Bcl2l10 Mediates the Proliferation, Invasion and Migration of Ovarian Cancer Cells

INTERNATIONAL JOURNAL OF ONCOLOGY 56: 618-629, 2020 Bcl2l10 mediates the proliferation, invasion and migration of ovarian cancer cells SU-YEON LEE, JINIE KWON, JI HYE WOO, KYEOUNG-HWA KIM and KYUNG-AH LEE Department of Biomedical Sciences, College of Life Sciences, CHA University, Seongnam-si, Gyeonggi-do 13488, Republic of Korea Received July 18, 2019; Accepted December 2, 2019 DOI: 10.3892/ijo.2019.4949 Abstract. Bcl2l10, also known as Diva, Bcl-b and Boo, is a homology (BH) domains (1,3). These proteins are grouped member of the Bcl2 family of proteins, which are involved in into 3 categories: i) Anti-apoptotic proteins, including Bcl-2, signaling pathways that regulate cell apoptosis and autophagy. Bcl-xL, Bcl-w, NR-13, A1 and Mcl-1; ii) multi-domain Previously, it was demonstrated that Bcl2l10 plays a crucial role pro-apoptotic proteins, such as Bax, and Bak; and iii) BH3 in the completion of oocyte meiosis and is a key regulator of domain-only proteins, such as Bad, Bim, Bid and Bik (1,4). Aurora kinase A (Aurka) expression and activity in oocytes. Interactions between pro-apoptotic and anti-apoptotic Bcl-2 Aurka is overexpressed in several types of solid tumors and family proteins play important roles in controlling and has been considered a target of cancer therapy. Based on these promoting apoptosis (1,3). However, Bcl2l10 reportedly previous results, in the present study, the authors aimed to has contradictory functions in different apoptotic cells or investigate the regulatory role of Bcl2l10 in A2780 and SKOV3 tissues and is recognized for its both pro-apoptotic (5-7) and human ovarian cancer cells. The protein expression of Bcl2l10 anti-apoptotic activities (8-10). was examined in human cancer tissues and cell lines, including Previously, it was reported that Bcl2l10 was highly the ovaries, using a tissue microarray and various human expressed in mouse ovaries and oocytes and that it was associ- ovarian cancer cell lines. It was found that Bcl2l10 regulated ated with cytoskeletal proteins (11,12). It was demonstrated the protein stability and activities of Aurka in ovarian cancer that Bcl2l10-depleted oocytes arrested at the metaphase I cells. Although apoptosis was not affected, the cell cycle was stage, exhibiting abnormal spindle and chromosome forma- arrested at the G0/G1 phase by Bcl2l10 knockdown. Of note, tion (12). These results revealed that Bcl2l10 plays a crucial cell viability and motility were markedly increased by Bcl2l10 role in oocyte meiosis. By using microarray analysis, it was knockdown. On the whole, the findings of this study suggest that found that Bcl2l10 was associated with cytoskeletal proteins, Bcl2l10 functions as tumor suppressor gene in ovarian cancer. such as targeting protein for xenopus kinesin-like protein 2 (Tpx2) and centrosomal protein of 192 kDa (Cep192), which Introduction are well-known cofactors of Aurora kinase A (Aurka) (13). Notably, Bcl2l10 was subsequently demonstrated to directly Bcl2l10, also known as Diva, Bcl-b or Boo, is a member of the interact with Tpx2 in meiotic spindles and to further regulate Bcl-2 family of proteins, which are central mediators of apop- the expression and activity of Aurka (14). tosis and autophagy (1,2). The Bcl-2 family proteins contain In somatic cells, Tpx2 directly interacts with Aurka at up to 4 conserved amino acid sequences, known as Bcl-2 spindle microtubules and protects Aurka from degrada- tion (15-17). Aurka controls multiple aspects of mitotic cell division and plays important roles in centrosome maturation and bipolar spindle formation in somatic cells (18). Aurka expression and activity are upregulated in several types of Correspondence to: Professor Kyung-Ah Lee, Department of solid tumors. Additionally, Aurka overexpression has been Biomedical Sciences, College of Life Sciences, CHA University, shown to increase cancer cell malignancy (19-22), promptly 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, impair cell cycle progression and cause chromosomal insta- Republic of Korea bility, supernumerary centrosomes and multipolar spindles. E-mail: [email protected] These properties have led to the consideration of Aurka as Abbreviations: AURKA, Aurora kinase A; BH, Bcl-2 homology; an oncogene and a target of cancer therapy (23-25). Based RNAi, RNA interference; siRNA, small-interfering RNA; Tpx2, on our previous finding that Bcl2l10 is an upstream regulator targeting protein for xenopus kinesin-like protein 2 of Aurka and considering that Aurka is considered a crucial oncogenic factor (14), it was hypothesized that Bcl2l10 plays Key words: Bcl2l10, Aurka, ovarian cancer, cell proliferation, an important role in cancer cells. If Bcl2l10 is functionally migration, invasion connected with Aurka in cancer cells, Bcl2l10 may be a novel effective therapeutic target that can be used with Aurka inhibitors as an anticancer drug. LEE et al: Bcl2L10 FUNCTIONS AS A TUMOR SUPPRESSOR IN OVARIAN CANCER CELLS 619 The functions of Bcl2l10 in cancers have been previously Cell transfection. Bcl2l10 small-interfering RNA (siRNA) reported. Bcl2l10 plays roles as an oncogene and as a tumor was synthesized by Shanghai GenePharma Co., Ltd. and suppressor gene depending on the type of cancer. In gastric negative control siRNA was purchased at Bioneer. One day and lung cancer cells, Bcl2l10 exhibits a low-level expression prior to transfection, the A2780 and SKOV3 cancer cells were relative to its expression in adjacent normal tissues, and the seeded in 6-well plates. Before transfection, the medium was overexpression of Bcl2l10 induces apoptosis and cell growth removed and replaced with 1.5 ml of fresh growth medium. inhibition (26-29). By contrast, Bcl2l10 acts as an oncogene in All siRNAs were diluted in 0.25 ml of OPTI-MEM to a myelodysplastic syndromes (MDS), acute myeloid leukemia final concentration of 100 nM, and Lipofectamine 3000 (AML), glioma and breast cancers (9,30,31). However, (Invitrogen; Thermo Fisher Scientific) was diluted in 0.25 ml although Bcl2l10 is highly expressed in human ovaries, the of OPTI-MEM. The solutions were incubated individually for biological functions of Bcl2l10 in ovarian cancer have yet to 5 min at room temperature and then combined and incubated be reported. Therefore, the aims of this study were to deter- for an additional 20 min at room temperature. The mixture mine whether Bcl2l10 regulates Aurka and to investigate the was then added to each well containing cells, which were function of Bcl2l10 in A2780 and SKOV3 ovarian cancer maintained in a 5% CO2 atmosphere at 37˚C for 48 h. cells. Total RNA extraction and cDNA synthesis. After the cultured Materials and methods cells were washed twice with PBS, 1 ml of TRIzol reagent (Takara Bio) and 0.2 ml of chloroform were added, and the Tissue microarray. Slides including 20 different types of mixture was incubated at room temperature for 10 min. human cancer tissues (brain, esophagus, larynx, thymus, The cells were centrifuged at 12,000 x g for 20 min at 4˚C, thyroid, breast, stomach, pancreas, tongue, lung, lymph node, and the supernatants were transferred to new tubes and liver, kidney, ovary, cervix, testis, colon, rectum, skin and resuspended in 0.5 ml of isopropanol. After the tubes were soft tissue) were purchased from AccuMax (ISU ABXIS). centrifuged at 12,000 x g for 10 min at 4˚C, the superna- Slides made from formalin‑fixed, paraffin‑embedded blocks tants were discarded, and the precipitates were dried. The and stained with hematoxylin and eosin were used to define pellets were washed with 75% ethanol, dried, and dissolved the most morphologically representative, well-fixed cells. in 0.1% diethylpyrocarbonate (DEPC)‑treated water. To Single-tissue cores (1 mm in diameter) were sampled from synthesize first‑strand cDNA, total RNA (2 µg) was added each paraffin block and assembled into a recipient paraffin to DNase I (New England Biolabs) and DNase I buffer block using a tissue microarray instrument (AccuMax Array, (New England Biolabs), and the total volume was adjusted to ISU ABXIS). To detect Bcl2l10 in cancer tissues, immuno- 11 ml with DEPC-treated water. The mixture was incubated histochemical staining was performed with an anti-Bcl2l10 at room temperature for 15 min, 1 ml of 25 mM EDTA were antibody. Briefly, antigen retrieval was conducted by boiling added, and the mixture was incubated at 65˚C for 15 min. the slide in 1X citrate buffer for 10 min, and blocking was Subsequently, 1 ml of oligo dT was added, and the mixture then performed in hydrogen peroxide solution for 10 min. was incubated at 70˚C for 10 min. M‑MLV RNase (Promega), The slide was incubated with the anti-Bcl2l10 antibody 5X buffer (Promega), RNase inhibitor (Promega) and 10 mM (anti-BCL2L10, 1:100 dilution, ab151419, Abcam) for 1 h at dNTP were then added, and the reaction was performed at room temperature and incubated with a horseradish peroxi- 42˚C for 1 h and 94˚C for 2 min. dase (HRP) polymer solution (Lab Vision; Thermo Fisher Scientific) for 15 min. Thereafter, the slide was stained with Western blot analysis. Proteins were extracted from the a 3,3'-diaminobenzidine-tetrahydrochloride (DAB) solution treated cells using RIPA lysis buffer with a 1% protease (Vector Laboratories) at room temperature for 3 min and coun- inhibitor cocktail (Thermo Fisher Scientific) and 0.5 M terstained with hematoxylin (Sigma-Aldrich; Merck KGaA) EDTA (pH 8.0). Protein concentrations were estimated using for 5 min. the Bio-Rad protein assay reagent (Bio-Rad) according to the manufacturer's instructions. Protein extracts (30 µg) were Cells and cell culture. Six ovarian cancer cell lines were used separated using 10% SDS‑PAGE and then transferred onto in this study. The CAOV-3, SKOV3 and OVCAR-3 cells were PVDF membranes (Amersham Biosciences).

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