Original Article Silencing of TMSG1 Enhances Metastasis Capacity by Targeting V-Atpase in Breast Cancer

Int J Clin Exp Pathol 2015;8(2):1312-1320 www.ijcep.com /ISSN:1936-2625/IJCEP0004348

Original Article Silencing of TMSG1 enhances metastasis capacity by targeting V-ATPase in breast cancer

Yuan Zi, Wenjian Zhao, Jun Zhou, Hanjiang He, Ming Xie

Department of Pathology, Xiangnan University, East of Wangxian Mountain, Chenzhou 423000, Hunan Province, China Received December 2, 2014; Accepted January 28, 2015; Epub February 1, 2015; Published February 15, 2015

Abstract: TMSG1, as a novel tumor metastasis suppressor gene, has been demonstrated to closely relate to the metastasis and drug-resistant of breast cancer. However, its molecular mechanism is still unclear. In this study, we explored the effect of small interference RNA (siRNA) targeting TMSG1 on the invasion of human breast carcinoma cell line MCF-7 and its molecular mechanisms associated with the extracellular pH. qRT-PCR and Western blot analysis revealed dramatic reduction of the levels of TMSG1 mRNA and protein after transfection of siRNA in MCF-7 cells. Cell migration and invasion were obviously increased by TMSG1 siRNA treatment. The activity of vacuolar ATPase (V-ATPase) and MMP-2 was signiﬁcantly increased in MCF-7 cells transfected with the TMSG1 siRNA compared with the controls. Furthermore, acidic intracellular environment signiﬁcantly increased the MMP-2 activity and the capacity of cell migration and invasion. In conclusion, silencing of TMSG1 increased V-ATPase activity, decreased extracellular pH and in turn the activation of secreted MMP-2, which ultimately promoted metastasis capacity of breast cancer cell.

Keywords: Breast cancer, TMSG1, V-ATPase, MMP-2, extracellular pH

Introduction pressed in many types of metastatic cancers and positively correlated to their invasion and Breast cancer is the most common malignancy, metastasis by decreasing extracellular pH and threatening the health of women around the increasing secretion and activation of degrada- world. Although diagnostic and therapeutic tive enzymes, such as matrix metalloproteinas- methods have greatly improved over the last es (MMPs) [6, 9]. Furthermore, low extracellular decade, more than 1.3 million women world- pH may promote the degradation and remold- wide are diagnosed with breast cancer and ing of ECM through proteolytic enzyme activa- about half-a-million women still die because of tion, thus contributing to cancer invasion and breast cancer each year [1, 2]. The high figures metastasis [10]. In breast cancer cells, the of incidences and mortality, even with the abundance of V-ATPase on the plasma mem- advancement of primary screening and diagno- brane correlates with an invasive phenotype sis, increase the urgency to study the metasta- [11], and V-ATPase inhibitors reduce cell migra- sis mechanism of breast cancer and explore tion in cancer cells with high levels of plasma the genes and treatment target associated membrane V-ATPase [12, 13]. with the metastasis of breast cancer. TMSG1 gene (tumor metastasis suppressor Accumulating evidence suggests a key role of gene 1) is a novel tumor metastasis suppressor tumor acidic microenvironment in cancer devel- gene, which plays a prominent role in suppres- opment, progression, and metastasis [3-5]. sion of tumor invasion and metastasis [14]. It V-ATPase, as a newly identified ATP-dependent has been found that TMSG1 is negative correla- proton pump, pumps H+ out of the cell mem- tion with tumor metastatic potential. Silencing brane and thus play an important role in forma- of TMSG1 gene in PC-3M-2B4 cells enhanced tion and maintenance of the extracellular acidic invasion and metastasis of PCA cell [15]. The microenvironment [6-8]. V-ATPases are overex- roles played by TMSG-1 in suppression of tumor Silencing of TMSG1 enhances breast cancer cell metastasis capacity invasion and metastasis have been reported to CCGAACGUGUCACGUTT, and reverse is, 5’- be achieved through various mechanisms. ACGUGACACGUUCGGAGAATT-3’; Recent studies showed that TMSG1 could regulate V-ATPase activity and extracellular pH by Transfection targeting the C subunit of V-ATPase, thereby induce apoptosis of tumor cells [16, 17]. In Twenty-four hours before transfection cells addition, TMSG1 also promotes cell apoptosis were seeded, TMSG1 siRNA and Negative through the synthesis of ceramide, which in Control siRNA were transfected with Lipo- turn induces cell cycle arrest and affects the fectamine 2000 (Invitrogen, Carlsbad, CA) activity of telomerase as well as cell life span according to the manufacturer’s instructions. [18, 19]. Recently, it has been reported that low Twenty-four or forty-eight hours later, MCF-7 expression of LASS2 was associated with poor cells were collected and subjected to further prognosis in patients with breast cancer [20]. analysis. Total RNA or protein was extracted However, the molecular mechanism by which from the indicated cells for analysis. The ex- TMSG1 on breast cancer metastasis is unclear periment was done in triplicates. More than at present. nine wells were treated with the same kind of siRNA. In this study, small interfering RNAs (siRNAs) targeting TMSG1 will be adopted to silence the RNA extraction and qRT-PCR gene expression in human breast cancer cell Total RNA was isolated from cells with the line MCF-7. Meanwhile, we further elucidated TRIzol reagent (Invitrogen) according to the the molecular mechanism of TMSG1 involved in manufacturer’s protocol. Real-time polymerase the regulation of tumor metastasis by investi- chain reaction was performed with the StepOne gating the activity of V-ATPase, the expression Plus sequence detection system (Applied and activity of matrix metalloproteinase 2 Biosystems, Foster City, CA). To ensure the (MMP-2) in MCF-7 cells, then further revealed reproducibility of the results, all the genes the key role of V-ATPase induced extracellular were tested in triplicate. ITGB1 generated using acidic microenvironment in metastasis of sense (5’-gaagccagctggagattcac-3’) and anti- breast cancer. sense (5’-gacatcagaggcaatgctga-3’) primers. β-action sense primers: 5’-cattaaggagaagctgt- Materials and methods gct-3’ and antisense primers: 5’-gttgaaggtagt- Cells and cell culture ttcgtgga’. Western blot analysis MCF-7 breast cancer cells (American Type Culture Collection, Manassas, VA, USA; HTB- Whole cell extracts were prepared with a cell 22) was cultured at 37°C in a humidified 5% lysis reagent (Sigma-Aldrich, St. Louis, MO, CO2 atmosphere in RPMI-1640 medium with USA) according to the manual, and then, the 10% fetal calf serum (Gibco, Invitrogen, protein was quantifed by a BCA assay (Pierce, Carlsbad, CA, USA), 100 IU/ml penicillin G, and Rockford, IL, USA). Then, the protein samples 100 mg/ml streptomycin sulfate (Sigma-Ald- were separated by SDS-PAGE (10%) and detect- rich, St Louis, MO, USA). ed by Western blot using polyclonal goat anti- TMSG1 antibody (Santa Cruz Bio-technology) or Construction of siRNAs rabbit anti-MMP-2 antibody (Santa Cruz Bio- technology) and monoclonal mouse anti-β- We designed and purchased four different actin (Santa Cruz Bio-technology) as a control. siRNA duplexes of TMSG1 from GenePharma Goat anti-rabbit IgG (Pierce, Rockford, IL, USA) company (Shanghai, China). siRNA-1 primers secondary antibody conjugated to horseradish sense is 5’-CUGCCUUCUUUGGCUAUUATT, and peroxidase and ECL detection systems reverse is, 5’-UAAUAGCCAAAGAAGGCAGTT-3’; (SuperSignal West Femto, Pierce) were used for siRNA-2 primers sense is 5’-GCUGCCCUCUU- detection. GAACAUAATT, and reverse is, 5’-UUAUGUUCA- AGAGGGCAGCTT-3’; siRNA-3 primers sense is Activity of V-ATPase 5’-GAGUCAGCCAAGAUGUUUATT, and reverse is, 5’-UAAACAUCUUGGCUGACUCTT-3’; siRNA- Assays V-ATPase activity was performed as negative control primers sense is 5’-UUCU- described previously by Fan et al [21].

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Figure 1. TMSG1 siRNAs silenced the expression of TMSG1 in MCF-7 cells. A. qRT- PCR analyzed the expression of TMSG1 after transfection with different siRNA frag- ments in MCF-7 cells. B. The western blot analysis showed the expression of TMSG1 protein after interference. Control, untreated cells. siRNA NC, transfection with negative control siRNA. siRNA1, transfection with TMGS1 siRNA 1. siRNA2, transfection with TMGS1 siRNA 2. siRNA3, transfection with TMGS1 siRNA 3. Data are the mean ± SD of duplicates from a representative experiment of three independent experiments. *P < 0.01 vs. control and siRNA NC group.

Gelatin zymography Wound healing assay

MMP Zymography assay kit (for MMP-2 For the wound healing assay, cells were seeded and MMP-9) (Applygen Technologie, China) in 12-well plates and grown to 90% confluence. was used to detect the activity of MMP2 and Monolayers in the center of the wells were MMP9. Protein extracts and positive mixture scraped with pipette tips and washed with PBS. were mixed with an equal volume of 2X SDS- Cell movement into the wound area was moni- PAGE non-reducing buffer, and electropho- tored and photographed at 0 and 24 h using a resed on 8% SDS polyacrylamide gels contain- light microscope. The migration distance ing 2 mg/ml of gelatin. Gels were then washed between the leading edge of the migrating cells twice for 30 min in buffer A at room tempera- and the edge of the wound was compared as ture, and incubated for 4 hours at room tem- previous work [22]. perature in incubation buffer B. Gels were then Statistical analysis stained for 2 hour with 0.25% Coomassie bril- liant blue and then destained in destaining buf- Each experiment was repeated at least three fer (10% acetic acid and 20% methanol) for 60 times. Data were shown as mean ± s.d and min. analyzed using SPSS 18.0. Statistical compari- sons between groups were analyzed using Invasion assay Student’s t-test and a two-tailed P < 0.05 was considered to indicate statistical significance. Cells were cultivated to 80% confluence on the 12-well plates. Then, we observed the Results procedures of cellular growth at 24 h. All the experiments were repeated in triplicate. The Effects of TMSG1 siRNAs on the expression of transwell invasion chambers were used to eval- TMSG1 in MCF-7 cells uate cell invasion. Then cells invading across the membrane were counted under a light The MCF-7 cells were transfected with TMSG1 microscope. siRNAs and with negative control siRNA con-

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Figure 2. TMSG1 siRNA affects the migration and invasion of MCF-7 cells. A. Transwell assays showed that the TMSG1 siRNA transfected cells displayed dramatically increased invasion ability compared with the untreated cells or Negative Control siRNA transfected cells. B. In wound migration assay, the cell-free wound gaps of MCF-7 cells monolayers healed slowly in the untreated cells or Negative Control siRNA transfected cells. Data are the mean ± SD of duplicates from a representative experiment of three independent experiments. *P < 0.01 vs. control and siRNA NC group.

siRNA-2, 60.1% with siRNA-3, and 12.2% with siRNA-1 in the levels of TMSG1 mRNA after transfection of siRNA in MCF-7 cells, compared with TMSG1 negative control siRNA or the control (untransfected) group (P < 0.05; Figure 1). Thus, the cells transfected with TMSG1 shRNA2 were used for further experiments.

TMSG1 siRNA promotes the migration and invasion of MCF- 7 cells

Figure 3. Effects of TMSG1 siRNA on V-ATPase activity. Data are the mean As shown in Figure 2A, tran- ± SD of duplicates from a representative experiment of three independent experiments. *P < 0.01 vs. control and siRNA NC group. swell assays without Matrigel demonstrated that TMSG1 siRNA could signifcantly pro- taining scrambled random sequence, respec- moted migration of MCR-7 cells when com- tively. We examined four siRNAs to target pared with siRNA NC group and control (untreat- human TMSG1 as described above. qRT-PCR ed) group. To further confrm this observation, analysis showed that siRNA-2 effectively we also determined the migration ability of blocked the expression of TMSG1 mRNA. qRT- breast cancer cells in the condition of TMSG1 PCR revealed dramatic reduction of 88.1% with silencing using a wound migration assay. The

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Figure 4. Effects of TMSG1 siRNA on MMP-2 protein. A. Western blot analysis was used to detect the expression levels of MMP-2 protein. B. The activity of MMP-2 was examined by Gelatin zymography. Data are the mean ± SD of duplicates from a representative experiment of three independent experiments. *P < 0.01 vs. control and siRNA NC group.

Figure 5. Effects of extracellular pH on MMP-2 protein. A. Western blot analysis was used to detect the expression levels of MMP-2 protein. B. The activity of MMP-2 was examined by Gelatin zymography. Data are the mean ± SD of duplicates from a representative experiment of three independent experiments. results showed that the cell-free wound gaps of inhibiting the V-ATPase activity through binding MCF-7 cells monolayers healed slowly in the to ATP6V1H, we examined the infuence of untreated cells or Negative Control siRNA trans- TMSG1 siRNA on the V-ATPase activity by fected cells. However, in TMSG1 siRNA-2 trans- GEMEND’s V-ATPase activity assay kit. The fected cells, the closure of the wounded area activity of V-ATPase = OD of sample/(concen- was signiﬁcantly accelerated (Figure 2B). tration × 31.1) × 103 U/mg. As shown in Figure 3, the V-ATPase activity was greatly increased TMSG1 siRNA increase the activity of V-ATPase in MCF-7 cells in TMSG1 siRNA-2 treated MCF-7 cells (2.53 ± 0.031) compared with untreated cells (1.43 ± To explore whether TMSG1 siRNA suppress 0.022) and siRNA-NC treated cells (1.39 ± breast cancer cell migration and invasion by 0.014).

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Figure 6. Extracellular pH affects the invasion and migration of MCF-7 cells. A. Transwell assays showed the invasion ability at extracellular PH 7.4, PH 7.0 and PH 6.6 respectively. B. Wound migration assay showed the migration ability at extracellular PH 7.4, PH 7.0 and PH 6.6 respectively. Data are the mean ± SD of duplicates from a representative experiment of three independent experiments.

Effects of TMSG1 siRNA on the expression and of MMP-2 was signifcantly increased by TMSG1 activity of MMP-2 protein siRNA. These results indicated that TMSG1 didn’t have obvious effects on the expression V-ATPases pumps proton out of the cell mem- levels of MMP-2 protein, but have signifcant brane and thus plays an important role in for- effects on the activity of MMP-2 protein in the mation and maintenance of the extracellular MCF-7 cells. acidic microenvironment, which positively are positively correlated to secretion and activation Extracellular pH affects the activity of MMP- of degradative enzymes, such as matrix metal- 2 protein and the migration and invasion of loproteinases (MMPs) [6, 9]. Here, we deter- MCF-7 cells mined the expression levels and activities of MMP-2, which are closed related to cancer Accumulating studies show a key role of tumor invasion and metastasis [23]. Western blot acidic microenvironment in cancer develop- showed that TMSG1 siRNA didn’t have obvious ment, progression, and metastasis. The acidic effect on the expression of total MMP-2 protein tumor extracellular microenvironment induces in the MCF-7 cells (Figure 4A). Furthermore, the the increased secretion and activation of prote- supernatant of cultured cells was collected and ases, and promotes the degradation and the gelatinase activity was assayed with gelatin remolding of extracellular matrix (ECM) through zymography. As shown in Figure 4B, the activity proteolytic enzyme activation, thus contribut-

1317 Int J Clin Exp Pathol 2015;8(2):1312-1320 Silencing of TMSG1 enhances breast cancer cell metastasis capacity ing to cancer invasion and metastasis. In this gene expression of TMSG1 in human breast study, we further analyzed the effects of acidic cancer cell line MCF-7. Furthermore, we extracellular microenvironment on MMP-2 pro- confirmed that siRNA2 could signifcantly tein in MCF-7 cells. As shown in Figure 5A, decreased the TMSG1 expression at the mRNA extracellular pH didn’t affect obviously the level and protein level, reduced 88.1% TMSG1 expression of total MMP-2 protein in the MCF-7 mRNA and 93.9% TMSG-1 protein. Then, we cells using western blot analysis. Furthermore, further analyzed the effects of LASS2 silencing the gelatinase activity assayed the revealed on invasion and migration of breast cancer. Our the positive correlation between extracellular results showed that silencing of TMSG1 could pH and MMP-2 activity. As shown in Figure 5B, promote the migration and invasion of MCF-7 the activity of MMP-2 was signifcantly cells, which in turn suggested that TMSG1 gene increased in a PH-dependent manner. These function as tumor metastasis suppressor. results indicated that extracellular pH didn’t have obvious effects on the expression levels It is well-known that extracellular pH is usually of MMP-2 protein, but have signifcant effects low in solid tumors, in contrast to the approxi- on the activity of MMP-2 protein in the MCF-7 mately neutral intracellular pH. VATPase, as cells. ATPdependent hydrogen ion pump, pumps out H+ through transmembrane transportation and Furthermore, we demonstrated the effects of plays important roles in formation and mainte- extracellular PH on invasion and migration of nance of extracellular acidic microenvironment MCF-7 cells. As shown in Figure 6A, acidic and metastasis [9]. Recently, Sennoune et al. extracellular microenvironment (PH 6.6) dra- showed the correlation of V-ATPases located at matically increased invasion ability compared the plasma membrane with the metastatic with the PH 7.0 and PH 7.4 extracellular micro- potential of breast cancer cells [12]. The prefer- environment. The wound migration assay fur- ential expression of V-ATPase at the cell sur- ther confrm this observation that acidic extra- face is important for the acquisition of invasive- cellular microenvironment greatly increased ness and metastasis of the breast cancer cells migration ability compared with the PH 7.0 and [12]. Moreover, TMSG1, as a suppressor of PH 7.4 extracellular microenvironment (Figure V-ATPase, inhibited the V-ATPase activity by 6B). interacting with ATP6V0C, the c subunit of V-ATPase proton pump [16]. Here, in order to Therefore, acidic extracellular microenviron- investigate the breast tumor metastasis sup- ment significantly increased the migration and pression mechanism of TMSG1, we studied the invasion ability of MCF-7 cells. interaction between TMSG1 and V-ATPase. As shown in Figure 3, the activity of V-ATPase was Discussion greatly increased byTMSG1 siRNA in MCF-7 Tumor metastasis suppressor gene 1 (TMSG1), cells. The findings were also confirmed in previ- also known as LASS2, is a newly found tumor ous work that TMSG1 siRNA and shRNA could metastasis suppressor gene. Accumulating evi- enhance the activity of V-ATPase and increase + dences showed that it not only suppressed extracellular H concentration in prostate can- tumor growth but was closely related to tumor cer cells [15, 25]. These results suggested that metastasis. Recently, it has been reported that TMSG-1 could inhibit the activity of V-ATPase, expression levels of TMSG-1 was altered in suppress the proton transmembrane secretion + breast cancer and TMSG-1 overexpression and thus decrease extracellular H concentra- inhibited the proliferation, invasion and apopto- tion in breast cancer cells. The promoting effect sis of metastatic breast cancer cells signifcant- of V-ATPase on cancer invasion and metastasis ly [20, 24]. However, as a novel TMSG, the pre- mainly relies on its maintaining acidic pH of cise molecular mechanisms of TMSG1 on extracellular microenvironment, which is relat- breast cancer metastasis are unclear. ed to the activation, secretion, and cellular distribution of many proteases involved in the Therefore, to further investigate the molecular digestion of ECM [11]. The pH-sensitive prote- mechanisms of the antitumor effect of TMSG-1 ases include cathepsin and MMPs (MMP-2, on metastasis of breast cancer, small interfer- MMP-9, MMP-3, etc.). Here, we demonstrated ence RNA (siRNA) was adopted to silence the that TMSG1 siRNA could increase the activity of

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MMP-2 in the supernatant of the MCF-7 cells, Wangxian Mountain, Chenzhou 423000, Hunan but had no effect on the expression and secre- Province, China. Tel: +86-18973579656; E-mail: tion of MMP-2 protein, which was consistent [email protected] with previous studies by Xu et al [15, 25]. These results indicated that migration of tumor cells References might be regulated by MMP activity, rather than [1] Cancer Genome Atlas Network. Comprehen- by MMP expression. sive molecular portraits of human breast tu- To reveal the relationship among V-ATPase- mours. Nature 2012; 490: 61-70. [2] Jabbarzadeh Kaboli P, Rahmat A, Ismail P and induced acidic extracellular microenvironment, Ling KH. Targets and mechanisms of berber- tumor metastasis and MMP2 protein, we fur- ine, a natural drug with potential to treat can- ther analyzed invasion and migration capacity cer with special focus on breast cancer. Eur J of MCF-7 cells at PH 6.6, PH 7.0 and PH 7.4 Pharmacol 2014; 740: 584-595. extracellular microenvironment. As shown in [3] Navratilova J, Hankeova T, Benes P and Smar- Figure 5, MMP2 expression level in MCF-7 cells da J. Acidic pH of tumor microenvironment en- was not affected by different PH cultured condi- hances cytotoxicity of the disulfram/Cu2+ tions, however, the MMP2 activity was complex to breast and colon cancer cells. Che- signiﬁcantly upregulated from pH 7.4 and pH motherapy 2013; 59: 112-120. 6.6 cultured conditions. Furthermore, acidic [4] Tong Z, Luo W, Wang Y, Yang F, Han Y, Li H, Luo extracellular microenvironment (PH 6.6) dra- H, Duan B, Xu T, Maoying Q, Tan H, Wang J, matically increased invasion and migration Zhao H, Liu F and Wan Y. Tumor tissue-derived ability compared with the PH 7.0 and PH 7.4 formaldehyde and acidic microenvironment synergistically induce bone cancer pain. PLoS extracellular microenvironment. TGMS1 si- One 2010; 5: e10234. lencing- induced invasion and migration were [5] Liotta LA and Kohn EC. The microenvironment reversed by high extracellular pH (PH 7.4). of the tumour-host interface. Nature 2001; These data also indicated that V-ATPase play- 411: 375-379. ed an important role in breast cancer [6] Sennoune SR, Luo D and Martinez-Zaguilan R. metastasis. Plasmalemmal vacuolar-type H+-ATPase in cancer biology. Cell Biochem Biophys 2004; In conclusion, silencing of TMSG1 promoted 40: 185-206. the formation of the extracellular acidic micro- [7] Rojas JD, Sennoune SR, Maiti D, Bakunts K, environment by targeting V-ATPase and promot- Reuveni M, Sanka SC, Martinez GM, Seftor EA, ing the V-ATPase activity in breast cancer. The Meininger CJ, Wu G, Wesson DE, Hendrix MJ acidic extracellular microenvironment activated and Martinez-Zaguilan R. Vacuolar-type H+- degradative enzymes, such as matrix metallo- ATPases at the plasma membrane regulate pH proteinases (MMPs) and degradation of ECM, and cell migration in microvascular endothelial ultimately accelerated tumor’s invasion and cells. Am J Physiol Heart Circ Physiol 2006; 291: H1147-1157. metastasis. Our ﬁndings suggest that TGMS1, [8] Torigoe T, Izumi H, Ishiguchi H, Uramoto H, Mu- as a novel tumor metastasis suppressor gene, rakami T, Ise T, Yoshida Y, Tanabe M, Nomoto is a promising therapeutic target for advanced M, Itoh H and Kohno K. Enhanced expression breast cancer. of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents. Acknowledgements J Biol Chem 2002; 277: 36534-36543. [9] Fais S, De Milito A, You H and Qin W. Targeting This work was supported by a grant of Youth vacuolar H+-ATPases as a new strategy against Foundation of Education Bureau of Hunan cancer. Cancer Res 2007; 67: 10627-10630. Province, China (NO 13B111) and a grant of [10] Rofstad EK, Mathiesen B, Kindem K and Innovation Center Foundation of the Higher Galappathi K. Acidic extracellular pH promotes Education Institutions of Hunan Province, China experimental metastasis of human melanoma (NO 13K113). cells in athymic nude mice. Cancer Res 2006; 66: 6699-6707. Disclosure of conflict of interest [11] Chung C, Mader CC, Schmitz JC, Atladottir J, Fitchev P, Cornwell ML, Koleske AJ, Crawford None. SE and Gorelick F. The vacuolar-ATPase modu- lates matrix metalloproteinase isoforms in hu- Address correspondence to: Dr. Ming Xie, Depart- man pancreatic cancer. Lab Invest 2011; 91: ment of Pathology, Xiangnan University, East of 732-743.

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