Tumor Biol. DOI 10.1007/s13277-015-3207-7

RESEARCH ARTICLE

USP2 promotes cell migration and invasion in triple negative breast cancer cell lines Qing Qu & Yan Mao & Gang Xiao & Xiaochun Fei & Jinglong Wang & Yuzi Zhang & Junjun Liu & Guangcun Cheng & Xiaosong Chen & Jianhua Wang & Kunwei Shen

Received: 7 September 2014 / Accepted: 3 February 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is often associated with a poor prognosis. The aim of our study was to identify biomarkers predictive of TNBC progression. Primary TNBC breast tissue samples including four with metastasis and six without metastasis were subjected to Affymetrix GeneChip® analysis (human genome U133). Ubiquitin-specific protease 2 (USP2) was identified as an upregulated gene in the metastatic group, and its expression was analyzed by immunohistochemistry in 121 primary breast cancers, 13 paired normal tissues, and 13 paired metastatic lesions. Survival analysis was performed using the log-rank test and Cox regression hazard model. Matrigel migration and invasion assays in USP2silenced and USP2-overexpressed breast cancer cell lines were used to investigate the mechanisms of USP2 in vitro.

Positive immunostaining for USP2 was detected in breast tumors and was correlated with estrogen receptor (ER) and progesterone receptor (PR) statuses and TNBC subtype. USP2 was overexpressed in distant metastatic lesions compared with primary breast cancers. Survival analyses demonstrated that positive USP2 is a poor prognostic factor for disease-free survival. Silencing of USP2 expression decreased migration and invasion in LM2-4175 and SCP46 cells in association with the downregulation of matrix metalloproteinase-2 (MMP2) expression, whereas overexpression of USP2 in MDA-MB468 and MDA-MB-231 cells enhanced migration and invasion and upregulated the expression of MMP2. The present study showed that USP2 expression is associated with TNBC cell line’s invasiveness and poor survival of breast cancer patients and may serve as a prognostic biomarker and therapeutic target for TNBC. Keywords Triple negative breast cancer . USP2 . Metastasis . MMP2

Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3207-7) contains supplementary material, which is available to authorized users. Q. Qu Department of Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Y. Mao : Y. Zhang : J. Liu : X. Chen : K. Shen (*) Comprehensive Breast Health Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No. 197, Ruijin 2 Road, Shanghai 200025, People’s Republic of China e-mail: [email protected] G. Xiao : J. Wang : G. Cheng : J. Wang Department of Biochemistry and Molecular & Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China X. Fei Department of Pathology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Introduction Breast cancer (BC) is one of the most common malignancies among women worldwide. It represents a heterogeneous group of tumors with diverse biological characteristics and clinical behavior, outcome, and response to therapy. Gene expression profiling led to the classification of breast cancers into five subtypes, luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpressing, and normallike and basal-like based on estrogen receptor (ER) and progesterone receptor (PR) statuses and/or epithelial cellular origin (basal or luminal) [1–3]. DNA microarray and immunohistochemical (IHC) analyses have shown that 70 to 80 % of

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triple negative tumors are basal-like, and their clinical behavior is similar to that of the basal-like subtype. Triple negative breast cancers (TNBCs), which are characterized by the lack of ER, PR, and HER2 gene amplification, represent approximately 12–15 % of all breast cancers and are frequently associated with a poor prognosis compared to other breast cancer subtypes. TNBCs are aggressive tumors that frequently affect younger patients and are more prevalent among AfricanAmerican women [4–6]. TNBC constitutes one of the most challenging types of breast cancer given its high rate of recurrence and poor prognosis [4, 7]. Metastasis is the most frequent reason of breast cancer treatment failure. TNBC metastatic progression is marked by early relapse with a predominance of visceral and central nervous system metastases and a lower rate of bone metastasis [7]. The risk of early recurrence is highest within the first 2 to 3 years after surgery, with the majority of deaths occurring in the first 5 years [4, 5, 8]. TNBC was recently shown to be a heterogeneous group of tumors that can be divided into six subtypes with different treatment responses, namely basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, and luminal androgen receptors [9]. Clinically, disease-free survival periods show marked variation among TNBC patients. Therefore, identifying new biomarkers predictive of TNBC progression may prove beneficial to prevent metastasis and for the design of effective therapeutic strategies for this disease. In the present study, high-throughput GeneChip® analysis was used to identify genes contributing to early metastasis in TNBC patients by comparing primary tumors with and without metastasis during the first 2 years after treatment. We identified ubiquitin-specific protease 2 (USP2) as an upregulated gene associated with early metastasis. USP2 is a member of the ubiquitin-specific protease (USP) family, whose members show limited homology outside of the active catalytic sites, suggesting the specificity of their interaction with defined substrates [10]. USPs are cysteine proteases that belong to a class of proteolytic enzymes known as deubiquitinating enzymes. USPs function in the Bediting^ or disassembly of polyubiquitin chains by catalyzing the removal of ubiquitin moieties and can generate free ubiquitin from polyubiquitin chains released after proteasomal activity. USP2 is overexpressed in human prostate adenocarcinomas, as determined by IHC, and its oncogenic behavior when overexpressed in vitro and in vivo is related to its antiapoptotic activity [11]. However, the role of USP2 in breast cancer remains unclear. In the present study, we investigated the role of USP2 in the progression of breast cancer and explored the underlying mechanisms. The effects of USP2 on increasing the migration and invasion of TNBC cell lines indicated that it has

oncogenic properties and suggest USP2 as a specific marker of aggressive BC subtypes such as TNBC.

Materials and methods Ethics statement The present study was conducted after obtaining approval from the institutional review board (IRB) at Ruijin Hospital, who also waived the need for consent. Consent was not needed for this study since there was no interaction with patients enrolled and was based on their existing, available medical information, and that information was used in a de-identified fashion. Materials Fresh BC tissue samples were obtained from ten patients (mean age 53.20 years; range 44–65 years) who were pathologically diagnosed with BC of histological grade II (n=3) and grade III (n=7) and classified as stage II (n=5) and stage III (n=5) and immediately stored at −80 °C for microarray analysis. Patients were identified from the breast cancer database and tumor bank of our institution, and USP2 expression levels in the primary tumors were assessed using immunohistochemistry (IHC). The samples analyzed were obtained from 121 primary breast cancers (supplementary Table), 13 paired adjacent breast tissues not containing tumor (referred to as Bnormal^), and 13 paired metastatic lesions (seven lung metastases, three liver metastases, two chest wall recurrences, one contralateral lymph node metastasis) collected from the Comprehensive Breast Health Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Archived formalin-fixed paraffin-embedded breast cancer blocks were collected from all the participants and used to construct a tissue microarray (TMA). All patients underwent breast cancer surgery between 2003 and 2008 (Table 1). All the breast cancer cases were followed from the date of diagnosis until May 31, 2013 or death, whichever came first. The median follow-up was 6.2 years. The small interfering RNA (siRNA) oligonucleotides were designed by GenePharma (Shanghai, China) and annealed according to the manufacturer’s instructions. The antibodies used in this study were anti-USP2 (Proteintech, USA), anti-matrix metalloproteinase-2 (MMP2) (Santa Cruz Biotechnology, USA), and antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Kangchen, China). The secondary antibodies used were goat anti-rabbit and goat anti-mouse from Jackson ImmunoResearch (USA).

Tumor Biol. Table 1

Characteristics of patients included in the GeneChip® analysis

No.

Age

With metastasis 1 48 2 60 3 44 4 60 Without metastasis 5 59 6 48 7 49 8 65 9 44 10 55

Menopause status

Pathology

Grade

T stage

N stage

Metastasis location

DFS

Pre Post Pre Post

IDC IDC IDC IDC

II III III III

T1 T2 T2 T2

N2 N3 N0 N0

Bone Brain Lung Lung

10 8 11 20

m m m m

Post Pre Pre Post Pre Post

IDC IDC IDC IDC IDC IDC

II III II III III III

T1 T1 T2 T2 T2 T2

N2 N2 N0 N3 N1 N0

– – – – – –

36 41 45 45 43 44

m m m m m m

Methods

Cell culture and transfections

Gene chip

Human breast adenocarcinoma MDA-MB-468 and MDAMB-231 cells were obtained from American Type Culture Collection. LM2-4175, SCP2, SCP28, and SCP46 cells were gifts from the Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, China. Cells were cultured at 37 °C with 10 % CO2 in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 4.5 g/l glucose, 10 % fetal bovine serum (FBS, GIBCO), 2 mM glutamine, and antibiotics. Transfections were performed using Lipofectamine 2000 (Invitrogen) in OPTI-MEM (Invitrogen) media as directed by the manufacturer. Approximately 4–6 h after transfection, media were supplemented with DMEM containing 10 % FBS without antibiotics.

Total RNA was extracted from cancer tissues using the TRIzol Reagent (Cat#15596-018, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol and checked for a RIN number to inspect RNA integrity using an Agilent BioAnalyzer 2000 (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified using an RNeasy Micro Kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79524, QIAGEN, GmBH, Germany). RNA samples were stored at −80 °C. RNA (250 ng) was reverse transcribed into complementary RNA (cRNA) using GeneChip® 3′ IVT Express Kit (Affymetrix, Santa Clara, CA, USA) and purified using a BioMek® FX workstation (Beckman Coulter, CA, USA). The purified cRNAs were fragmented into 35- to 200-ntlong fragments and hybridized to the GeneChip® H Human Genome U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA, USA) using standard protocols. The chips were washed in the GeneChip® Fluidics Station (Affymetrix) and subsequently scanned using the GeneChip® Scanner 3000 (Affymetrix). Raw data were obtained using GCOS software and analyzed using R2.9.0 software and the Bioconductor 2.4.

Tissue microarray and immunohistochemistry Immunostaining was performed on formalin-fixed paraffinembedded samples (TMA blocks). The primary antibody used was anti-USP2 (Proteintech, USA) at 1:50 dilution. For interpretation of the IHC staining results, the IHC tests for USP2 were categorized as negative (0), B1+,^ B2+,^ or B3+^ under a high-power field (×200 magnification) according to the intensity of cytoplasmic staining in every case.

USP2 downregulation by shRNA The sequences of the synthetic oligonucleotides (GenePharma, China) used for USP2 siRNA constructs were as follows: 5′CAGAUUGUGGUUACUGUUCUA-3′, 5′-CAGGAGAA UGGCACACUUUCA-3′, and 5′-CCGCGCUUUGUUGG CUAUAAU-3′; the control siRNA was 5′-UGAACACCAC CAGGGAUCUTT3-′. LM2-4175 and SCP46 cells were transfected with USP2 or control siRNA and 48–72-h posttransfection; the downregulation of USP2 was assessed by Western blotting for the protein expression level or by quantitative reverse transcription polymerase chain reaction (RTPCR) for the messenger RNA (mRNA) expression level. USP2 overexpression constructs To restore the expression of USP2 in TNBC cells, human fulllength USP2 complementary DNA (cDNA) was inserted into pcDNA™6/myc-His A, B, and C. MDA-MB-468 and MDA-

Tumor Biol.

MB-231 cells were transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 48–72 h, the overexpression of USP2 was assessed by Western blotting for the protein expression level or by quantitative RT-PCR for the mRNA expression level.

blindly, without any information regarding clinical parameters or outcome. USP2 positivity was assigned for scores B2+^ and B3+.^

Reverse transcriptase-PCR

Data are presented as mean±SD. Values with p50 years ≤50 years Tumour size >2 cm ≤2 cm Lymph node status Metastasis No metastasis Grade Grade I Grade II Grade III ER status Positive Negative PR status Positive Negative Subtype TNBC Non-TNBC

USP2 negative n=69 (%)

USP2 positive n=52 (%)

p value

41 (59.4) 28 (40.6)

42 (80.8) 10 (19.2)

0.017

34 (49.3) 35 (50.7)

28 (53.8) 24 (46.2)

0.714

21 (30.4) 48 (69.6)

22 (42.3) 30 (57.7)

0.186

29 (42.0) 28 (40.6) 12 (17.4)

16 (30.8) 26 (50.0) 10 (19.2)

52 (75.4) 17 (24.6)

30 (57.7) 22 (42.3)

0.05

45 (65.2) 24 (34.8)

24 (46.2) 28 (53.8)

0.043

13 (18.8) 56 (81.2)

20 (38.5) 32 (61.5)

0.023

0.455

USP2 ubiquitin-specific protease 2, ER estrogen receptor, PR progesterone receptor, HER2 human epidermal growth factor receptor 2

MB-231 BC cell lines. These results indicated that USP2 expression may be related to the aggressiveness of TNBC (Fig. 1b).

72 h, the expression of both USP2 and GAPDH was analyzed by Western blotting. Analysis of the effect of USP2 knockdown on the invasive behavior of BC cells showed that silencing of USP2 expression by siRNA decreased migration and invasion in LM2-4175 (migration, p=0.046; invasion, p= 0.010) and SCP46 cells (migration, p=0.010; invasion, p= 0.003) (Fig. 4). An important process associated with migration and invasion during tumor progression is the degradation of the extracellular matrix, usually through the action of matrix metalloproteinases (MMP). The effect of USP2 on the expression of MMP2 was assessed by Western blotting, which showed that silencing of USP2 downregulated the expression of MMP2 (Fig. 4). USP2 overexpression increases migration and invasion in triple negative breast cancer cells To further examine the effect of USP2 on the invasive behavior of BC cells, USP2 was overexpressed in the different BC cell lines. The expression of USP2 and GAPDH was analyzed by Western blotting after 72 h of transfection. Our results showed that USP2 overexpression increased the migration and invasion of MDA-MB-468 (migration, p=0.025; invasion, p=0.005) and MDA-MB-231 BC cells (migration, p= 0.017; invasion, p=0.001) (Fig. 5). To confirm the effect of MMP2 on USP2, the expression of MMP2 was measured in BC cells overexpressing USP2. Our results showed that USP2 overexpression upregulated the expression of MMP2, suggesting that USP2 is likely involved in the regulation of MMP2 expression (Fig. 5).

Discussion USP2 silencing decreases migration and invasion in triple negative breast cancer cells To examine the function of USP2, its expression was knocked down by siRNA in the different breast cancer cell lines. After Fig. 3 Kaplan–Meier analysis of USP2 expression. Survival was compared between the USP2positive and USP2-negative groups. The p values were calculated using the log-rank test. a Disease-free survival (p=0.023). b Overall survival (p=0.269)

BC is a heterogeneous disease, and different molecular subtypes have been identified. TNBC exhibits dismal survival rates because of its highly aggressive and metastatic behavior, which represents a significant obstacle to the development of

Tumor Biol.

Fig. 4 Analysis of USP2 expression in two breast cancer cell lines (LM24175 and SCP46) by USP2 silencing. a Western blot analysis: LM2-4175 cells transfected with siUSP2 showed reduced USP2 and MMP2 levels. b Western blot analysis: SCP46 cells transfected with siUSP2 showed reduced USP2 and MMP2 levels. c Silencing of USP2 suppressed the migration potential of LM2-4175 cells (p=0.046, after 18 h). d Silencing

of USP2 suppressed the migration potential of SCP46 cells (p=0.010, after 18 h). e Silencing of USP2 suppressed the invasion potential of LM2-4175 cells (p=0.010, after 22 h). f Silencing of USP2 suppressed the invasion potential of SCP46 cells (p=0.003, after 22 h). Asterisk means p50

members known to date [16]. Certain USPs play key regulatory roles in a variety of biological and pathological processes, such as cellular growth pathways, stabilization/degradation of the p53 protein [17–19], and DNA repair [20] and only rarely display oncogenic properties [21–23]. USP2, a cysteine protease and a member of the ubiquitin-specific protease family, is oncogenic when overexpressed in prostate cancer cell lines and represents a therapeutic target in human prostate cancer [11]. However, the role of USP2 in human breast cancer remains unclear. To our knowledge, this is the first comprehensive analysis of the role of USP2 in breast cancer. We used immunochemistry techniques to demonstrate that USP2 is overexpressed in almost 50 % of breast cancers when compared with adjacent normal tissues. Furthermore, we showed that USP2 is overexpressed in the majority of metastatic lesions compared with primary breast tumors. Our present clinical study provides further evidence supporting the findings of preclinical in vivo studies, and

Tumor Biol.

Fig. 5 Analysis of USP2 expression in two breast cancer cell lines (MDA-MB-468 and MDA-MB-231) overexpressing USP2. a Western blot analysis: MDA-MB-468 cells with USP2 overexpression showed increased USP2 and MMP2 levels. b Western blot analysis: MDA-MB231 cells with USP2 overexpression showed increased USP2 and MMP2 levels. c Overexpression of USP2 enhanced the migration potential of

MDA-MB-468 cells (p=0.025, after 22 h). d Overexpression of USP2 enhanced the migration potential of MDA-MB-231 cells (p=0.017, after 22 h). e Overexpression of USP2 enhanced the invasive potential of MDA-MB-468 cells (p=0.005, after 24 h). f Overexpression of USP2 enhanced the invasive potential of MDA-MB-231 cells (p=0.001, after 24 h)

our IHC analysis identified prognostic factors most frequently associated with the development of metastasis in breast cancer. We found a significant association between USP2, hormone receptor expression, and TNBC subtype in our tissue samples. The results from this study demonstrate, for the first time, that USP2 is an independent prognostic marker in breast cancer patients. Adjusted HRs of USP2 for DFS were 1.415 (95 % CI, 1.049–1.909, p =0.023), and 1.270 (95 % CI, 0.831–1.939, p = 0.269) for OS, indicating that USP2positive breast cancer patients showed a poor DFS rate. But we did not find the same prognostic impact on survival among the luminal or TNBC subtype breast cancer patients probably because of the small sample size. We also found the upregulation of USP2 expression in metastatic lesions compared with the primary breast tumors, which supported a positive association between USP2 expression and tumor progression or metastasis. To further confirm these results in vitro and to examine the mechanisms underlying the effect of USP2 in TNBC, we

performed basic studies and found that USP2 plays an integral role in the promotion of invasiveness in basallike breast cancer cells. Knockdown of USP2 resulted in phenotypic and physiological changes in BC cell lines; the most striking of which was the inhibition of migration and invasion, as measured by Matrigel assay in LM2-4175 and SCP46 breast cancer cell lines. Conversely, overexpression of USP2 promoted the migratory and invasive potential of MDA-MB-231 and MDA-MB468 breast cancer cells. The role of USP2 as a metastasis promoter gene was supported by the following observations: (1) USP2 is overexpressed in breast cancer tissues and metastatic lesions, (2) USP2 is required and sufficient to drive cancer cell migration and invasion, and (3) the metastasis promoting activity of USP2 is further enhanced by the upregulation of MMP2 activity. Taken together, these findings suggest that USP2 plays a role in tumor metastasis by modulating the activity or

Tumor Biol.

expression of MMP2. It is noteworthy that USP2 showed the capacity to increase aggressiveness, suggesting that it could be a candidate marker of breast cancer. Our findings in breast cancer are corroborated by similar findings in prostate cancer, where Priolo et al. [11] reported that USP2 was significantly correlated with advanced human prostate cancer, further strengthening a role for USP2 as a useful biomarker of cancer progression and metastasis. Our data support a therapeutic approach in which intracellular USP2 could be an effective molecular target for the improvement of survival in breast cancer patients. Furthermore, our findings are clinically important because they provide a novel potential target candidate for TNBC, since USP2 is frequently highly expressed in this subtype. Although our study had several limitations, including the retrospective study design and the small sample size, our findings provide preclinical and clinical evidence that the contribution of USP2 to metastasis in an experimental setting can be translated into poor survival outcomes in breast cancer patients. In summary, results from IHC analysis of tumors demonstrated that USP2 is an independent marker for metastatic disease in patients with breast cancer. Our in vitro findings showed that USP2 expression promotes migration and invasion in TNBC cell lines. Taken together, our results suggest that USP2 expression is associated with TNBC cell line’s invasiveness and poor survival of breast cancer patients and may serve as a prognostic biomarker and therapeutic target for TNBC. Acknowledgments We thank Dr. Guohong Hu from the Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, China, for providing LM2-4175, SCP2, SCP28, and SCP46 cell lines. Research in the authors’ laboratory is supported by the Scientific Research Project of Shanghai Municipal Health Bureau (20134Y039) and the National Natural Funding of China (81202087, 81172520, 81202088). Conflicts of interest None

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USP2 promotes cell migration and invasion in triple negative breast cancer cell lines.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is often associated with a poor prognosis. The aim of our study wa...
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