Lin et al. Cell Death and Disease (2021) 12:451 https://doi.org/10.1038/s41419-021-03729-1 Cell Death & Disease ARTICLE Open Access MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis Chia-Liang Lin1,Tsung-HoYing2,3, Shun-Fa Yang 1,4, Hui-Ling Chiou5, Yong-Syuan Chen1,Shao-HsuanKao 1,4 and Yi-Hsien Hsieh1,4 Abstract Metastasis-associated protein 2 (MTA2) is a transcription factor that is highly associated with matrix metalloproteinase 12 (MMP12). Thus, we hypothesized that MTA2 may regulate MMP12 expression and is involved in cervical cancer metastasis. Results showed that MTA2 and MMP12 were highly expressed in cervical cancer cells, and MTA2 knockdown reduced MMP12 expression and inhibited the metastasis of cervical cancer cells in xenograft mice. MMP12 knockdown did not influence the viability of cervical cancer cells but clearly inhibited cell migration and invasion both in vitro and in vivo. MMP12 was highly expressed in cervical tumor tissues and correlated with the poor survival rate of patients with cervical cancer. Further investigations revealed that p38 mitogen-activated protein kinase (p38), mitogen-activated protein kinase kinase 3 (MEK3), and apoptosis signal-regulating kinase 1 (ASK1) were involved in MMP12 downregulation in response to MTA2 knockdown. Results also demonstrated that p38-mediated Y-box 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; binding protein1 (YB1) phosphorylation disrupted the binding of AP1 (c-Fos/c-Jun) to the MMP12 promoter, thereby inhibiting MMP12 expression and the metastatic potential of cervical cancer cells. Collectively, targeting both MTA2 and MMP12 may be a promising strategy for the treatment of cervical cancer. Introduction molecular mechanism of cervical carcinogenesis must be Cervical cancer is one of the most life-threatening comprehensively explored to develop more specific and malignancy in women in the developing countries1,2. effective treatments to this disease. Various types of treatments combining surgeries, radio- Distal metastasis is often found in tumor recurrence, therapy, and chemotherapy have been developed to heal and it is the major cause of death for most patients with precancerous lesions and eradicate aggressive and malig- cancer. Compared with the mechanisms for tumor nant cervical carcinomas3. However, many patients with initiation and regulation of tumor growth, the molecular cervical cancer still suffer from recurrence and distal process of metastasis remains unclear. Many dynamic metastasis of cancer after treatments. Therefore, the cellular processes are involved in the dissemination of cancer cells from the primary site to distant sites, including changes in cytoskeletal structure, cell–cell and Correspondence: Shao-Hsuan Kao ([email protected]) or Yi- cell–extracellular matrix (ECM) interaction, and ECM Hsien Hsieh ([email protected]) 4 1Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan degradation . Matrix metalloproteinases (MMPs) belong 2Department of Obstetrics and Gynecology, School of Medicine, College of to the zinc protease family and play a pivotal role in tumor Medicine, Chung Shan Medical University, Taichung, Taiwan metastasis mainly by inducing ECM degradation and sti- Full list of author information is available at the end of the article 5 These authors contributed equally: Chia-Liang Lin, Tsung-Ho Ying mulating tumor growth . Edited by S. Tait © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Lin et al. Cell Death and Disease (2021) 12:451 Page 2 of 12 Metastasis-associated protein 2 (MTA2) is a transcrip- reaction (RT-PCR) was conducted using GoScript Reverse tional regulator belonging to the metastasis tumor- Transcriptase (Madison, WI, USA). Quantitative RT-PCR associated family. Previous studies demonstrated that (qRT-PCR) was performed using the StepOne Real-Time MTA2 plays an important role in cytoskeletal organization PCR System (Applied Biosystems, Foster City, CA, USA). and transcription, as well as in the promotion of the The primers used for qRT-PCR were: MTA2, (F) 5ʹ-TGT metastatic potential of tumor cells6,7. However, the role of ACC GGG TGG GAG ATT AC-3ʹ,(R)5ʹ- TGA GGC TAC MTA2 in the metastatic ability of cervical cancer cells TAG AAA TGT CCC TG-3ʹ;MMP12,(F)5ʹ-CATGAA remains poorly understood. Here, we showed that MTA2 is CCG TGA GGA TGT TGA -3ʹ,(R)5ʹ-GCA TGG GCT highly expressed in cervical tumor tissues and is associated AGG ATT CCA CC-3ʹ; and glyceraldehyde 3-phosphate with the poor prognosis of cervical cancer. To explore the dehydrogenase (GAPDH), (F) 5ʹ-CAT CAT CCC TGC CTC role of MTA2 in the metastatic potential of cervical cancer TAC TG-3ʹ,(R)5ʹ-GCC TGC TTC ACC ACC TTC-3ʹ cells, we established MTA2-knockdown cervical cancer (Mission Biotech, Taipei, Taiwan). Relative quantitation of cells to investigate the effects of MTA2 knockdown on cell gene expression was normalized with endogenous GAPDH ΔΔ motility, invasiveness, and in vivo metastasis. via the 2- Ct method. Materials and methods Cell culture and gene knockdown by shRNA lentivirus Reagents and antibodies infection Chemicals and antibodies without specific indication Human cervical cancer cell lines HeLa, SiHa, and C33A were purchased from Sigma-Aldrich (St. Louis, MO, USA). were cultured in Dulbecco’s modified Eagle’s medium The antibodies source and dilution factor: MTA2 (sc- (DMEM) containing 10% v/v fetal bovine serum (FBS, 55566; 1:1000), ERK (sc-514302; 1:1000), p-p38 mitogen HyClone, Thermo Fisher, Waltham, MA, USA), 50 U/mL activated protein kinase (p-p38) (sc-166182; 1:1000), p38 penicillin, and 50 μg/mL streptomycin (Sigma-Aldrich) in (sc-7972; 1:1000), p-JNK (sc-6254; 1:1000), JNK (sc-571; a humidified incubator supplied with 5% CO2. MTA2 and 1:1000), ASK1 (sc-5294; 1:1000), p-MEK3 (sc-8407; MMP12 knockdown was achieved by the transfection of 1:1000), MEK3 (sc-960; 1:1000), c-Jun (sc-74543; 1:1000), specific shRNA lentiviral constructs (shMTA2 and c-Fos (sc-8047; 1:1000), lamin B (sc-374015; 1:2000), YB1 shMMP12, National RNA Interference Core Facility, (sc-18057; 1:1000), β-actin (sc-69879; 1:2000), and Institute of Molecular Biology, Academia Sinica, Taipei, peroxidase-conjugated antibodies against mouse IgG or Taiwan). The target sequences of shMTA2 and shMMP12 rabbit IgG (1:10000) were obtained from Santa Cruz Bio- were 5ʹ-AGGGAGTGAGGAGTGAATTAA-3ʹ and 5ʹ- technology (Santa Cruz, CA, USA). MMP12 (ab66157; CTTGCTTGACTCTACTATTAA-3ʹ, respectively. Luci- 1:1000) were purchased from Abcam (Cambridge, UK). ferase lentiviral construct (shLuc) was used as control. The Phospho(p)-ASK1 (#3764; 1:1000), phospho(p)-ERK1/2 transfection assay was conducted according to the guide- (#9101; 1:1000) and phospho(p)-YB1 (#2900; 1:1000) were lines of the Institutional Biosafety Committee of Chung purchase from Cell Signaling Technology (Beverly, MA, Shan University. HEK293T cells were used for target USA). Human cervix cancer array (CR805) were pur- lentivirus production. Both HeLa and SiHa cells with chased from US Biomax, Inc (Rockville, MD, USA). stable MTA2- and MMP12-knockdown were generated by lentiviral infection and then followed by puromycin Immunohistochemical staining selection for 2 weeks. Inhibition efficiency of the shRNA Tissues were fixed, blocked, sliced, and incubated with constructs was confirmed via Western blot and qRT-PCR. primary antibodies against human MTA2 or anti- MMP12. After washing with PBS, the reacted sections Western blot analysis were incubated with peroxidase-conjugated second anti- Cells were washed with PBS, collected, and then lysed bodies and then reacted with diaminobenzidine solution with RIPA buffer (Cell Signaling, Beverly, MA, USA) for for signal visualization as previously described8. Protein protein extraction. The extracted proteins were separated expression in the immunohistochemical staining was by a 10% SDS–polyacrylamide gel and then transferred quantified according to the immunoreactivity score. onto a PVDF membrane (Immobilon P, Merck Millipore, Billerica, MA, USA). The membrane was blocked with 2% Reverse transcription and real-time quantitative skimmed milk and then incubated with primary anti- polymerase chain reaction bodies, peroxidase-conjugated secondary antibody (Santa Cells were harvested, and total RNA was extracted using Cruz Technology), and with a chemiluminescence sub- Isol-RNA-Lysis Reagent (Gaithersburg, MD, USA). Com- strate (GE Healthcare, London, UK) as previously plementary DNA was acquired via reverse-transcription of described9. Chemiluminescent signals were acquired and total RNA by using