MAPK4 Silencing Together with a PARP1 Inhibitor As a Combination Therapy in Triple‑Negative Breast Cancer Cells

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MAPK4 Silencing Together with a PARP1 Inhibitor As a Combination Therapy in Triple‑Negative Breast Cancer Cells MOLECULAR MEDICINE REPORTS 24: 548, 2021 MAPK4 silencing together with a PARP1 inhibitor as a combination therapy in triple‑negative breast cancer cells XIAOQI ZENG1*, SHANSHAN JIANG2*, SIMIN RUAN1*, ZHAOZE GUO1, JINGYUN GUO1, MINFENG LIU1, CHANGSHENG YE1 and JIANYU DONG1 1Breast Center, Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515; 2Institute of Hematological Research, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi 710000, P.R. China Received December 14, 2020; Accepted April 23, 2021 DOI: 10.3892/mmr.2021.12187 Abstract. Triple‑negative breast cancer (TNBC) is the most of HCC1937 cells. MAPK4 was highly expressed in TNBC common type of cancer among females worldwide and is asso‑ patient tissues and cell lines. Moreover, overexpression of ciated with poor prognosis. Poly ADP‑ribose polymerase‑1 MAPK4 could promote HCC1937 cell proliferation. Treatment (PARP1) inhibitors are effective against TNBC with mutations of HCC1937 cells with the combination of siMAPK4 and a in the breast cancer type 1 susceptibility protein (BRCA1) PARP1 inhibitor olaparib decreased their proliferation and and/or BRCA2 genes; however, the development of resistance migration and increased their apoptosis. The protein expression to PARP1 inhibitors limits their use. Thus, identifying strate‑ levels of the DNA repair‑related proteins p‑DNA‑dependent gies to overcome this resistance is urgently required. The aim protein kinase catalytic subunit (DNA‑PK) and RAD51 of the present study was to investigate the potential func‑ recombinase (RAD51) were inhibited in the siMAPK4 tion and mechanism of small interfering (si)RNA‑MAPK4 and siMAPK4 + olaparib groups. However, the marker of a (siMAPK4) in enhancing the efficacy of a PARP1 inhibitor double‑stranded break γH2AX showed increased protein and reducing the resistance. In the present study, data on the expression in the siMAPK4 + olaparib group. As MAPK4 mRNA expression level of MAPK4 in normal breast tissues and could phosphorylate AKT at threonine 308 (AKTT308), the TNBC tissues were obtained from The Cancer Genome Atlas current study restored p‑AKTT308 using a constitutively active database. The mRNA and protein expression levels of MAPK4 AKT plasmid (AKT‑CA). p‑DNA‑PK and RAD51 showed in normal breast cells and TNBC cells were analyzed using high expression and γH2AX exhibited lower protein expres‑ reverse transcription‑quantitative PCR and western blotting, sion in the AKT‑CA group. The present findings suggested respectively. The phosphorylated (p) histone H2AX (γH2AX) that siMAPK4 can enhance the sensitivity of TNBC cells to protein expression was assessed via immunofluorescence. Cell PARP1 inhibitors. Counting Kit‑8, wound healing and TUNEL assays were used to determine the proliferative, migratory and apoptotic abilities Introduction Breast cancer is the most commonly diagnosed type of cancer and the third leading cause of cancer‑related mortality among Correspondence to: Dr Jianyu Dong or Dr Changsheng Ye, females worldwide (1). In 2020, 2.65 million new cases and Breast Center, Department of General Surgery, Nanfang Hospital, 685,000 deaths occurred, accounting for the second highest Southern Medical University, 1838 North Guangzhou Avenue, incidence of cancer and fifth highest rate of cancer‑related Guangzhou, Guangdong 510515, P.R. China mortality (2). Moreover, the incidence and mortality rates E‑mail: [email protected] of breast cancer are rising annually (2). Triple‑negative E‑mail: [email protected] breast cancer (TNBC), which is defined by a lack of expres‑ sion of estrogen receptor, progesterone receptor and HER‑2, * Contributed equally accounts for 11‑20% of all breast carcinomas (3,4). Notably, targeted therapy has a poor effect on TNBC (5). Therefore, Abbreviations: TNBC, triple‑negative breast cancer; PARP1, poly the discovery of novel therapies for the treatment of TNBC is ADP‑ribose polymerase‑1; BRCA1/2, breast cancer type 1 and 2 susceptibility protein; TCGA, The Cancer Genome Atlas; AKT‑CA, urgently required. constitutively active AKT; RT‑qPCR, reverse transcription‑quantitative MAPK4, also termed ERK4, p63 and Prkm4, is an PCR; casp‑3, caspase‑3 atypical MAPK as it is not phosphorylated by the dual serine/threonine and tyrosine MAPK kinase due to a lack Key words: TNBC, MAPK4, PARP1 inhibitor, AKT of the threonine‑X‑tyrosine activation motif (6). Thus far, phosphorylation, DNA repair MAPK4 has been associated with several diseases, such as numerous types of cancer and methamphetamine addiction leading to psychosis (7). According to The Cancer Genome 2 ZENG et al: LOSS OF MAPK4 ENHANCES THE SUPPRESSIVE EFFECT OF A PARP1 INHIBITOR IN TNBC Atlas (TCGA), lung adenocarcinoma, low‑grade glioma, Ltd., while the anti‑mouse IgG (H + L) F(ab')2 Fragment thyroid carcinoma and urothelial carcinoma with upregulation (Alexa Fluor® 488 Conjugate; cat. no. 4408S) for immu‑ of MAPK4 are associated with poor prognosis (8). Moreover, nofluorescence was from Cell Signaling Technology, Inc. MAPK4 phosphorylates AKT at threonine 308 (AKTT308) and The RNeasy Mini kit (cat. no. 74104), QuantiTect Rev serine 473 (AKTS473), thereby promoting cell proliferation and Transcription kit (cat. no. 205311) and miScript SYBR inhibiting apoptosis (9). It has been verified that microRNA Green PCR kit (cat. no. 218073) were obtained from (miR)‑767‑5p upregulates MAPK4, and that the circular Qiagen GmbH. siMAPK4‑1 (5'‑GGU GAG CUG UUC (circ)RNA circ_0000190 inhibits the progression of multiple AAG UUC AGC‑3'), siMAPK4‑2 (5'‑GGA AGG UCG myeloma by regulating MAPK4 (10). Furthermore, circRNA CUG UGA AGA AGA ‑3'), siMAPK4‑3 (5'‑GGU UGG UAA MAPK4 inhibits apoptosis by regulating miR‑125a‑3p, which CAA AGU GGU ACC ‑3') and scramble siRNA (5'‑GGC GTG inhibits the MAPK signaling pathway (11). This evidence indi‑ AAT GGG TCT AAC CTT ‑3') were purchased from Addgene, cates the role of MAPK4 as an oncogene. It has been shown Inc. Overexpression‑MAPK4 plasmid (cat. no. C05008), the that the AKT signaling pathway is associated with the DNA pcDNA 3.1 empty vector (cat. no. C06002), which was used damage repair mechanism (12,13). Hence, we hypothesized as the overexpression‑negative control (NC), and constitu‑ that MAPK4 could regulate DNA damage repair by activating tively active AKT (AKT‑CA) plasmids (cat. no. 53583) were the AKT signaling pathway. designed and purchased from Shanghai GenePharma Co., In recent years, the DNA damage repair mechanism has Ltd. The mRNA expression level of MAPK4 from 116 TNBC been utilized in the treatment of cancer (14). Poly ADP‑ribose samples and 114 normal samples was obtained from UALCAN polymerase‑1 (PARP1) serves vital roles in DNA damage (http://ualcan.path.uab.edu) (19), which could provide mRNA repair of single‑strand and double‑strand breaks by catalyzing expression data regarding cancer and normal samples in the formation of poly ADP‑ribose and maintaining genome TCGA. integrity (15). PARP inhibitors, such as olaparib and rucaparib, have been approved by the US Food and Drug Administration, Cell culture. MCF‑10A (normal human mammary epithelial and it has been shown that these drugs are effective for cell line) cells were purchased from The Cell Bank of Type the treatment of ovarian and breast cancer, particularly Culture Collection of Chinese Academy of Sciences and TNBC (16,17). However, the wide use of PARP inhibitors cultured in MCF‑10A‑specific medium (Procell Life Science can lead to the development of resistance (18). Thus, there is & Technology Co., Ltd.). MDA‑MB‑468 and MDA‑MB‑231 an urgent requirement to discover novel targets or drugs that (breast adenocarcinoma), HCC1937 (ductal breast carcinoma) can be combined with PARP1 inhibitors. cells are TNBC cell lines (all from The Cell Bank of Type As aforementioned, MAPK4 was identified to be associ‑ Culture Collection of Chinese Academy of Sciences) and were ated with several cancer types, and can promote DNA repair cultured in RPMI‑1640 medium containing 10% FBS and by increasing AKT phosphorylation. Thus, small interfering 1% 100 IU/ml penicillin‑streptomycin at 37˚C in an incubator (si)RNA MAPK4 (siMAPK4) may be a novel molecule to with 5% CO2. combine with PARP1 inhibitors. Therefore, the present study aimed to investigate the potential function and mechanism Cell transfection. According to the instructions provided by of siMAPK4 in enhancing the efficacy of a PARP1 inhibitor, the manufacturer of Lipofectamine® 2000 (Thermo Fisher which could reduce the possibility of PARP1 inhibitor resis‑ Scientific, Inc.), HCC1937 cells were seeded in 6‑well plates tance in TNBC and provide a new therapeutic strategy for the at the density of 1.2x106 cells/well. The following day, 10 µl treatment of TNBC. Lipofectamine 2000 and 4 µg plasmids were diluted with 500 µl Opti‑MEM (Thermo Fisher Scientific, Inc.) and Materials and methods incubated at room temperature for 20 min. Subsequently, the Lipofectamine 2000 and plasmids solution was added to Materials and reagents. RPMI‑1640 medium the cells. Following 6 h of incubation at 37˚C, the medium (cat. no. 11875093), FBS (cat. no. 10100139C) and 100 IU/ml was changed with fresh RPMI‑1640 medium. After 24 h, the penicillin‑streptomycin (cat. no. 15140‑122) were purchased subsequent experiments were performed with the transfected from Gibco; Thermo Fisher Scientific, Inc. MCF‑10A‑specific HCC1937 cells. medium (cat. no. CM‑0525) was from Procell Life Science & Technology Co., Ltd. The PARP1 inhibitor olaparib Western blotting. Western blotting was performed as previ‑ (cat. no. HY‑10162) was purchased from MedChemExpress. ously described (20,21). Briefly, total protein of HCC1937 cells Antibodies against MAPK4 (cat. no. ab211501), p‑AKTT308 was extracted using RIPA lysis buffer (Beyotime Institute of (cat. no. ab38449), p‑AKTS473 (cat. no. ab8932), DNA‑dependent Biotechnology), quantified using an Enhanced BCA Protein protein kinase catalytic subunit (DNA‑PK; cat. no. ab44815), assay kit (Beyotime Institute of Biotechnology), separated via p‑DNA‑PK (cat. no. ab18192), RAD51 (cat. no. ab63801), Bcl‑2 10% SDS‑PAGE (20 µg total protein/lane) and transferred to (cat.
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