistance [4]. Applying a more global array-based including 13 LMP tumors and 104 HG ovarian tumors. technology, several studies have demonstrated that DNA Thirteen normal ovarian tissue samples were also included methylation changes in ovarian cancer are cumulative with as controls. Table 1 shows the major clinical characteristics disease progression and chemotherapy (CT) resistance of these patients for whom extensive follow-up clinical [5–7]. Using a similar approach (methylated DNA data (up to 5-years) were available. The age ranged from immunoprecipitation coupled to CpG island tiling arrays) 41 to 83 years (median: 66 years). High-grade tumors we have recently shown that DNA hypermethylation were all grade 3 (100%) including stage III (69%) and occurs in all (including less invasive/early) stages of stage IV (31%) tumors. The majority of patients (93%) ovarian tumorigenesis. Interestingly, advanced EOC was received a combination of platinum and paclitaxel. The exclusively associated with DNA hypomethylation of a median baseline CA125 was around 800. Forty percent of number of oncogenes, implicated in cancer progression, the patients had a progression or a recurrence within the invasion/metastasis and probably chemoresistance [8]. first 6 months of follow-up; for 39% of the patients the The cytosolic form of the branched chain amino-acid progression-free survival (PFS) interval was in the range transaminase 1 (BCAT1) was among the genes identified of 7 to 24 months, and 21% of the patients displayed PFS to be notably hypomethylated in low-malignant potential values higher than 25 months (Table 1). (LMP) and high grade (HG) serous EOC tumors [8]. As seen from Figure 1, BCAT1 displayed BCAT enzymes comprise two isoforms: BCAT1 (cytosolic) significantly higher expression in LMP tumors and HG and BCAT2 (mitochondrial). Both isoforms regulate serous EOC tumors, when compared to normal tissues the first step in degradation of branched-chain amino (p = 0.0003 and p = 0.0014, respectively), which correlates acids (BCAAs) including leucine, isoleucine and valine, with BCAT1 hypomethylation status in these tumor types. which are essential for cellular metabolism and growth However, we did not observe any significant differences [9]. While BCAT2 is expressed in most tissues, BCAT1 between the levels of BCAT1 expression and patients’ PFS expression is rather restricted to some highly specialized values (p = 0.0901; see Supplemental Figure 1), which tissues, including brain, ovary and placenta [9]. Several suggests that staining intensity for BCAT1 in pre-treatment groups have confirmed that BCAT1 is involved in cell surgical specimens is not predictive of PFS. proliferation, cell cycle progression, differentiation and apoptosis [10, 11]. Recent studies were indicative Phenotype analysis of BCAT1 suppression in for the important role of BCAT1 in the progression of EOC cells: possible implications in EOC cell several malignancies, including medulloblastomas, proliferation, cell cycle control, migration and nonseminomas, colorectal and nasopharyngeal carcinomas invasion and glioblastomas [12–16]. Moreover, the role of BCAAs metabolism in cancer pathogenesis has been also a topic Next, we decided to verify if short-hairpin RNA of interest [17]. (shRNA)-mediated BCAT1 gene knockdown could This prompted us to further investigate if BCAT1 produce any cancer-related phenotypic changes in EOC displays elevated expression levels in serous EOC tumors cells. We tested several EOC cell lines for endogenous with different malignant potential, and whether this gene BCAT1 protein expression by Western blot analysis is functionally implicated in EOC dissemination. Here we (see Supplemental Figure 2). Among these, the SKOV3 present experimental data indicative for strong BCAT1 and the A2780s cell lines displayed strong BCAT1 overexpression in LMP and HG serous EOC tumors, expression and were further used to generate stably which probably correlates with its hypomethylated transfected shRNA-BCAT1 clones. Clone selection for status. We also show that blocking BCAT1 expression further analyses was based on semi-quantitative (sq) RT- inhibits proliferation, migration, invasion and peritoneal PCR and Western blot validation of the BCAT1 mRNA/ dissemination, possibly through altering EOC metabolism. protein expression in selected clones, compared to empty vector-transfected clones. Among the clones analyzed, the RESULTS SKOV3 shRNA-BCAT1 knockdown clones sh-B1 and sh-B2 and the A2780s clones sh-A1 and sh-A2 displayed Overexpression of BCAT1 in both LMP and HG significant decrease ofBCAT1 expression levels compared serous EOC tumors to the mock-transfected control (Supplemental Figure 2) and were selected for further analyses. Previously, we have identified the BCAT1 gene We investigated the impact of the BCAT1 gene as hypomethylated in LMP and HG EOC tumors, when suppression on SKOV3 cell proliferation, cell cycle compared to normal tissues [8]. Here, we further evaluated control, migration, invasion, and sensitivity to cisplatin BCAT1 protein expression by immunohistochemistry and paclitaxel (drugs, conventionally used for first- (IHC) in serous EOC tumors and ovarian normal tissue line EOC therapy). The BCAT1 gene knockdown led to www.impactjournals.com/oncotarget 31523 Oncotarget Table 1: Detailed patients’ clinicopathological characteristics. Variable Range n/total % Age (years) ≥ 65 64/130 49 < 65 66/130 51 Median 66 Tissue/tumor type Normal 13/130 10 LMP 13/130 10 High-grade 52/130 40 OM 52/130 40 Grade 3 104/104 100 Stage III 72/104 69 IV 32/104 31 Chemotherapy1 platinum+taxol 97/104 93 Other 13/104 7 CA125 ≥ 800 47/104 45 < 800 53/104 55 PFS (months)2 0–6 41/103 40 7–24 40/103 39 > 25 22/103 21 1All patients were subjected to adjuvant therapy. 2Extended follow-up, including PFS values, were available for 103 patients.