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USP4 Promotes Invasion of Breast Cancer Cells Via Relaxin/TGF-Β1/Smad2/MMP-9 Signal

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USP4 Promotes Invasion of Breast Cancer Cells Via Relaxin/TGF-Β1/Smad2/MMP-9 Signal European Review for Medical and Pharmacological Sciences 2016; 20: 1115-1122 USP4 promotes invasion of breast cancer cells via Relaxin/TGF-β1/Smad2/MMP-9 signal W.-H. CAO1, X.-P. LIU2, S.-L. MENG3, Y.-W. GAO3, Y. WANG1, Z.-L. MA1, X.-G. WANG1, H.-B. WANG1 1Department of Breast Surgery, the Affiliated Hospital of Qingdao University, Qingdao, China 2Department of Central Laboratory, the Affiliated Hospital of Qingdao University, Qingdao, China 3Department of Gynaecology, People’s Hospital of Zhangqiu, Jinan, China Abstract. – OBJECTIVE: Ubiquitin-specific = dimethyl sulfoxide; BB94, batimastat; PBS, phosphate protease 4 (USP4) is a deubiquitinating enzyme buffered saline; FBS, fetal bovine serum; DMEM, Dul- with key roles in the regulation of TGF-β1 signal- becco’s modified Eagle medium; IMDM, Iscove’s modi- ing, suggesting its importance in tumorigenesis. fied Dulbecco’s medium. However, the underlying mechanisms causing this are not entirely clear. In the present study, we investigated the effect of USP4 on invasion Introduction and tumorigenesis of breast cancer cells, and explored its mechanism. MATERIALS AND METHODS: Breast cancer is the second-fourth leading cause Effects of USP4 of cancer-related deaths in women in the world, overexpression or USP4 silencing by small inter- fering RNA (USP4 siRNA) on invasion of breast suggesting that early diagnosis and prevention of 1 cancer MDA-MB-231 and T47D cells in vitro was this disease is urgently needed . Currently, breast detected. Using siRNAs and inhibitors to exam- cancer is treated with surgery, chemotherapy, and ine the USP4 signaling pathway. radiation therapy or combined modalities with RESULTS: The migration and invasion assays remarkable success. In addition, patients with showed that USP4 promotes human breast can- breast cancer or preneoplastic lesions are also cer cell migration and invasion by USP4 overex- pression, and knockdown of USP4 by siRNA in- treated with hormonal therapy either for treatment hibits human breast cancer cell migration and in- or prevention purposes. Although these treatment vasion. Treatment with RLX siRNAs, TGF-β1 siR- modalities are successful, a significant number of NAs, Smad2 siRNAs or BB94 (MMPs inhibitor) to patients either do not respond to therapy, or the tu- USP4-overexpressing breast cancer cells revealed mor may recur during therapy and develop metas- that USP4- induced RLX via TGF-β1 pathway pro- tasis, for which there is limited curative therapy2,3. motes the cell migration and invasion. Further This inadequate outcome strongly suggests that studies demonstrated that USP4-mediated TGF-β1 activation not only enhances the phosphorylation the evaluation of novel targeted therapeutic agents of Smad2 through TGF-β, but also directly upreg- is urgently needed to improve the treatment out- ulate matrix metalloproteinase (MMP)-9-mediated come of patients diagnosed with this disease. cell migration and invasion of breast cancer cells. Ubiquitin-specific proteases (USPs) regulate CONCLUSIONS: Therapies targeting the USP4 post-translational modification of proteins by inhibits invasion of breast cancer cells via Re- inhibiting ubiquitination of its targets, affecting laxin/TGF-β1/Smad2/MMP-9 signal. These re- multiple biological processes such as cell cycle, sults indicate that USP4 is an attractive target 4 for breast cancer therapy. DNA repair, and cell signaling pathways . USP4, a member of USP subfamily, is the first deubiq- Key Words: uiting enzymes that have been identified in mam- Breast cancer, Ubiquitin-specific protease 4, Relax- malian cells. TGF-β, a multifunctional cytokine, in, TGF-β1. plays a tumor suppressive role in normal epithelia cells and precancerous tissues by inhibiting cell Abbreviations proliferation and inducing apoptosis, but acceler- ates the progression of established cancers by pro- BC = breast cancer; TGF-b = transforming growth fac- moting cell proliferation, invasion, and metasta- tor-beta; MMP-9 = matrix metalloproteinases-9; DMSO sis5-8. TGF-β initiates signaling by binding to type Corresponding Author: Hai-Bo Wang, MD; e-mail: [email protected] 1115 W.-H. Cao, X.-P. Liu, S.-L. Meng, Y.-W. Gao, Y. Wang, Z.-L. Ma, X.-G. Wang, H.-B. Wang I and type II receptor serine/threonine kinases on USP4 siRNA Transfection the cell surface, receptor-mediated Smads activa- MDA-MB-231 were seeded in six-well plates, tion to regulate gene expression, which is a classi- grown to 50-80% confluence. Cells were trans- cal pathway for TGF-β signaling transduced from fected with USP4 siRNA (2 μg) in OptiMEM the cell membrane to the nucleus, and resulting in (Gibco, BRL, Grand Island, NY, USA) for 48 hs tumor suppression. Recently, it has been shown using Lipofectamine 2000 (Invitrogen, Carlsbad, that USP4overexpression facilitates tumor cell in- CA, USA) according to the manufacturer’s in- vasion, migration, and aggressiveness by enhanc- structions. ing transforming growth factor-β (TGF-β) sig- naling9-10. However, the underlying mechanisms USP4 Plasmids Construction causing this are not entirely clear. Moreover, the and Transfection contribution of USP4 to breast cancer progression Human USP4 cDNA was cloned and amplified has not been determined. from leukocytes. The polymerase chain reaction Relaxin (RLN) is a small peptide hormone ex- (PCR) primers were: forward, 5′- GAA GGC CCT pressed in several cancers of reproductive and endo- GGA TGT GAT GGT G-3′ and reverse, 5′- CAT crine organs. Increased expression of RLN in breast TTC TTC CTG GGC TGC TTA TC-3′ with the cancer correlates with aggressive cancer. Previous restriction enzymes KpnI and XhoI link. The cD- studies have found that RLN was the downstream NA was then subcloned into a linearized PMD-18T of the TGF-β1/Smad2 signal, which could in- vector [Takara Biotechnology Co., Ltd., Dalian, duce MMP-9 expression, suggesting that RLN plays China], digested and released with KpnI and XhoI a key role in breast cancer tumorigenesis11,12. (Fermentas, Burlington, Ontario, Canada), and then In this study, we use USP4 gene-silenced and cloned into pcDNA3.1 (+) vector (Invitrogen Life overexpressing breast cancer cells to determine Technologies, Carlsbad, CA, USA). After amplifi- if USP4 can promote the migration and invasion cation and DNA sequence confirmation, this vector of breast cancer cells and clarify whether RLN/ was designated as pcDNA3.1-USP4 and used for TGF-β/Smad2/MMP-9 are involved in the mech- the overexpression of USP4 in breast cancer cells. anisms that USP4 promotes the migration and in- pcDNA3.1 and pCDNA3.1- USP4 plasmids were vasion of breast cancer cells, providing a novel separately transfected into T47Dcells using Li- therapeutic target for the pathogenesis and gene pofectamine 2000 (Invitrogen Life Technologies, therapy of breast cancer. Carlsbad, CA, USA) according to the manufactur- er’s instructions, and stable cell lines were selected with 600 μg/ml G418 (Invitrogen Life Technolo- Materials and Methods gies). Gene expression was confirmed by Western blot analysis. To determine the signaling pathways Cell Line and Culture involved in the production of RLX, TGF-β1, TGF- MDA-MB-231 and T47D human breast can- βRI, Phospho-Smad2 (Ser-465), MMP-9. The sta- cer cells were cultivated in HAM’s F12 medium ble pcDNA3.1 or pCDNA3.1- USP4 transfected (Biochrom, Shanghai, China) substituted with T47D cells were transfected with RLX siRNA or 10% fetal calf serum (FCS), 2 mmol L-glutamine TGF-β1 siRNA or Smad2 siRNA for 48 hs or treat- (Gibco, Invitrogen, Hangzhou, China), 6 ng/ ml ed withBB94 for 24 hs. insulin (Gibco; Invitrogen Corp, Hangzhou, Chi- na), 3.75 ng/ml hydrocortisone (Sigma, Oakville, Western Blot Analysis Shanghai, China). Cells were washed with ice-cold PBS and whole cell extracts were prepared using cell lysis Agents buffer [20 mmol/L Tris (pH 7.5), 0.1% Triton-X, The primary antibodies against USP4, RLX2, 0.5% deoxycholate, 1 mmol/L phenylmethylsul- TGF-β1, Phospho-Smad2 (Ser-465), MMP-9 fonyl fluoride, 10 ug/mL aprotinin, and 10 ug/ and actin were all purchased from Cell Signaling mL leupeptin] and cleared by centrifugation at Technology, Inc. (Shanghai, China). USP4 siR- 12,000 x g, 4oC. Total protein concentration was NA, RLX2 siRNA, TGF-β1 siRNA, Smad2 siR- measured using the bicinchoninic acid assay kit NA, BB94 (MMPs inhibitor) were from Cell Sig- (Sigma, St. Louis, MO, USA) with bovine serum naling Technology. Cells incubated with culture albumin as a standard. Cell lysates containing 30 medium or culture medium with DMSO served as mg total protein were analyzed by immunoblot- controls. ting. Chemoluminescent detection (Upstate, Lake 1116 USP4 promotes invasion of breast cancer cells via Relaxin/TGF-β1/Smad2/MMP-9 signal Placid, NY, USA) was done in accordance with were measured by Image-Pro Plus 6.0 software. the manufacturer’s instructions. The changes in migration were determined by comparing the difference in wound-healing areas Invasion Assay within 48 h (n = 6). The upper chamber of each Transwell was coated with Matrigel (BD Biosciences, Franklin Statistical Analysis Lakes, NJ, USA) 1:6 diluted with DMEM at 37°C Student’s t-test was used to assess the signifi- for 3 h. Cells (2×104) were seeded in upper cham- cance of differences among the different groups. bers in DMEM and incubated in 24-well-plates Differences were expressed as mean ±SD with with 10% FBS supplemented DMEM. After 36 h p-values < 0.05 being considered as statistically of incubation, cells remaining on the upper sur- significant. face of the membrane were removed with a cot- ton swab. Cells that invaded through the Matri- gel-precoated membrane filter were fixed, stained Results and counted using a microscope. Effect of siRNA on USP4 Expression Wound Healing Assay in MDA-MB-231 Cells Cells (3 × 105 cells/well) were seeded into More USP4 protein expression was shown in 24-well plates at 60-80% confluency, and the cell the MDA-MB-231 cells.
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