Cancer Letters xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Downregulation of MCT1 inhibits tumor growth, metastasis andenhances chemotherapeutic efficacy in osteosarcoma through regulationof the NF-jB pathway

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.08.042

⇑ Corresponding authors. Tel.: +86 20 8775 5766; fax: +86 20 8733 2150(J. Wang), tel.: +86 20 8734 3183; fax: +86 20 8734 3170 (T. Kang).

E-mail addresses: Kangtb@mail.sysu.edu.cn (T. Kang), 2004wjhf@163.com(J. Wang).

1 These authors contributed equally to this work.

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhibits tumor growth, metastasis and enhances chemotherapeutic effiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.08.042

Zhiqiang Zhao a,1, Man-si Wu b,1, Changye Zou a, Qinglian Tang a, Jinchang Lu a, Dawei Liu c,Yuanzhong Wu b, Junqiang Yin a, Xianbiao Xie a, Jingnan Shen a, Tiebang Kang b,⇑, Jin Wang a,⇑a Department of Musculoskeletal Oncology, First Afflicted Hospital, Sun Yat-Sen University, Guangzhou 510080, Chinab State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou 510060, Chinac Department of Pathology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China

a r t i c l e i n f o

Article history:Received 23 March 2013Received in revised form 25 August 2013Accepted 28 August 2013Available online xxxx

Keywords:MCT1NF-jB pathwayOsteosarcomaCHC

a b s t r a c t

Monocarboxylate transporter isoform 1 (MCT1) is an important member of the proton-linked MCT familyand has been reported in an array of human cancer cell lines and primary human tumors. MCT1 expres-sion is associated with developing a new therapeutic approach for cancer. In this study, we initiallyshowed that MCT1 is expressed in a variety of human osteosarcoma cell lines. Moreover, we evaluatedthe therapeutic response of targeting MCT1 using shRNA or MCT1 inhibitor. Inhibiting MCT1 delayedtumor growth in vitro and in vivo, including in an orthotopic model of osteosarcoma. Targeting MCT1greatly enhanced the sensitivity of human osteosarcoma cells to the chemotherapeutic drugs adriamycin(ADM). In addition, we observed that MCT1 knockdown significantly suppressed the metastatic activityof osteosarcoma, including wound healing, invasion and migration. Further mechanistic studies revealedthat the antitumor effects of targeting MCT1 might be related to the NF-jB pathway. Immunochemistryassay showed that MCT1 was an independent positive prognostic marker in osteosarcoma patients. Inconclusion, our data, for the first time, demonstrate that MCT1 inhibition has antitumor potential whichis associated with the NF-jB pathway, and high MCT1 expression predicates poor overall survival inpatients with osteosarcoma.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Osteosarcoma is the most common primary malignant bonecancer diagnosed in the first two decades of life [1]. In the past, pa-tients with only localized disease have been treated with surgeryalone, leading to a 20% survival rate, which may be the result ofoverlooking the micrometastasis likely present at diagnosis [2].Currently, the combined treatment of localized tumor with neoad-juvant chemotherapy has increased the 5-year survival rate toapproximately 60–70% [3]. Unfortunately, the 5-year survival rateof those patients with metastatic tumor is still only 30% [4,5].Moreover, effective chemotherapy has been hampered by thedevelopment of chemoresistance, especially multi-drug resistance(MDR), after prolonged therapy, and it is unlikely to gain furtherrefinement with cytotoxic chemotherapy regimens [6]. Therefore,

it is imperative that new therapeutic targets and approaches forthe treatment of osteosarcoma patients are sought out.

A distinctive feature of tumor cells is the alteration of the cellu-lar metabolism [7]. Malignant cancer cells increase glucose con-sumption and metabolism by glycolysis and produce largeamounts of lactate even in the presence of oxygen, which was firstreported by Warburg and was termed aerobic glycolysis [8,9]. Toallow tumor cell proliferation and survival and avoid death in-duced by lactic acid, the lactate must be transported across theplasma membrane by monocarboxylate transporters (MCT). Inmammals, at least 14 isoforms of MCT are known [10,11]. Of those,MCT1, which differs from MCT4, transports lactate into cancer cellsand is taken up by oxygenated tumor cells [12,13]. Moreover,CD147, an integral plasma membrane protein of the immunoglob-ulin superfamily that associates closely with MCT1 and MCT4 [14],is critical for the growth and metastasis of glycolytic tumors[15,16]. It is known that many human cancers have high MCT1 lev-els, and studies have documented that the reduction of MCT1markedly delays tumor growth and reduces tumor metastasis,indicating that MCT1 is important for tumor progression [17,18].

cacy in

2 Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx

The NF-jB pathway is a central regulator of immune andinflammatory responses as well as oncogenesis. The deregulatedactivation of NF-jB is associated with many human diseases,including cancer. It has been shown to regulate many tumor cellcharacteristics, including survival, proliferation, and metastasisthrough controlling the expression of several genes relevant tothe tumorigenic process [19–21]. For instance, NF-jB regulatesthe expression of genes encoding antiapoptotic proteins and in-duces growth factors (interleukin-2 and interleukin-8) and cell cy-cle regulators (cyclin D1 and c-myc). Notably, a new mechanism ofthe NF-jB pathway that contributes to the switch to tumor glycol-ysis and energy production has been recently uncovered [22,23]. Inosteosarcoma, there is evidence that the hyperactivation of NF-jBsignaling enhances tumor cell survival and metastasis and in-creases resistance to chemotherapy. Recent data from our labora-tory has also revealed a novel mechanism that the NF-jBpathway plays a key role in the process of GSK-3b-mediated regu-lation of cell survival in osteosarcoma cell lines. Therefore, it hasbeen proposed that the regulation of NF-jB could be an adjuvanttherapy for cancer [24].

Here, we explore the roles and mechanisms of MCT1 in the pro-gression of osteosarcoma. Targeting MCT1 inhibits proliferation,enhances sensitivity to chemotherapeutic drugs and reduces met-astatic activity of osteosarcoma. Furthermore, studies concerningthe roles of NF-jB in the human cancers and our previous workhave demonstrated the importance of the NF-jB pathway in oste-osarcoma. We further revealed that targeting MCT1 showed anti-tumor potential by suppressing the NF-jB pathway in humanosteosarcoma cells.

2. Materials and methods

2.1. Cell culture

The human osteosarcoma cell lines U2OS, MG63, SAOS2 and MNNG/HOS werepurchased from the American Type Culture Collection and were cultured accordingto the instructions. U2OS/MTX cells and the primary cell cultures ZOS and ZOS-Mwere described previously [25]. All osteosarcoma cell lines and the primary cell cul-tures were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, Invit-rogen), supplemented with 10% fetal bovine serum (Invitrogen), 1% L-glutamine,and 1% penicillin/streptomycin. All cells were incubated in a 5% CO2 atmosphereat 37 �C. For hypoxic stimulation, the cells were cultured with a gas mixture of1% O2, 5% CO2, and 94% N2 in a modular incubator (Billups-Rothenburg, Del Mar,CA).

2.2. Antibodies and reagents

Antibodies against MCT1 and MCT4 were purchased from Millipore. Antibodiesagainst Phospho-IkappaB-alpha (Ser32/36) and Phospho-NF-jB p65 (Ser536) wereobtained from Cell Signaling Technology. Antibodies against IjBa and p65 werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA). a-Cyano-4-hydroxycin-namate (CHC) was obtained from Sigma–Aldrich and was dissolved with DMSO.

2.3. Silencing experiments

Cells were transfected with siRNA or shRNA according to the manufacturer’sprotocol (Invitrogen). The specific siRNA against human MCT1 had the following se-quences:sense, 50-CAAAGAGAUUGAAGGUAUA dTdT-30; antisense, 30-dTdT GUUU-CUCUAACUUCCAUAU-50 . The sequences of the MCT1 shRNA were as follows: top,50-CACCGCAGTATCCTGGTGAATAAATCGAAATTTATTCACCA-GGATACTGC-30; bot-tom, 50-AAAAGCAGTATCCTGGTGAATAAATTTCGATTTATTC-ACCAGGATACTGC-30 .The sequences of the siRNA against p65 were as follows: sense, 50-CCAUCAACUAU-GAUGAGUUdTdT-30; antisense, 50-AACUCAUCAUAGUUGAUGGTdGd-30 . The oligo-nucleotides were synthesized by Invitrogen. Plasmids encoding human p65 werekindly provided from Dr. Jiong Deng [26] (Shanghai Jiaotong University, Shanghai,China).

2.4. Western blotting analysis

The procedure was detailed previously [27,28]. Cells were washed twice withPBS and subsequently lysed in RIPA buffer (1X PBS, 1% Nonidet P40, 0.5% sodiumdeoxycholate, 0.1% SDS) after the addition of 0.1 M PMSF plus 1:100 protease inhib-itor cocktail (P2714, Sigma–Aldrich, Germany). After the removal of the cell debris

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

by centrifugation (12,000g, 5 min), the concentration of the protein in the superna-tant was measured using the BCA Protein Assay Kit (Pierce, Germany). Equalamounts of protein were boiled for 10 min in sample buffer, separated by 10%SDS–PAGE and transferred to a PVDF membrane. Nonspecific reactivity was blockedin 5% nonfat dry milk in TBST [10 mmol/L Tris–HCl (pH7.5), 150 mmol/LNaCl, and0.05% Tween20] for 1 h at room temperature. The membrane was then immuno-blotted with various antibodies, as described above, overnight at 4 �C, followedby 3 washes in Tris–buffered saline with Tween-20 (TBST) for 10 min. Subse-quently, the membranes were incubated with horseradish peroxidase-conjugatedsecondary antibody for 1 h at room temperature. The protein bands were detectedon X-ray film using an enhanced chemiluminescence detection system.

2.5. Cell viability assay

The MTT assay was performed to determine cell viability. Briefly, 2500 cellswere seeded in 96-well microplates. After culturing for 12 h, the cells were treatedwith varying concentrations of CHC and/or ADM for the indicated time period. Fol-lowing the incubation period, 20 ll of MTT (5 mg/ml in PBS) was added into eachwell and incubated for 4 h. Subsequently, the supernatant was removed, and200 ll of DMSO was added to each well. The dark-blue MTT-formazan crystals weredissolved by shaking the plates at room temperature for 10 min, and the absorbancewas subsequently measured on a Bio-Rad Microplate Reader (Bio-Rad, Hercules, CA)using a test wavelength of 490 nm. Each experiment was performed in triplicate.

2.6. Colony formation assay

Cells were plated in a six-well plate (Corning Costar) at a density of 100 cells perwell and were cultured with Dulbecco’s modified Eagle’s medium supplementedwith 10% fetal bovine serum for approximately 10 days. After most cell cloneshad expanded to >50 cells, the cells were washed twice with PBS, fixed in methanolfor 15 min, and dyed with crystal violet for 15 min at room temperature. Afterwashing out the dye, we counted the number of colonies that contained >50 cellsand compared the results. The colony formation efficiency was the ratio of the col-ony number to the planted cell number.

2.7. Wound healing assays

Cells were plated in 6-well plates. When the cells reached 90% confluency, ascratch was made using a sterile yellow 100 ll pipette tip, and the detached cellswere removed by washing with PBS. For CHC treatment, 10 nM CHC was addedto the medium after washing with PBS. Phase contrast images were taken in thesame field at the indicated time points. The experiments were performed intriplicate.

2.8. In vitro migration and invasion assays

Generally, 5.0 � 104 osteosarcoma cells in 200 ll of serum-free DMEM wereadded to the upper compartment with an 8-lm microporous filter without extra-cellular matrix coating (Becton Dickinson Labware, Bedford, MA). Then, 500 lL ofDMEM containing 10% FBS was added to the bottom chamber. After a 24 h incuba-tion at 37 �C, the cells on the lower surface of the filter were fixed, stained andexamined under a microscope. The average number of migrated cells from five ran-dom optical fields (�100 magnification) and triplicate filters was determined.

For in vitro invasion assays, the inserts of the chambers, which had been seededwith cells, were coated with Matrigel (Becton Dickinson Labware, Bedford, MA). Thenumber of invading cells in five random optical fields (�100 magnification) or eachfilter from triplicate inserts was averaged. For the migration screen, 5 � 104 cellswere seeded onto the cell culture inserts. After 24 h of incubation at 37 �C, the mi-grated cells on the lower surface of filters were trypsinized, propagated, and re-screened twice.

2.9. In vivo animal models

All animal model experiments were approved by the Institutional Review Boardof Sun Yat-sen University. We used 4- to 6-week-old male BALB/c nude mice(Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China). Osteosarcoma cellswere harvested, washed and resuspended in PBS. The cells were injected subcuta-neously near the scapula of nude mice. After one week, the mice were randomlyseparated into the appropriate groups. Vehicle or CHC (25 lmol in 200 ll) was in-jected i.p. each day. ADM (6 mg/kg) was injected intraperitoneally once a week. Thesize of the transplanted tumors was measured at the indicated time, and the tumorvolume was calculated using the following formula; V = 1/2 (width2 � length). Themice were sacrificed 30 days postinoculation.

We used the primary osteosarcoma ZOS-M cells as an orthotopic model ofosteosarcoma. The mice were anesthetized with isoflurane. The needle (30 gauge)was inserted to the proximal tibia through the cortex of the anterior tuberosity.We confirmed that the needle was in the medullary cavity of proximal tibia. Finally,20 ll of the ZOS-M cell suspension was slowly injected. After 10 days, the micewere randomly separated into appropriate groups. Vehicle or CHC (25 lmol in

bits tumor growth, metastasis and enhances chemotherapeutic efficacy inttp://dx.doi.org/10.1016/j.canlet.2013.08.042

Fig. 1. MCT1 is expressed in human osteosarcoma cell lines. (a) Protein expression of MCT1 in human osteosarcoma cell lines. (b) Osteosarcoma cell lines were cultured witha gas mixture of 1% O2, 5% CO2, 94% N2 in a modular incubator for 48 h, and the expression of MCT1 was detected in the U2OS and MNNG/HOS cell lines. (c) Western blottinganalysis of U2OS cells stably transfected with shRNA targeting MCT1. (d) U2OS cells were treated with 1–20 nM CHC for 48 h or 10 nM CHC for 0–48 h as indicated, andwestern blotting was performed.

Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx 3

200 ll) were injected i.p. each day. The tumors were measured in 2 perpendiculardimensions (D1, D2) with a caliper every 2 days, and the tumor volume was calcu-lated using the formula

V = 4/3 p[1/4(D1 + D2)]2, as described previously [29].

2.10. Immunohistochemistry staining

This procedure was described previously [30]. Immunohistochemical analysiswas performed on 3 lm sections. The primary antibodies against MCT1 was diluted1:200 and were incubated at 4 �C overnight in a humidified container. After wash-ing with PBS three times, the tissue slides were treated with a non-biotin horserad-ish peroxidase detection system according to manufacturer’s instructions (Dako).The results of immunohistochemistry staining were evaluated by two independentpathologists and discordant cases were discussed in a double-head microscope inorder to determine a final score. Immunoreaction extent was scored semiquantita-tively using the criteria previously described [31,32]: 0: 0% of immunoreactive cells,1:<5% of immunoreactive cell, 2: 5–50% of immunoreactive cells, and 3:>50% ofimmunoreactive cells. Also, intensity of staining was scored semi-qualitatively as0: negative, 1: weak, 2: intermediate, and 3: strong. Immunoreaction final scorewas defined as the sum of both parameters (extent and intensity), and groupedas negative (scores 0 and 2) and positive (3–6).

2.11. Statistical analysis

All experiments were repeated 3 times. Mean values and standard deviationswere calculated, and the statistical significance was determined using Student’s t-test. The Kaplan–Meier method was used to estimate the overall survival, and thelog-rank test was used to evaluate the differences between survival curves. Com-parisons in which P < 0.05 were considered statistically significant. All statisticalanalyses were performed using the SPSS13.0 software.

3. Results

3.1. Expression of MCT1 in osteosarcoma cell lines and MCT1downregulation by shRNAs or CHC

MCT1 has been detected in a variety of human cancer cell linesand tissue sections, including human colon, breast, head and neck,and lung cancers, and was associated with poor prognosis [17,30].Similarly, western blotting revealed that MCT1 is expressed in anarray of human osteosarcoma cell lines available to us (Fig. 1a).MCT1 expression appeared to be much higher in the U2OS/MTXor ZOS-M cell lines (relative high-grade osteosarcoma comparedwith parent cell line) than in the U2OS or ZOS cell lines. Previousstudies have reported that O2 regulates the expression of manykey enzymes in metabolic pathways [17]. To confirm this observa-tion in osteosarcoma cell lines, we cultured osteosarcoma cell lines

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

in the hypoxic condition (1% O2, 48 h) and found that hypoxia in-creased the expression of MCT1 (Fig. 1b), which has been demon-strated to play a key role in glycolysis, growth properties andtumor maintenance of many cancer cells. Those results indicatedthat MCT1 might be more important to cancer cells in hypoxic con-ditions, which could provide a suitable therapeutic target in oste-osarcoma. In summary, MCT1 is expressed in humanosteosarcoma cell lines and much higher in relative high-gradeosteosarcoma compared with parent cell lines.

In addition, short hairpin RNA (shRNA) was used for suppress-ing the expression of MCT1. As shown in Fig. 1c, MCT1 was mark-edly reduced compared with the untransfected or vector-transfected cells. Moreover, we used a highly selective inhibitorof MCT1 to study the function of MCT1 [17]. As expected, we ob-served a dose- and time-dependent down-regulation of MCT1 afterCHC treatment in osteosarcoma cells (Fig. 1d).

3.2. MCT1 suppression shows antitumor effects in osteosarcoma

Targeting MCT1 has shown promising therapeutic effects forhuman cancers that display MCT1 expression, such as neuroblas-toma [17,33]. We also found that MCT1 was expressed in an arrayof osteosarcoma cell lines, and the effect of MCT1 suppression oncellular proliferation was evaluated in several osteosarcoma celllines. We performed an MCT1 knockdown in cells, and the effectof MCT1 on osteosarcoma cell growth was investigated by colonyformation and MTT assays. The MCT1 knockdown in U2OS andMNNG/HOS cells significantly reduced the number of colonies(Supplementary Fig. 2a and b). MTT assay demonstrated that thereis a decrease in osteosarcoma cell proliferation after treatmentwith CHC, including U2OS, U2OS/MTX and ZOS. As expected, cellproliferation was inhibited by the MCT1 inhibitor in all osteosar-coma cell lines tested (Fig. 2a). Moreover, the percent of the viableU2OS cell also decreased with CHC treatment in a dose-dependentmanner (Supplementary Fig. 1).

To further confirm the antitumor potential of downregulatingMCT1, we detected the effects of MCT1 inhibition in vivo, we usedCHC alone to inhibit the tumor growth of U2OS/MTX or ZOS cells inxenograft models (Fig. 2b and c). U2OS/MTX and ZOS cells (1 � 106

in 200 ll PBS) were injected near the scapula of nude mice. Sixdays after the cells injection, a palpable tumor developed, andthe mices were separated into two groups in random. CHC

bits tumor growth, metastasis and enhances chemotherapeutic efficacy inttp://dx.doi.org/10.1016/j.canlet.2013.08.042

Fig. 2. MCT1 inhibition suppressed growth of osteosarcoma in vitro and in vivo. (a) The effect of MCT1 inhibition on osteosarcoma cell proliferation, including U2OS, U2OS/MTX and ZOS cells, was measured by MTT. The growth of osteosarcoma cells was suppressed compared with the control. (b and c) An antitumor assay in the nude mousesubcutaneous model using U2OS/MTX or ZOS cells for 21 or 24 days. The tumor volumes were monitored beginning on the sixth day. Tumors were measured every 3 days,and the results were expressed as the mean tumor volume ± SD of 5 or 6 mice from the control group and CHC-treated group (daily doses of 25 lmol in 200 ll i.p.). ⁄P < 0.05indicates a statistically significant decrease in tumor volume in CHC-treated samples compared to the control. (d) Antitumor assay in an orthotopic model using ZOS-M (20 lLof the ZOS-M cell suspension was slowly injected) cells for 40 days. The tumor volumes were monitored at day 12 and then every 4 days, as indicated. The tumor volumeswere monitored beginning on the sixth day and measured every 3 days.

4 Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx

(25 lmol in 200 ll) or the vehicle control (200 ll) was injectedintraperitoneally every day. As indicated in Fig. 2b and c, MCT1inhibition sustainably delayed U2OS/MTX and ZOS tumor growthcompared with vehicle-injected control animals. To more closelymodel the human condition, we also developed an orthotopic mod-el to further evaluate the antitumor effects of MCT1 inhibition. Inthe orthotopic model, 20 ll of the ZOS-M cell suspension wasslowly injected in the medullary cavity of proximal tibia. The int-ratibial injection of human ZOS-M cells in these nude mice inducedorthotopic osteosarcoma formation after 2 weeks inoculation fol-lowed CHC or vehicle injection. As shown in Fig. 2d, the MCT1inhibitor also resulted in an inhibition of tumor growth. Intrigu-ingly, the antitumor efficacy of MCT1 inhibition is restricted to tu-mor cells expressing MCT1 at the plasma membrane. Furthermore,off-target effects or a major response to the MCT1 inhibitor by thehost cells was not observed [17,34]. Likewise, there were noremarkable side effects with this regimen in our animal model.These observations indicate that MCT1 is critical for tumor growthin osteosarcoma.

3.3. Targeting MCT1 enhances chemotherapy efficacy for osteosarcoma

We examined the expression of MCT1 in U2OS and U2OS/MTXcells. We found that the MCT1 levels were markedly increased in

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

U2OS/MTX cells compared to parental U2OS cells (Fig. 1a). Thisindicated that drug resistance may be correlated with the in-creased MCT1 expression. Adriamycin (ADM) is commonly usedchemotherapeutic drug for patients with osteosarcoma. We deter-mined whether inhibition of MCT1 could show synergistic effectsor partially reverse the resistance of osteosarcoma cells when com-bined with chemotherapy. To confirm this, MCT1 inhibition orADM as single or combined treatment was performed using theMTT assay after the indicated times. As shown in Fig. 3a, in vitroCHC-mediated MCT1 inhibition combined with ADM treatmentsignificantly enhanced the cell death of osteosarcoma cells overADM treatment alone. Similar assays were performed in U2OS cellswith shRNA-mediated knockdown of MCT1, and these cells alsoshowed increased sensitivity to chemotherapeutic drugs.

We next evaluated the ability of MCT1 inhibition to enhancechemotherapy efficacy against osteosarcoma in vivo. We utilizeda subcutaneous model in which U2OS/MTX or ZOS human osteosar-coma cells were injected into 6-week-old nude mice. AlthoughMCT1 inhibition did not show a greater antitumor effect than othersingle therapeutic modalities, such as ADM, MCT1 inhibition didprevent obvious mouse weight loss, which indicated that CHC canachieve similar efficacy in suppressing osteosarcoma growth with-out severe side effects of ADM (Supplementary 3). Notably, the con-sequences of combined CHC/ADM treatment were significantly

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Fig. 3. Targeting MCT1 enhanced the sensitivity of osteosarcoma cells to chemotherapeutic agents. (a) U2OS, U2OS/MTX or ZOS cells were treated with CHC (10 nM), ADM(4 ng/ml), or both for 5 days, and cell viability was measured by MTT. (b and c) U2OS/MTX or ZOS tumor bearing mice were treated with CHC (25 lmol in 200 ll), ADM(6 mg/kg) or both after dividing the mice into four groups. The tumor volumes were monitored beginning on the sixth day. The tumors were measured every 3 days, and the resultsare expressed as the mean tumor volume ± SD of 5 or 6 mice from the control group and the CHC-treated group (daily doses of 25 lmol in 200 ll i.p.).

Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx 5

increased compared with any single therapy (Fig. 3b and c). More-over, the benefit of combining MCT1 inhibition with radiotherapyfor treating Lewis lung carcinoma has previously been demon-strated [17]. Taken together, our results suggest that targetingMCT1 not only inhibits tumor growth but also enhances the sensi-tivity of chemotherapeutic drugs for osteosarcoma.

3.4. Inhibition of MCT1 suppresses the migration and invasion ofosteosarcoma

We have previously established two novel human osteosar-coma cell lines, ZOS and ZOS-M, from a primary tumor and meta-static lesion [35]. We also found that ZOS-M, which expressesincreased levels of MCT1, possesses higher metastatic ability com-pared with ZOS. Those datas suggested that MCT1 may correlatewith osteosarcoma metastasis. To explore this connection, weindependently examined the effects of MCT1 on cell motility usingwound healing and transwell migration assays. As shown in Fig. 4a,MNNG/HOS cells were plated in 6-well plates and transfected withshRNA targeting MCT1. We made a scratch when the cells reached90% confluency. After 24 h, MNNG/HOS cells with the MCT1 knock-down had an obvious delay in the wound healing migration assaycompared with the control, and the wound healing migration delaywas correlated with the expression of MCT1 between shMCT1-1and sh-MCT1-2. The exposure of MNNG/HOS cells to CHC (5 nM)also reduced cell migration through the wounded area. MCT1 inhi-bition also suppressed the invasion ability of osteosarcoma cells in

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

the transwell invasion assay. In this assay, 5.0 � 104 osteosarcomacells in 200 ll of serum-free DMEM were added to the upper com-partment with an 8-lm microporous filter with or without extra-cellular matrix coating. After 24 h, the cells on the lower surfaceof the filter were fixed, stained, and examined under a microscope.The MCT1 knockdown in MNNG/HOS cells significantly suppressedthe migration ability (Fig. 4c). The invasion ability of MNNG/HOScells was also significantly reduced by MCT1 knockdown(Fig. 4d). Our results indicate that MCT1 may correlate with theinvasion and migration abilities of osteosarcoma cells.

3.5. Targeting MCT1 suppresses the activity of the NF-jB pathway inosteosarcoma cells

The NF-jB pathway is a central regulator of the immune andinflammatory responses as well as oncogenesis [36]. We have doc-umented that the inhibition of the NF-jB pathway affected cellsurvival in osteosarcoma cells, which is regulated by GSK-3b.Importantly, the NF-jB pathway contributes to cancer cell malig-nancy, including osteosarcoma cells, by altering tumor cell metab-olism [22,23]. Therefore, we examined whether MCT1 inhibitionregulated the activity of the NF-jB pathway thereby altering oste-osarcoma cell malignancy. As shown in Fig. 5a, the level of p65 andphosphorylated p65 was downregulated in U2OS treated with CHCin a time- and dose-dependent manner. It has been reported thatlactate could stimulate NF-jB activity in an MCT1-dependent man-ner in endothelial cells [37].

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Fig. 4. Suppression of MCT1 inhibited the migration and invasion abilities of MNNG/HOS cells. MNNG/HOS cells were transduced with shRNA targeting MCT1. (a and b) Thesuppression of MCT1 dramatically reduced the migration ability of the cells as determined by a wound-healing assay. Yellow dashed lines denote the margins of the wound.(c and d) A 2-chamber assay was used to further evaluate the invasion/migration ability of the MNNG/HOS cells. The suppression of MCT1 significantly reduced the migrationand invasion abilities of osteosarcoma cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

6 Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx

To further investigate the molecular mechanism mediating theantitumor effects of MCT1, we demonstrated that directly targetingp65 by siRNA suppressed the invasion of osteosarcoma cells (Sup-plementary Fig. 4) and previous results showed that PDTC (NF-jBinhibitor) could impaired the growth of osteosarcoma cells. Wethen asked whether the function of inhibited MCT1 is mediatedthrough the inactivation of the NF-kB pathway in osteosarcoma.As shown in Fig. 5b, the inhibitory effect of CHC on tumor growthwas partially reversed in U2OS cells that overexpress p65. Like-wise, the invasion ability was also partially rescued by the knock-down of MCT1 in p65-overexpressing MNNG/HOS cells comparedto MCT1-knockdown MNNG/HOS cells (Fig. 5c and d). Taken to-gether, our results indicated that the NF-jB pathway may play akey role in MCT1-mediated tumor growth and metastasis.

3.6. MCT1 expression predicts clinical outcome of patients withosteosarcoma by IHC

Given the above results, we further examined the clinical signif-icance of MCT1 expression in primary human osteosarcoma usingosteosarcoma tissue microarray. To this end, samples from 61cases of osteosarcoma collected for IHC were treated with standardprotocol, successfully followed up, and most of them were grade

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

IIB according to Ennecking staging system. Positive MCT1 expres-sion mainly observed in plasma membrane was detected in a highpercentage of cases (Fig. 6a). Among them, 41 cases (67.3%) werepositive, whereas 20 cases (32.7%) were negative for MCT1, dem-onstrating that overexpression of MCT1 was detected in the major-ity of patients with osteosarcoma. Kaplan–Meier survival analysiswas used for comparing the clinical characteristics of low-stainingand high-staining osteosarcomas, which showed that the outcomefor patients in the MCT1 high-staining group was significantlyworse than for those in the MCT1 low-staining group (at a medianfollow-up of 44 months, OS for patients whose tumors had positiveMCT1 = 45.6 months, OS for patients whose tumors have negativeMCT1 = 63.8 months, P = 0.014, Fig. 6b). Our results indicate thatincreased expression of MCT1 correlates with poor prognosis in pa-tients with osteosarcoma. Thus, MCT1 may serve as a prognosticmarker for osteosarcoma survival.

4. Discussion

In this study, we show that MCT1 is expressed in a variety ofhuman osteosarcoma cell lines, sustains osteosarcoma cell prolifer-ation and metastasis, and is implicated in cellular sensitivity to

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Fig. 5. MCT1 inhibition suppressed the activity of NF-jB pathway in osteosarcoma cells. (a) The level of p65 and phosphorylated p65 was detected in U2OS cells that hadbeen treated with 0–10 mM CHC for 48 h or 10 nM CHC for 0–48 h as indicated. Western blotting was performed. (b) Western blotting confirmed that the siRNA targeted p65and p65 overexpression (PcDNA3.0/P65.U2OS cells). The cells were treated with or without CHC (10 nM), and cell viability was analyzed. (c) MNNG/HOS cells weretransfected with shRNA targeting MCT1 or p65-overexpression, and the p65 expression was detected by western blotting. (d) MNNG/HOS cells were transfected with shRNAtargeting MCT1 or a p65-overexpression plasmid, and the MNNG/HOS migration ability was analyzed.

Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx 7

chemotherapeutic drugs. We also demonstrated that downregulat-ing MCT1 with specific MCT1 inhibitors or suppressing MCT1 withRNAi is critical for regulating the activity of the NF-jB pathway,which might represent a novel strategy for the treatment ofosteosarcoma.

MCT1 exports lactate in cancer cells and is known to be in-volved in cancer cell aerobic glycolysis, which is a hallmark ofmalignant tumors. In previous reports, it has been demonstratedthat an array of primary human tumors express MCT1, such as hu-man colon, breast, head and neck, and lung cancers. Furthermore,CD147, as an integral plasma membrane protein of the immuno-globulin superfamily, has shown a strong interaction with MCT1.It has been shown that a CD147-targeting siRNA may inhibit pro-liferation and metastasis in some cancer types, such as malignantmelanoma [16,38]. In human lung cancer cells, MCT1 has beendemonstrated to be involved in invasion. However, the role ofMCT1 in tumor progression and survival is still unclear, especiallyin human osteosarcoma. Here, our results show comprehensivein vitro and in vivo evidence that targeting MCT1 (via RNAi or spe-cific MCT1 inhibitors) is a promising therapy in osteosarcoma. Ourstudy has demonstrated that MCT1 inhibition showed antitumoreffects without obvious side effects. Most notably, chemotherapeu-tic drugs combined with MCT1 inhibition exert an additive sup-pression of osteosarcoma in vitro and in vivo. We alsodemonstrated that MCT1 may correlate with the invasion andmigration abilities of osteosarcoma cells in vitro. Moreover, the

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

study first illustrated the expression of MCT1 in primary osteosar-coma tissues, followed by demonstrating the association betweenthe MCT1 expression and prognosis in 61 osteoarcoma samples.Our results showed that the outcome for patients in the MCT1high-staining group was significantly worse than for those in theMCT1 low-staining group.

Constitutive NF-jB activity has been reported in a number ofmalignant tumors, which involves cell growth, apoptosis, adhesion,chemoresistance and angiogenesis of normal and pathological sys-tems [38,39]. We also demonstrated that targeting p65 with siRNAcould reduce the invasion ability. In a recent study, NF-jB signal-ing was reported to be a physiological regulator of cellular bioen-ergetic pathways in normal and cancer cells [22,23]. Moreover,our recent study has revealed the role of NF-jB signaling in theprocess of GSK-3b-promoted tumorigenicity of osteosarcoma,which is critical for cell survival in osteosarcoma cell lines. Inter-estingly, the role of MCT1 in the lactate/NF-jB/IL-8 pathway sug-gests an important link between tumor metabolism andangiogenesis, which further supports the current interest for newcancer treatments targeting metabolic pathways [37]. The underly-ing mechanism regarding the efficacy of MCT1 inhibition has rarelybeen discussed. In this study, we showed that the inhibition ofMCT1 provided antitumor effects by suppressing the NF-jB path-way in human osteosarcoma cells, including cell proliferationand invasion. The p65 protein, an indicator of NF-jB transcrip-tional activity, was decreased in U2OS cells treated with CHC in a

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Fig. 6. MCT1 expression predicts clinical outcome of patients with osteosarcoma by IHC (a) Immunohistochemical analysis was performed on 3-lm sections from paraffin-embedded tissues of 61 patients with osteosarcoma using the primary antibodies against MCT1, as described in ‘‘Section 2.’’ Representative images of differentimmunohistochemistry staining intensities and percentage of MCT1 are shown in osteosarcoma tissues. (b) Overall survival rates of patients with osteosarcoma positive ornegative for MCT1 were estimated with the Kaplan–Meier method by log-rank test; n = 61, P = 0.014).

8 Z. Zhao et al. / Cancer Letters xxx (2013) xxx–xxx

time- and dose-dependent manner. In addition, the proliferationand invasion activity of cells ectopically transfected with p65 werereversed by treatment with CHC. Our results support a role forMCT1 inhibition in mediating antitumor effects through the NF-jB pathway.

In summary, the present results have revealed that MCT1 playsan important role in many human tumors. This study providesvaluable evidence in support of targeted therapy capable of inhib-iting MCT1, and future studies may reveal for the mechanism thatlinks MCT1 inhibition and tumors.

Conflicts of Interest

No conflicts of interest to disclose.

Acknowledgements

This study was supported by grants from the National NaturalScience Foundation of China (No. 81072193 & No. 81272940 to

Please cite this article in press as: Z. Zhao et al., Downregulation of MCT1 inhiosteosarcoma through regulation of the NF-jB pathway, Cancer Lett. (2013), h

Jin Wang) and Talent Grant of Sun Yat-Sen University (No.09kpy43 to Jin Wang). The funders had no role in study design,data collection and analysis, decision to publish, or preparationof the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.canlet.2013.08.042.

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