Cancer Letters 328 (2013) 126–134

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Cancer Letters

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Adenovirus vector-mediated expression of TMEM166 inhibits human cancercell growth by autophagy and apoptosis in vitro and in vivo

Ying Chang a,b, Yanjun Li a,b, Jia Hu a,b, Jinhai Guo c, Dong Xu a,b, Hong Xie a, Xiaodong Lv a, Taiping Shi c,Yingyu Chen a,b,⇑a Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Science Center, Beijing, Chinab Peking University Center for Human Disease Genomics, Beijing, Chinac Chinese National Human Genome Center, Beijing, China

a r t i c l e i n f o

Article history:Received 4 April 2012Received in revised form 24 August 2012Accepted 24 August 2012

Keywords:TMEM166Adenovirus vectorAutophagyApoptosisAnti-tumor activity

0304-3835/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.canlet.2012.08.032

⇑ Corresponding author at: Key Laboratory of MedHealth, Peking University Health Science Center, 38 XChina. Tel./fax: +86 10 82801149.

E-mail address: yingyu_chen@bjmu.edu.cn (Y. Che

a b s t r a c t

TMEM166 is a novel programmed cell death-related molecule. In this report, we constructed a recombi-nant adenovirus 5-TMEM166 vector (Ad5-TMEM166) and evaluated its expression and anti-tumor activ-ities in vitro and in vivo. Cell viability analysis revealed that the adenovirus-mediated increase ofTMEM166 inhibited tumor cell growth in a dose- and time-dependent manner. This inhibitory effectwas mediated by both autophagy (via inhibition of mTOR and activation of p70S6K) and apoptosis (viacaspase-3 activation), both of which contributed to cell death and suppression of tumorigenicity. Ourdata indicated that Ad5-TMEM166 may be a novel gene therapy candidate for cancer.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Autophagy is an evolutionarily conserved cellular process innature, which is tightly controlled by complex regulatory mecha-nisms. It has been observed under various cell conditions, includ-ing the degradation of proteins in response to nutrientdeprivation, differentiation, aging and cancer therapy [1–4]. Thisprocess is a major catabolic pathway for delivery of proteins andorganelles to lysosomes or the vacuole, where they are degradedand recycled. The signaling pathways involved in autophagy havebeen studied intensively in the last several years due to their rolesin many diseases including cancers, infection, autoimmune dis-eases and neurodegenerative diseases [5–8].

There are ample biochemical and genetic lines of evidence toindicate that autophagy is suppressive to the initiation and pro-gression of tumors. The exact nature of this role, however, is com-plex [9,10]. An anti-cancer role for autophagy is implied from itsactivation by tumor suppressor proteins, including Beclin-1[11,12], BH3-only proteins [13,14], death associated protein-kinase1 (DAPK1) [15], alternate reading frame (ARF) [16] and UV-irradi-ation resistance-associated gene (UVRAG) [17,18]. Reduced levels

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n).

(or the complete absence) of the aforementioned proteins thatare required for autophagy have also been associated with in-creased tumor progression, both in animal models and in humans.Heterozygous knock-out mice for beclin-1 are predisposed to mul-tiple tumor types, including B cell lymphoma, hepatocellular carci-noma and lung adenocarcinoma [11,19]. Beclin-1 is also mono-allelically deleted in a subset of human tumors of the breast, ovary,brain and prostate [20]. Like Beclin-1, UVRAG is mono-allelicallymutated in human cancer. ARF is also mutated or lost in multipletypes of human cancers including leukemia, lymphoma and breastcarcinoma. Meanwhile, Bax-binding protein-1 (Bif-1), another po-sitive regulator of autophagy, is downregulated in colon adenocar-cinomas [21]. These findings further suggest a role for autophagyin tumor suppression.

Transmembrane protein 166 (TMEM166), also known as familywith sequence similarity 176 member A (FAM176A), is a novel hu-man gene involved in programmed cell death [22]. Over expressionof TMEM166 induces both cell autophagy and apoptosis. Kineticanalysis has revealed that the appearance of autophagy-relatedbiochemical parameters precedes the cellular apoptosis inTMEM166-transfected HeLa cells. Furthermore, suppression ofTMEM166 expression by small interference RNA (siRNA) inhibitsstarvation-induced autophagy in HeLa cells. Immunohistochemicalor tissue chip analysis has revealed that the levels of TMEM166protein are down regulated or negatively expressed in most cancertissues [23]. In this study, we further investigated the bioactivity of

Y. Chang et al. / Cancer Letters 328 (2013) 126–134 127

human TMEM166 by constructing an adenovirus expression vector(Ad5-TMEM166) and testing its expression in various tumor celllines. Both in vitro and in vivo experiments were conducted to eval-uate the Ad5-TMEM166-mediated its anti-tumor effects andinduction of cancer cell autophagy and apoptosis. Our results sug-gest that adenovirus-mediated TMEM166 gene transfer may pres-ent a new therapeutic approach for cancer treatment.

2. Materials and methods

2.1. Cell culture

U2OS, Saos-2 were cultured in McCoy’s 5A medium (Life Technologies, USA)supplemented with 10% fetal bovine serum (Hyclone, USA), 2 mmol/L glutamine,100 units/mL penicillin and 100 lg/mL streptomycin. The BGC823 cell line was cul-tured in Dulbecco’s Modified Eagle’s medium (Life Technologies, USA) supple-mented as above. The KYSE150 cell line was cultured in RPMI1640 medium (LifeTechnologies, USA) supplemented as above. All the cells were maintained at 37 �Cin a humidified chamber with 5% CO2.

2.2. Adenoviral vectors

All of the recombinant adenovirus is based on type 5 (E1/E3 deficient) adenovi-rus. Ad5-null, Ad5-GFP and Ad5-GFP-LC3 were purchased from SinoGenoMax (Bei-jing, China). The complete coding sequence of the TMEM166 was subcloned into theBamHI and EcoRI sites of the pShuttle-CMV vector (SinoGenoMax). Then theexpression cassette of TMEM166 was transfered into the Adenoviral backbone vec-tor pAdxsi, the recombinant clones of shuttle plasmid and viral-backbone plasmidof TMEM166 were confirmed by DNA sequencing. Then the recombinant viral plas-mid of TMEM166 (Ad5-TMEM166) was linearized by PacI digestion and packaged inHEK293 cell. All of the viral particles were purified by cesium chloride density gra-dient centrifugation and titered by TCID50 method.

2.3. Cell viability assay

Cell viability assays were performed using the Cell Counting Kit-8 (CCK8, Dojin-do, Kumamoto, Japan). Briefly, U2OS, Saos-2, BGC823 and KYSE150 cells were in-fected by Ad5-Null or Ad5-TMEM166 at the indicated multiplicity of infection(MOI) for different times. Cells were then added to the CCK-8 solution accordingto the manufacturer’s protocols. Absorbance readings at 450 nm of reduced WST-8 were recorded using an EL-311SX ELISA Reader (Bio-Tec Instruments, USA). Cellviability was calculated as follows: cell viability = absorbance of test group/absor-bance of uninfected cell group � 100%.

2.4. Fluorescence and confocal microscopy

U2OS, Saos-2, BGC823 and KYSE150 cell lines were plated on glass coverslipsand then infected with both Ad5-GFP–LC3 and Ad5-TMEM166, or Ad5-GFP–LC3combined with Ad5-Null at the indicated MOI. After 20 h, cells were observed usingfluorescence microscopy and imaged by a Leica SP2 confocal system (Germany). Itwas considered a positive accumulation of GFP–LC3 punctate distribution in thecells when the number of dots exceeded 10 in a cell. Percentages of cells with punc-tate distribution of GFP–LC3 were determined in ten non-overlapping fields, andstatistical analysis was performed on data obtained from three repeatedexperiments.

2.5. Flow cytometry

For detecting the efficiency of adenovirus infection, cells were treated with Ad5-GFP at the indicated MOI for 24 h before harvesting and analysis on a FACSCaliburflow cytometer (Becton Dickinson, USA). An FITC–Annexin V staining Detection kit(Beijing Biosea Biotechnology Co., Ltd., Beijing, China) was used to analyze apopto-sis levels. Briefly, cells were infected by either Ad5-TMEM166 or Ad5-Null at differ-ent MOI for various times. Treated cells were trypsinized and resuspended in 100 llbinding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM MgCl2, 5 mM KCl,2.5 mM CaCl2). FITC-conjugated Annexin V was added to a final concentration of0.5 lg/ml according to the manufacturer’s instructions. After incubation for30 min in the dark, the samples were immediately analyzed on a FACSCalibur flowcytometer (Becton Dickinson, USA).

2.6. Assay for caspase-3 activity

Caspase-3 activity was measured using a Caspase-3/CPP32 Fluorometric AssayKit (Biovision, USA). All procedures were carried out according to the manufac-turer’s instructions. Caspase-3 mediated cleavage of DEVD-AFC into free AFC wasquantified using a FLUOSTAR fluorometer (BMG Lab-technologies, Germany) withan excitation filter of 380 nm and emission filter of 460 nm.

2.7. Western blot analysis

Treated cells were collected and extracted using a RIPA lysis buffer (50 mM Tris,pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and withfreshly added proteinase inhibitor cocktail). Protein concentrations in cell lysateswere measured using the BCA protein assay reagent (Pierce, USA). Equal amountsof protein were separated by 12.5% SDS–PAGE and transferred onto PVDF mem-branes. Membranes were blocked and incubated with the indicated antibodies.The protein bands were visualized using an IRDye 800CW-conjugated secondaryantibody, and the fluorescence image was obtained using an Odyssey infrared imag-ing system (Li-Cor Bioscience, Germany).

2.8. Ex vivo and in vivo treatments with adenoviruses

A nude mouse xenograft model was established using 6–8 week-old femaleBALB/c nude mice (Experimental Animal Center, Peking University Health SciencesCenter, Beijing, China). Mice were housed and maintained in a pathogen-free facil-ity, and all experimental procedures and protocols were approved by the Institu-tional Authority for Laboratory Animal Care of Peking University. For ex vivotreatments, adenovirus-infected KYSE150 cells were injected subcutaneously intothe left axilla of BALB/c nude mice in a total volume of 100 ll (2 � 106 cells), andtumor sizes were monitored with calipers every 2 days. The tumor volume for eachmouse was determined (in cubic millimeter) by measuring in two dimensions andcalculated as tumor volume = length � (width)2/2. Animals showing tumorsexceeding 15 mm in two perpendicular diameters or 20 mm in one diameter weresacrificed by CO2 inhalation.

For in vivo treatments, 2 � 106 KYSE150 cells were inoculated subcutaneouslyinto the left axilla of each nude mouse. When the tumor volume reached100 mm3, all mice were divided randomly into three groups (six mice per group)and treated every 3 days with multiple-center intratumor injection of 50 ll ofAd5-Null or Ad5-TMEM166 at 200 MOI per animal (adenovirus stock solution con-tained 10 mmol/L Tris, pH 8.0, 4% sucrose, 2 mmol/L MgCl2) or mock treated. Thexenograft tumor tissue and important organs were excised and prepared for routinepathological examination. Some tumor tissues were subjected to immunohisto-chemical analysis, and some were homogenized and the proteins extracted forWestern blotting with anti-p62 and anti-b-actin. For the duration of the experi-ment, mice were observed for general health, changes in weight and possible sideeffects of the treatments.

2.9. Immunohistochemistry

Tumor xenograft samples were fixed in 10% buffered formalin solution andembedded in paraffin. Tumor xenograft slides were cut, deparaffinized in xyleneand rehydrated in descending grades of ethanol. Endogenous peroxidase activitywas blocked with 3% hydrogen peroxide in water. The slides were stained with arabbit polyclonal antibody to Ki-67 (Dako Corporation, USA) or rabbit IgG as a con-trol. Immunostaining was performed using a variation of the avidin–biotin-peroxi-dase method. Ten randomly selected fields were analyzed in the tumors of eachspecimen. The percentage of Ki-67 positive stained cells over the total number ofnucleated cells in a 40� field (average of ten 40� fields) was recorded.

An In Situ Cell Death Detection kit, POD (Roche Diagnostics, USA), was used toperform TUNEL assays and identify apoptotic cells present in xenograft tumor spec-imens. Briefly, after staining according to the manufacturer’s directions, six tumorspecimens were randomly selected from each treatment group and imaged usinga light microscope. The percentage of DAB-stained apoptotic cells was calculatedas the mean of ten different 40� fields from each specimen.

2.10. Statistical analysis

Results are presented as the mean ± standard error of mean. One-way ANOVAwas used for statistical comparisons among three groups; post hoc analysis by theleast squares difference method was used for statistical comparison between indi-vidual groups. The differences between groups were considered to be statisticallysignificant when the P value was <0.05.

3. Results

3.1. Ad5-TMEM166 induces growth arrest of cancer cells in vitro

We initially screened a variety of cancer cell lines to determinethe most appropriate cell lines to carry out the following experi-ment. Cell lines were infected by Ad5-GFP for 24 h at 100–1600MOI, followed by flow cytometric analysis to detect the infectionefficiency. The results showed that the proportion of Ad5-GFP po-sitive cells in U2OS, Saos-2, KYSE150 and BGC823 cell lines reachedup to 75% at 200 MOI after 24 h (Table 1). Western blot analysis

Table 1Infection efficiency of Ad5-GFP detected by flow cytometry.

Cell line Time (h) Ad5-GFP (MOI)

100 200 400 800 1600

BGC823 24 72.16a 77.66 87.45 92.25 92.47U2OS 24 54.35 75.24 85.02 91.86 93.73Saos2 24 76.58 85.42 91.3 95.92 97.9KYSE150 24 84.57 89.51 92.8 95.19 93.93

a Percentage of Ad5-GFP positive cells.

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showed that the TMEM166 levels in Ad5-TMEM166-infected cellssignificantly increased in a dose-and time- dependent manner(Fig. 1A and B).

Next, we analyzed the cell viability of the aforementioned celllines infected by either Ad5-Null or Ad5-TMEM166 at differentMOI and times using the CCK-8 assay. As shown in Fig. 2A and B,the inhibition of growth in cells infected by Ad5-TMEM166 wassignificantly greater than that with Ad5-Null treatment, especiallyin U2OS and Saos-2 cell lines, and the inhibition was time- anddose-dependent.

3.2. Ad5-TMEM166 induces cancer cell autophagy

Previous research has revealed that TMEM166 can induce hall-marks of autophagy early after over expression [22]. To furtherconfirm the effects of Ad5-TMEM166 on cell autophagy, U2OS,Saos-2, KYSE150 and BGC823 cell lines were infected by eitherAd5-TMEM166 or Ad5-Null at 200 MOI combined with Ad5-GFP–LC3. After 20 h, the extensive punctate GFP–LC3 distribution inAd5-TMEM166-infected cells could be observed by confocal andfluorescence microscopy, in contrast to the diffuse pattern inAd5-Null-infected cells (Fig. 3A). Quantification of the punctateGFP–LC3 cells from three independent experiments showed thatthe difference of punctate GFP cells/total GFP cells between theAd5-Null and Ad5-TMEM166 infected groups was statistically sig-nificant (Fig. 3B). Immunoblots using anti-LC3 antibody showedthat the membrane-bound LC3-phospholipid conjugate LC3-IIwas significantly increased in Ad5-TMEM166-infected cells, com-pared with Ad5-Null- infected cells (Fig. 3C).

Fig. 1. Western blotting analysis of TMEM166 expression in Ad5-TMEM166-infected tumTMEM166 at 100, 200, 400, 800 and 1600 MOI or Ad5-Null at 200 MOI for 24 h. The doSaos-2, BGC823 and KYSE150 cells were infected with either Ad5-TMEM166 or Ad5-Nuanalyzed by immunoblotting. Actin was detected as the protein loading control.

It has been widely recognized that the p62 protein serves as alink between LC3 and ubiquitinated substrates. It incorporates intothe completed autophagosome and is degraded in autolysosomes.Therefore, the level of p62 protein is a marker for detecting auto-phagic flux [24]. Our Western blot analysis demonstrated that withincreased TMEM166 protein levels in Ad5-TMEM166-infectedU2OS and KYSE150 cells, p62 protein levels decreased (Fig. 3D),indicating the increase of the autophagic flux and the occurrenceof autophagy.

3.3. Ad5-TMEM166-induced autophagy is mediated by the mTOR/p70S6K pathway

It has been reported that the mTOR signaling pathway plays acritical role in autophagy induction [25]. In this pathway, deactiva-tion of the downstream substrate of mTOR, p70S6K, is associatedwith autophagy induction. To dissect the mechanism of autophagyinduction by Ad5-TMEM166, U2OS, Saos2, BGC823 and KYSE150cell lines were infected with either Ad5-TMEM166 or Ad5-Null.After 20 h, Western blot analysis was carried out to detect thephosphorylation levels of mTOR and p70S6K at Thr389. As shownin Fig. 4, Ad5-TMEM166 but not Ad5-Null could decrease the phos-phorylation levels of both mTOR and p70S6K (T389) in the afore-mentioned cell lines, indicating the inactivation of the mTORsignal and p70S6K as well as the subsequent induction of autoph-agy. Additionally, the decreased levels of p62 in cells infected byAd5-TMEM166 (Fig. 4) demonstrated the increase in the autopha-gic flux. These results suggest that Ad5-TMEM166-inducedautophagy, at least partly is mediated by the mTOR/p70S6Kpathway.

3.4. Ad5-TMEM166 induces cancer cell apoptosis

Aside from autophagy, apoptosis is another factor affecting cellviability. Therefore, we detected the exposure of phosphotidylser-ine (PS), which is a key biochemical hallmark of apoptosis, in celllines infected with either Ad5-Null or Ad5-TMEM166 at variousMOI for 24 h. We found that Ad5-TMEM166 resulted in a signifi-cant dose-dependent increase in the number of apoptotic cellscompared with Ad5-Null (Fig. 5A). The percentage of increase inapoptosis after Ad5-TMEM166 infection ranged from 21.18 to

or cell lines. (A) U2OS, Saos-2, BGC823 and KYSE150 cells were infected with Ad5-se-dependent expression of TMEM166 was analyzed by immunoblotting. (B) U2OS,ll at 200 MOI for 24 and 36 h. Time-dependent expression of Ad5-TMEM166 was

Fig. 2. Ad5-TMEM166 suppresses tumor cell growth in a time- and dose-dependent manner. (A) U2OS, Saos-2, BGC823 and KYSE150 cells were infected by either Ad5-TMEM166 or Ad5-Null at 100, 200, 400 and 800 MOI for 48 h. (B) U2OS, Saos-2, BGC823 and KYSE150 cells were infected by either Ad5-TMEM166 or Ad5-Null at 200 MOI for24, 48 and 72 h. Percentage of cell viability was detected by CCK-8 assay. Each bar represents the mean ± SD from three independent experiments. �P < 0.05, ��P < 0.01,���P < 0.001, NS, not significance.

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55.39% in U2OS cells and from 16.33 to 41.97% in KYSE 150 cells,depending on the MOI from 100 to 800 (Fig. 5A, upper panel).The percentage of increase in apoptosis after Ad5-TMEM166 infec-tion ranged from 16.1 to 37.6% in Saos-2 cells and from 13.93 to28.32% in BGC823 cells, depending on the MOI from 100 to 400(Fig. 5A, lower panel). Meanwhile, there were no obvious changesin cells with Ad5-Null or mock. We also analyzed the changes ofapoptotic cells over time in the U2OS, KYSE150, BGC823 and Saos2cell lines. Cells treated with Ad5-TMEM166 at 200 MOI were de-tected by FITC-Annexin V staining at 24 and 48 h. As shown inFig. 5B, the proportion of apoptotic cells increased in a time-depen-dent manner compared with Ad5-Null-infected cells or Mock.

Caspase-mediated apoptosis is the best-defined cell death pro-gram counteracting tumor growth, and caspase-3 is the most di-rect effector molecule in its protein family. Therefore, caspase-3activity was quantitatively measured using a DEVD cleavage assayin U2OS and KYSE150 cell lines. As shown in Fig. 5C, Ad5-TMEM166-infected cells showed significantly elevated caspase-3activity compared with Ad5-Null, indicating that the activation ofcaspase-3 involved TMEM166-induced cell death (Fig. 5C).

3.5. Ad5-TMEM166 mediates inhibition of tumorigenicity ex vivo andin vivo

After verifying the anti-tumor activity of TMEM166 in vitro, wenext sought to determine whether exogenous TMEM166 affectedtumorigenicity when given ex vivo and in vivo. According to the re-sults of preliminary experiments, we found that KYSE150 cell linehas a better ability of tumorigenicity than other cell lines wetested. Therefore, we selected this cell line to finish the followinganimal experiments. For ex vivo treatment, KYSE150 cells were in-fected with 200 MOI of Ad5-Null or Ad5-TMEM166 for 24 h beforebeing injected into the axilla of BALB/c nude mice. As shown inFig. 6A, Ad5-TMEM166 treated cells nearly completely suppressedtumor growth compared with Ad5-Null or mock treatment. At20 days, mice were sacrificed and imaged (Fig. 6B). It was obviousthat the tumor size with Ad5-TMEM166 treatment was signifi-cantly smaller than the other control treatments. In addition, noevidence of toxicity was observed in any of the control or treat-ment groups throughout these studies as monitored by weight loss(Fig. 6C).

Fig. 3. Ad5-TMEM166 induces cancer cells autophagy. (A) U2OS, Saos-2, BGC823 and KYSE150 cell lines were infected with either Ad5-TMEM166 or Ad5-Null at 200 MOIcombined with Ad5-GFP–LC3 at 50 MOI for 20 h. Confocal microscopy was used to observe the punctated distribution of GFP–LC3 during autophagy. Representativefluorescence microphotographs from different cell lines are shown. (B) Statistical analysis of cells with GFP–LC3 punctated distribution. �P < 0.05, ��P < 0.01. (C) Cells weretreated as in (A). Accumulations of LC3-II protein in Ad5-GFP, Ad5-Null and Ad5-TMEM166-infected cell lines were analyzed by immunoblotting. (D) U2OS and KYSE150 cellswere infected with either Ad5-Null or Ad5-TMEM166 at 200 and 400 MOI for 20 h. Expressions of TMEM166 and p62 were analyzed by immunoblotting. Actin was detectedas the protein loading control.

Fig. 4. Ad5-TMEM166-induced autophagy is mediated by the mTOR/p70S6K pathway. U2OS, Saos-2, BGC823 and KYSE150 cell lines were infected with either Ad5-TMEM166or Ad5-Null at 200 MOI for 20 h. Equal amounts of protein per lane were separated by SDS–PAGE, transferred to a PVDF membrane and analyzed by immunoblotting usingphospho-mTOR, mTOR, phospho-p70S6K (Thr389), p70S6K, p62 and TMEM166 antibodies. Actin was detected as the protein loading control.

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Next, the in vivo anti-tumor activity of Ad5-TMEM166 was eval-uated using a KYSE150 xenograft model established in BALB/c nudemice. When the tumors reached a size of 100 mm3, Ad5-TMEM166,

Ad5-Null or Mock was administered locally by multiple-centerintratumor injection. For mice received Ad5-TMEM166 treatment,a significant suppression in tumor growth was detected on day 16,

Fig. 5. Ad5-TMEM166 induces cancer cell apoptosis. (A) U2OS and KYSE150 cells were infected with either Ad5-Null or Ad5-TMEM166 at 100, 200, 400 and 800 MOI for 24 h.BGC823 and Saos-2 cells were infected with Ad5-TMEM166 at 100, 200 and 400 MOI, or Ad5-Null at 400 MOI as a control for 24 h. Apoptotic cells were measured by FITC–Annexin V staining followed by flow cytometry analysis. (B) U2OS, KYSE150, Saos-2 and BGC823 cell lines treated with either Ad5-Null or Ad5-TMEM166 at 200 MOI weredetected by FITC–Annexin V staining at 24 h and 48 h, followed by flow cytometry analysis. (C) U2OS and KYSE150 cell lines were treated with either Ad5-Null or Ad5-TMEM166 at 200 MOI for 24 h. Caspase-3 activity was measured using a Caspase-3 Fluorometric Assay Kit and quantified using a POLARSTAR fluorometer. ���P < 0.001.

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18 and 20 (Fig. 6D) (P < 0.05 vs. Ad5-Null; P < 0.01 vs. mock). Fol-lowing tumor excision on day 20, the mean tumor weight for themock, Ad5-Null and Ad5-TMEM166 groups were counted. Asshown in Fig. 6E and F, the tumor weight of the Ad5-TMEM166treatment group was markedly lighter than that of the controlgroups (P < 0.01 vs. Ad5-Null and mock). Additionally, there wasno evidence of toxicity observed in any of the control or treatmentgroups throughout these studies as monitored by weight loss(same results as in Fig. 6C). It should be noted that during ourexperiments, we often observed that approximately half of the tu-mor tissues treated by Ad5-TMEM166, but not Ad5-Null, displayedsevere bleeding and liquefactive necrosis within the tumor mass,and although the tumors were well circumscribed (Fig. 6G).

3.6. Ad5-TMEM166 inhibits cell proliferation, increases cell autophagyand apoptosis in KYSE150 xenograft tumors

To investigate the mechanism of Ad5-TMEM166-induced inhi-bition of tumor growth, tumor specimens were obtained from tu-mor-bearing animals after death. The specimens were subjected

to the Ki-67 staining assay. The Ki-67 protein is strictly associatedwith cell proliferation. During interphase, the Ki-67 antigen can beexclusively detected within the cell nucleus, whereas in mitosismost of the protein is relocated to the surface of the chromosomes.Ki-67 protein is present during all active phases of the cell cycle(G1, S, G2, and mitosis), but is absent from resting cells (G0). Ourexperimental data were shown in Fig. 7A and B, the percentageof Ki-67 positive cells in the Ad5-TMEM166 group was signifi-cantly lower than that the Ad5-Null and Mock groups (P < 0.001).We further assessed whether tumor suppression was attributableto autophagy and apoptosis induced by Ad5-TMEM166. Westernblot analysis suggested that the level of p62 in proteins extractedfrom tumors of mice treated with Ad5-TMEM166 was downregu-lated compared with those of the Ad5-Null and Mock groups(Fig. 7C), indicating the occurrence of autophagy.

TUNEL analysis, which detects single- and double-strand DNAbreaks, was used to identify apoptotic cells present in tumor spec-imens. As shown in Fig. 7D and E, treatment with Ad5-TMEM166revealed a significantly high level of cell death compared withAd5-Null treatment and Mock (P < 0.001). Taken together, these re-

Fig. 6. Ad5-TMEM166 inhibits growth of KYSE150 cells ex vivo and in vivo. (A) KYSE150 cells infected with Ad5-TMEM166 or Ad5-Null were injected subcutaneously in BALB/c nude mice. Development of tumors (mean volume ± SD) was monitored using calipers every 2 days. (B) Excised xenograft tumors were imaged on day 20. (C) Body weight(mean ± SD) was determined over time in different treatment groups. (D) KYSE150 cells were inoculated subcutaneously into the left axilla in BALB/c nude mice. When thevolume of tumors reached 100 mm3, treatments were given as described in methods. Development of tumors (mean volume ± SD) was monitored using calipers every 2 days.�P < 0.05 vs. Ad5-Null; #P < 0.01 vs. mock. (E) Excised xenograft tumors were imaged on day 20. (F) Excised xenograft tumors were weighed on day 20. ��P < 0.01 vs. Ad5-Nulland mock. (G) Representative images of H&E stained tumor xenografts (original magnification, 200�).

132 Y. Chang et al. / Cancer Letters 328 (2013) 126–134

sults confirmed that Ad5-TMEM166 could exert strong anti-tumoractivities in vivo on KYSE150 cells and inhibited tumor growth byinduction of autophagy and apoptosis.

4. Discussion

TMEM166 is a novel gene originally characterized in our labora-tory [22]. It is conserved in humans, chimpanzees, rats, mice anddogs, indicating that it may have important functions in vertebrateanimals. In a previous study TMEM166 was shown to be a lyso-somal and endoplasmic reticulum-associated protein that can in-duce tumor cell death, exhibiting both autophagic and apoptoticcharacteristics.

The involvement of TMEM166 in both autophagy and apoptosisimplies that it may have an essential role in cell death. Neverthe-less, the molecular mechanism through which TMEM166 activatescell autophagy and apoptosis has remained elusive. Earlier wedemonstrated by RT-PCR analysis that TMEM166 mRNA is ex-pressed in a variety of normal and tumor tissues [22]. Tissue

microarray analysis has indicated that the expressions ofTMEM166 protein in most cancer tissues are negative or lowercompared with normal tissues [23]. In the present study, we con-structed an adenovirus expression vector expressing TMEM166to test its biological function both in vitro and in vivo.

In this study, we first evaluated adenovirus-mediated expres-sion of TMEM166 in some cancer cell lines. Western blotting dem-onstrated that Ad5-TMEM166 significantly increased TMEM166protein levels in U2OS, Saos-2, KYSE150 and BGC832 cell lines. Cellviability analysis showed that Ad5-TMEM166 caused significantgrowth arrest in these cell lines. Furthermore, we demonstratedthat Ad5-TMEM166 induced growth arrest through cell autophagyand apoptosis, which may have occurred independently in parallelpathways or cooperatively to lead to cell death. In ex vivo andin vivo assays, we evaluated the anti-tumorigenicity of Ad5-TMEM166 and observed notable growth inhibitory effects. Thesafety of Ad5-TMEM166 was also supported by the absence of tox-icity/morphological damage in hemotoxylin and eosin (H&E) anal-ysis of heart, liver, spleen, lung and kidney of nude mice infectedby Ad5-TMEM166 or control (data not shown). These data

Fig. 7. Ad5-TMEM166 inhibits cell proliferation, increases cell autophagy and apoptosis in KYSE150 tumor xenografts. (A) Representative images of immunohistochemicalstaining for Ki-67 in tumor xenografts from each treatment group (original magnification, 400�). (B) Quantification of Ki-67 expression as percentage of positive cells(mean ± SD). ���P < 0.001. (C) Western blot analysis of TMEM166 and p62 expression in proteins extracted from xenograft tumors of different treatment groups. Actin wasdetected as a loading control. (D) Representative images of TUNEL staining performed on KYSE150 xenografts from each treatment group (original magnification, 400�). (E)Results and statistical analysis of TUNEL assay (mean ± SD). ���P < 0.001.

Y. Chang et al. / Cancer Letters 328 (2013) 126–134 133

indicated that the adenovirus-mediated expression of TMEM166exerted strong anti-tumor activity, implying that it may be poten-tially used in gene therapy for cancer. At present, we are workingwith other researchers to explore the relationship between expres-sion of TMEM166 and clinical disease, such as cancer and endo-crine disease.

The signaling pathways responsible for induction of autophagyare considered to vary according to cell type and stimulus. mTOR isa negative regulator of autophagy induction. It is regulated throughthe action of a class I PI3-K that allows the membrane binding andsubsequent activation of Akt/PKB and 3-phosphoinositide-depen-dent protein kinase 1 (PDK1), which cause inhibition of autophagy[25,26]. On the other hand, over expression of PTEN, a negativeregulator of the PI3K/AKT pathway, induces autophagy [27]. TheTSC1–TSC2 complex acts as a GTPase-activating protein (GAP) forthe GTPase Rheb. Inhibition of TSC1–TSC2 by Akt stabilizes theGTP-bound form of Rheb, which stimulates mTOR [28]. Both mTORand PDK1 activate the ribosomal subunit S6 kinase (p70S6k) [29],and the suppression of S6k phosphorylation generally favorsautophagy induction [30,31]. In our study, TMEM166 could de-crease the phosporylation level of mTOR in the cell lines tested,implying that TMEM166 may be a negative regulator of mTOR sig-naling. In response to the inactivation of mTOR, the level of p70S6phosphorylation was downregulated accompanied by the subse-

quent high level of autophagy. Because the phosphorylation ofAkt was not affected by TMEM166 (data not shown), it is unlikelythat TMEM166 regulates phosphorylation of p70S6K by inhibitingthe PDK1/Akt signaling module. We cannot exclude the possibilityof TMEM166 regulating phosphorylation of p70S6K directly or byan alterative signaling mechanism [32,33], but this remains to betested.

Increased autophagy is often observed in tumor cells in re-sponse to chemotherapy and radiation. The majority of studiesindicate that autophagy inhibition sensitizes tumor cells to a widespectrum of cancer therapies, while others show that treatment-induced tumor cell death requires intact autophagic machinery.During our study, we used inhibitors of class III PI3K pathway, suchas 3-MA and LY294002 which inhibit autophagy at an early stage,to observe the impact of Ad5-TMEM166 on tumor cells. We foundthat after treatment with 3-MA or LY294002, apoptotic cell deathinduced by Ad5-TMEM166 was significantly increased in theU2OS, KYSE150 and BGC823 cell lines (Fig. S1), indicating that inhi-bition of autophagy sensitized the cells to Ad5-TMEM166 withapoptosis induction. The role of autophagy in the Ad5-TMEM166-mediated cell death was further studied by knocking down the be-clin 1 expression using specific shRNA. Beclin 1 is a component ofclass III phosphatidy-linositol 3-kinase complex essential for auto-phagosome formation [20]. Consistent with the results using 3-MA

134 Y. Chang et al. / Cancer Letters 328 (2013) 126–134

and LY294002, the degree of cell death induced by Ad5-TMEM166was also increased by silencing Beclin 1 expression when com-pared to the shRNA control (Fig. s2). These primary results implythat autophagy induced by Ad5-TMEM166 may confer protectionof tumor cells against apoptosis, and blockade of autophagy couldresult in potentiation of the proapoptotic effect of Ad5-TMEM166.Consequently, more detailed investigations are doing for under-standing the crosstalk between apoptosis and autophagy inducedby TMEM166. Alternatively, the action of other autophagy formscannot be excluded, such as chaperone-mediated autophagy ormicroautophagy, which could compensate for the loss of macro-autophagy and thus influence tumor cell killing in unexpectedways [34]. As LY294002 is also a cancer chemotheraputic drug,our data suggest that tumors overexpressing TMEM166 may bemore susceptible to chemotherapy, which put forth the possibilityof using Ad5-TMEM166 together with chemotherapeutic drugs forthe treatment of cancer.

Acknowledgements

We thank Qihua He for his help with confocal imaging. Thiswork was supported by the Grants from the National Key Projectfor Basic Research of China (973, 2011CB910103), and the NationalNatural Science Foundation of China (30771057).

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.2012.08.032.

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