RNA interference against cancer/testis genes ... - Wiley Online Library

RNA Interference Against Cancer/Testis Genes Identifies Dual Specificity Phosphatase 21 as a Potential Therapeutic Target in Human Hepatocellular Carcinoma Qing Deng,1,2* Kun-Yu Li,2* Hui Chen,1,2* Ji-Hong Dai,2 Yang-Yang Zhai,2 Qun Wang,1,2 Niu Li,1,2 Yu-Ping Wang,1,2 and Ze-Guang Han1,2,3 Cancer/testis (CT) antigens have been considered therapeutic targets for treating cancers. However, a central question is whether their expression contributes to tumorigenesis or if they are functionally irrelevant by-products derived from the process of cellular transformation. In any case, these CT antigens are essential for cancer cell survival and may serve as potential therapeutic targets. Recently, the cell-based RNA interference (RNAi) screen has proven to be a powerful approach for identifying potential therapeutic targets. In this study we sought to identify new CT antigens as potential therapeutic targets for human hepatocellular carcinoma (HCC), and 179 potential CT genes on the X chromosome were screened through a bioinformatics analysis of gene expression profiles. Then an RNAi screen against these potential CT genes identified nine that were required for sustaining the survival of Focus and PLC/PRF/5 cells. Among the nine genes, the physiologically testis-restricted dual specificity phosphatase 21 (DUSP21) encoding a dual specificity phosphatase was up-regulated in 39 (33%) of 118 human HCC specimens. Ectopic DUSP21 had no obvious impact on proliferation and colony formation in HCC cells. However, DUSP21 silencing significantly suppressed cell proliferation, colony formation, and in vivo tumorigenicity in HCC cells. The administration of adenovirus-mediated RNAi and an atelocollagen/siRNA mixture against endogenous DUSP21 significantly suppressed xenograft HCC tumors in mice. Further investigations showed that DUSP21 knockdown led to arrest of the cell cycle in G1 phase, cell senescence, and expression changes of some factors with functions in the cell cycle and/or senescence. Furthermore, the antiproliferative role of DUSP21 knockdown is through activation of p38 mitogen-activated protein kinase in HCC. Conclusion: DUSP21 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment. (HEPATOLOGY 2014;59:518-530)

G

enerally, cancer/testis (CT) genes are defined by a unique expression pattern in which they are expressed exclusively in gametes and trophoblasts in normal tissues and are also found in a

number of malignancies, although varying definitions of CT genes exist in the different literature.1-5 CT genes can be divided into two groups: CT genes that are on the X chromosome (CT-X genes) and CT genes

Abbreviations: Ad-shDUSP21, recombinant adenoviral vector harboring an shRNA against DUSP21; CCK-8, cell-counting kit-8; CT, cancer/testis genes; CT-X, cancer/testis genes on X-chromosome; DAPI, 40 ,6-diamidino-2-phenylindole; DEPC, diethypyrocarbonate; DUSPs, dual specificity phosphatases; DUSP21, dual specificity phosphatase 21; EST, expressed sequence tag; GEO, gene expression omnibus; HCC, hepatocellular carcinoma; MAPK, mitogen-activated protein kinase; MeV, multiexperiment viewer; MPSS, massively parallel signature sequencing; PI, propidium iodide; RNAi, RNA interference; RT-PCR, reverse transcriptionpolymerase chain reaction; SA-b-Gal, senescence-associated b-galactosidase; shRNA, short-hair pin RNA; siRNA, small interference RNA; STRING, a database of known and predicted protein interactions. From the 1Key Laboratory of Systems Biomedicine (Ministry of Education) of Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China; 3Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China. Received March 21, 2013; accepted July 29, 2013. Supported by grants from Chinese National Key Program on Basic Research (2010CB529200 and 2012CB722308), National Natural Science Foundation of China (81101875, 81071722, and 81272271), China National Key Projects for Infectious Disease (2012ZX10002012-008 and 2013ZX10002010-006), Shanghai Natural Science Foundation (11ZR1425300), and the Shanghai Commission for Science and Technology. *These authors contributed equally to this work. 518

HEPATOLOGY, Vol. 59, No. 2, 2014

that are not (non-X CT genes). Intriguingly, 10% of genes on the human X chromosome are CT-X genes.1,6 Most significant, some CT-X antigens are used in therapeutic vaccines against cancers.1 For example, phase II trials conducted postoperatively in early-stage, nonsmall-cell lung cancer patients demonstrated that the MAGE-A3 vaccine markedly reduced the recurrence rate.7 Furthermore, NY-ESO-1 (CTAG1B) has also been successfully targeted in trials of NY-ESO-1-specific adoptive T-cell therapy for melanoma.8 Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies, ranking as the sixth most common cancer worldwide and the third most common cause of cancer mortality.9 The prognosis of HCC patients remains very poor, so novel therapeutic approaches against drug targets are urgently needed. Some CT-X genes, such as MAGE, SSX, and GAGE, are detected in tumor tissues or the peripheral blood of HCC patients,10,11 indicating that certain CT genes are deregulated in HCC. Although CT-X antigens are the core focus in the development and clinical testing of experimental cancer vaccines, their biological functions in both germ cells and tumors have remained obscure. Recently, the cell-based RNA interference (RNAi) screen has proven to be a powerful approach for characterizing biological features by targeting genes of interest.12 In this study, to identify new CT antigens as potential therapeutic targets for HCC, we initially identified potential CT-X genes by comparing gene expression profiles and then performed a cell-based RNAi screen against these candidate CT-X genes to determine those genes that contribute to maintaining cell proliferation and survival in HCC cells. Of 179 CT-X genes, nine were crucial to the viability of HCC cells. Among these genes, dual specificity phosphatase 21 (DUSP21) encodes a member of the atypical dual specificity phosphatases (DUSPs) that are implicated as major modulators of critical signaling pathways13 and therapeutic targets.14 To our knowledge, we demonstrate for the first time in this study that DUSP21 is a potential therapeutic target for HCC.

DENG, LI, ET AL.

519

Materials and Methods In Silico Analysis of Gene Expression Profiling. Cancer/testis genes were retrieved from the CT database of the Ludwig Institute for Cancer Research,15 and massively parallel signature sequencing (MPSS) data of 31 human tissues extracted from the Gene Expression Omnibus database were compared to identify testis-enriched genes on the X-chromosome. In addition, expressed sequence tag (EST) profiles (NCBI Unigene) were analyzed to recognize testis-enriched genes on the X-chromosome. The following formula was used to calculate testis-enrichedgenes as previously described16: S5log 2 Pn

Eh11 Et2Eh11

. Here, S is the

t51

enrichment, E1 to En are the expression levels across all tissues, and Eh is the expression value observed in testis for a given gene. S values >2 would be considered testis-enriched genes. RNA Interference Screen. Briefly, 3,000 cells per well were seeded in a 96-well culture plate. Likewise, small interfering RNA (siRNA) duplexes were placed into a 96-well culture plate and delivered into Focus and PLC/PRF/5 cells by Lipofectamine 2000 (Invitrogen) transfection according to the manufacturer’s instructions. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories). These experiments were repeated three times independently. Raw data from each plate were normalized and analyzed according to the described method.17 Phenotypes were scored for their statistical significance. Colony Formation Assay. HCC cells, 5-10 3 104, were cultured in 10-cm plates in triplicate, then G418 (Life Technologies, Grand Island, NY) was added to the medium at a final concentration of 0.6-1 mg/mL. After 3 or 4 weeks, the remaining colonies were washed twice with phosphate-buffered saline (PBS) and stained with crystal violet. The colonies were counted according to the defined colony size. Cell Senescence Assay. Senescence-associated b-galactosidase (SA-b-Gal) staining was performed to evaluate senescence of Focus cells 24 hours after doxorubicin (1 lM) treatment as described.18 Using a

Address reprint requests to: Ze-Guang Han, M.D., or Qing Deng, Ph.D., Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China. E-mail: [email protected] or [email protected]; fax: 186-21-50800402. C 2013 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26665 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.

520

DENG, LI, ET AL.

HEPATOLOGY, February 2014

Fig. 1. The cell-based RNAi screen of the CT genes responsible for sustaining cell viability. (A) The potential CT genes on the human chromosome X, including the 126 known genes deposited in the database and 86 candidates proposed by in silico analysis of transcriptomic data. (B) The RNAi screen was performed in Focus and PLC/PRF/5 HCC cells transfected with 228 siRNA duplexes, the raw data output from the screen was normalized, and two statistical parameters, Z-score and P-value, were calculated. An overall profiling of the effect of these siRNAs on the viability of both HCC cells was outlined, where the log-transformed P-value served as the Y-axis and the Z-score served as the X-axis. The indicated genes (left) targeted by certain siRNAs in the range of Z < 21 and P < 0.05 were considered as candidates responsible for sustaining cell viability. (C) The efficiency of the siRNA-mediated knockdown of these targeted genes was evaluated using real-time PCR. (D) An independent assessment was performed to evaluate the effect of the knockdown of these candidate genes on cell viability, where si-NC was used as a negative control. The histogram shows the mean values of at least three independent experiments 6 SD.

magnification of 1003, SA-b-Gal staining-positive cells were counted in at least six random microscopic fields. Tumor Therapy Using a Xenograft Model. PLC/ PRF/5 cells (5 3 106 in 200 lL of PBS) and MHCC-97H cells (2 3 106 in 100 lL of PBS) were injected subcutaneously near the scapula of 6-week-old nude mice to establish an HCC xenograft model for antitumor assays. The adenoviral- and nonviral atelocollagen- (Koken, Tokyo, Japan) based delivery methods were used for antitumor therapy. After tumor xenografts reached 50 mm3, either Ad-shDUSP21 or the atelocollagen/siRNA complex was injected intratumorally every 3 days for a total of five times. The tumor formation kinetics were estimated by measuring the tumor size and volume at 3-day intervals. Additional methods and descriptions are provided in more detail in the Supporting Information.

Results RNAi Screen of Candidate CT Genes Responsible for Sustaining Cell Viability. To date, there are 33 CT-X gene families that contain 126 members in the CT database from the Ludwig Institute for Cancer Research. In this study, to define whether more genes on the human X chromosome could be designated as CT-X genes, we characterized the expression profile of 1,336 known genes localized on the X chromosome through an in silico analysis of the transcriptomic data deposited in the public database. The analysis of MPSS data obtained from the GEO database from 31 human tissues revealed 33 testis-enriched genes (Supporting Fig. 1A), and the analysis of EST data from 49 human tissues suggested 68 testis-enriched genes (Supporting Fig. 1B). Of these testis-enriched genes, only 19 overlapped in the two datasets. In addition,


DENG, LI, ET AL.

521

Table 1. Expression Pattern of Candidate Cancer/Testis Genes That are Required for HCC Cell Survival Expression Profiling Analysis

Symbol

DUSP21 CT45 ZCCHC13 PIH1D3 PNMA5 MPC1L MAGEA9 IL13RA1 MAGEB6

CT Identifier*

Gene Description

— CT45 — — — — CT1.9 CT19 CT3.4

dual specificity phosphatase 21 cancer/testis antigen family 45 zinc finger, CCHC domain containing 13 PIH1 domain containing 3 paraneoplastic Ma antigen family member 5 mitochondrial pyruvate carrier 1-like melanoma antigen family A, 9 interleukin 13 receptor, alpha 1 melanoma antigen family B, 6

Human Normal Tissuesy

testis-restricted testis-restricted testis-restricted testis-inclusive testis-inclusive testis-inclusive testis-inclusive testis-inclusive testis-restricted

Human HCC Specimensz

8/24 7/24 4/24 4/24 6/24 6/24 3/24 1/24 3/24

*CT identifier is the nomenclature included in the CT database of the Ludwig Institute for Cancer Research (http://www.cta.lncc.br), and “—” denotes no inclusion in the CT database. † The expression pattern of candidate genes in 14 human tissues, including brain, heart, lung, spleen, kidney, stomach, esophagus, small intestine, ovary, breast, prostate, pancreas, liver, and testis, were examined by RT-PCR; “testis-restricted” indicates candidate genes expressed exclusively in testis; “testis-inclusive” denotes these genes expressed in testis and a few extra tissues. ‡ The numbers of HCC patients with an up-regulation of a given gene in 24 human HCCs examined, where the mRNA levels were detected by RT-PCR.

four testis-specific genes were manually selected based on the published literature1,19 (Supporting Fig. 1C). A total of 86 testis-enriched genes, including 53 potential CT-X genes not deposited in the CT database, could be considered CT genes (Fig. 1A; Supporting Table 1). Of the integrated 179 genes, including 126 known CT-X genes from 33 families and 53 candidates, 128 genes lacking high homology with each other were further examined by reverse-transcription polymerase chain reaction (RT-PCR) using specific primers in 14 normal human tissues and a panel of 19 HCC cell lines. Among the 113 genes successfully examined, 87 (77.0%) were detected in two or more HCC cell lines (Supporting Fig. 1D, Supporting Table 2), of which 79 (69.9%) significantly exhibited relatively high expression in Focus and PLC/PRF/5 cells (P < 0.001). To investigate the role of these potential CT-X genes in HCC cell proliferation, we established a cell-based RNAi screen to identify the candidate genes responsible for sustaining cell proliferation by evaluating cell viability (Fig. 1B; Supporting Fig. 2). In this screen, 228 siRNA duplexes were designed against these 179 genes, where some CT-X gene families shared the same siRNAs (Supporting Table 3), and were then applied to both Focus and PLC/PRF/5 cells. Based on the RNAi screen, 19 siRNAs were considered to statistically significantly suppress cell proliferation at the cutoff value of Z < 21 and P < 0.05 (Fig. 1B). Subsequently, we performed a quantitative RT-PCR assessment of the RNAi knockdown efficiency of these 19 siRNAs against 11 genes in Focus and PLC/PRF/5 cells. The resulting data indicated that the messenger RNA (mRNA) levels of nine genes were efficiently and significantly decreased to less than 60% by their corre-

sponding siRNAs (Fig. 1C). An independent evaluation of the effect of these siRNAs on cell viability was also performed and confirmed that the knockdown of these nine genes inhibited cell viability (Fig. 1D). These collective data suggested that the nine candidate CT genes screened by the RNAi strategy could be required for sustaining HCC cell survival. Tissue Expression Patterns and Role of Candidate CT Genes in HCC Cells. The nine genes include known CT genes, such as CT45, MAGEA9, IL13RA1, and MAGEB6, and new candidates, including DUSP21, ZCCHC13, PNMA5, PIH1D3, and MPC1L (Table 1). To determine whether these genes could be assigned to the CT genes expressed in HCC, we first evaluated their expression patterns in human tissues using RT-PCR. The results showed that only DUSP21, ZCCHC13, CT45, and MAGEB6 were exclusively expressed in testis; MAGEA9, PNMA5, PIH1D3, and MPC1L were also expressed in a few extra tissues. In contrast, IL13RA1 was ubiquitously expressed in the 14 tissues tested (Fig. 2A). The data were generally consistent with the in silico analysis of the transcriptomic data deposited in public databases, including MPSS data from 31 normal human tissues and ESTs of 49 human tissues (Fig. 2B). We next evaluated the expression of these nine genes in HCC specimens through a bioinformatics analysis of human HCC sample data from three public microarray databases (49 samples from GSE4024, 91 from GSE1898, and 238 from GSE5975) and through detection in our HCC samples by RT-PCR. Here, the bioinformatics analysis indicated that DUSP21, MAGEA9, PNMA5, and PIH1D3 were significantly elevated in HCC specimens, which was

522

DENG, LI, ET AL.


Fig. 2. Tissue expression patterns and the effect of candidate cancer/testis genes on HCC cells. (A) RT-PCR results indicating the expression pattern of the nine candidate CT genes in 14 adult human tissues. (B) Heat map of expression patterns of these CT genes by way of massively parallel signature sequencing analysis on 31 human normal tissues (upper) and ESTs from 49 human tissues (lower). (C) Bar charts of expression intensities of CT genes from three published HCC datasets, i.e., GSE4024, GSE1898, and GSE5975, where each dataset was statistically analyzed for deviations from the Gaussian distribution using the D’Agostino and Pearson omnibus normality test. (D) The growth curves were described based on cell viability on different days after siRNAs against DUSP21 and ZCCHC13 were transfected into Focus and PLC/PRF/5 cells, with si-NC used as a negative control. (E) Representative dishes of colony formation in Focus and PLC/PRF/5 cells are shown, where cells were transfected with plasmids containing shRNAs against DUSP21 and ZCCHC13. sh-NC based in the pSUPER vector was used as a negative control. The numbers of colonies were counted and statistically analyzed using a two-tailed t test. The histograms represent the mean values of three independent experiments 6 SD. *P < 0.05; **P < 0.01; *** P < 0.001.

supported by at least two of the three datasets (Fig. 2C). In addition, our RT-PCR data revealed that six genes, DUSP21, CT45, MPC1L, PNMA5, PIH1D3, and ZCCHC13, were noticeably up-regulated in four or more of the 24 HCC samples compared with corresponding normal liver tissues (Table 1; Supporting Fig. 3).

To further confirm the role of these six CT genes in sustaining HCC cell proliferation, we performed an independent evaluation of the growth of Focus and PLC/PRF/5 cells by knocking them down by way of synthesized siRNAs. As expected, the knockdown of these genes resulted in the decreased growth of Focus and PLC/PRF/5 cells (Fig. 2D; Supporting Fig. 4A).


DENG, LI, ET AL.

523

Fig. 3. The expression of DUSP21 as a novel CT gene in HCCs. (A) The mRNA level of DUSP21 was measured in 40 HCC specimens and corresponding adjacent noncancerous liver tissues by RT-PCR, where b-actin was used as an internal reference. (B) Representative immunohistochemical staining of HCC samples with an anti-DUSP21 antibody. (C) The mRNA level of DUSP21 was evaluated in HCC cell lines by RT-PCR, where b-actin was used as an internal control. (D) The immunofluorescence assay indicated the subcellular location of DUSP21 in Focus cells. The nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI).

Moreover, we evaluated colony formation by way of recombinant pSUPER constructs carrying shRNAs against DUSP21 and ZCCHC13, which are two novel testis-restricted CT-X genes that are up-regulated in HCCs. The pSUPER-mediated RNAi against DUSP21 and ZCCHC13 significantly inhibited colony formation in Focus and PLC/PRF/5 cells (Fig. 2E; Supporting Fig. 4B,C). These observations suggested that these six CT genes exert a significant role in sustaining HCC cell growth and survival. DUSP21 Is a Novel CT Gene Frequently Upregulated in Human HCCs. Among the six CT genes we found up-regulated in HCC, only DUSP21, CT45, and ZCCHC13 were exclusively expressed in testis. CT45 is a member of a known CT gene family,20,21 whereas both DUSP21 and ZCCHC13 are considered novel CT genes. ZCCHC13, with six CCHC-type zinc fingers, functions as a transcription factor, and interestingly, DUSP21 encodes a member of the DUSP family, which serves as a potential therapeutic targets for cancer.22,23 Because it could be a potential target for cancer therapy, we thus focused on the role of DUSP21 in HCC.

To determine the frequency of DUSP21 upregulation in HCC samples, we also evaluated DUSP21 expression in 40 additional paired HCC samples by qualitative RT-PCR analysis. The data showed that DUSP21 was up-regulated in 15 (37.5%) of the 40 HCC specimens compared with the corresponding normal liver tissues (Fig. 3A). Furthermore, we performed immunohistochemical staining on a tissue array containing 78 pairs of HCC specimens with an anti-DUSP21 antibody. As expected, DUSP21 levels were up-regulated in 24 (30.8%) of the 78 HCC specimens (Fig. 3B; Supporting Fig. 5). In addition to PLC/PRF/5 and Focus cells, DUSP21 was detected in nearly half of the human HCC cell lines examined by RT-PCR (Fig. 3C). An immunofluorescence assay showed that DUSP21 is mainly localized to the cytoplasm of HCC cells (Fig. 3D; Supporting Fig. 6A), consistent with the results obtained in a previous study.23 DUSP21 is also localized to the mitochondria in rat testis.24 We thus examined the subcellular localization of human DUSP21 by transfecting a vector carrying C-terminal FLAG-tagged DUSP21 into Hep3B and Huh-7 cells; however,

524

DENG, LI, ET AL.


Fig. 4. The antiproliferative role of DUSP21 knockdown in HCC cells. (A) The anchorage-dependent colony formation assay was performed after pSUPER-mediated DUSP21 knockdown in Hep3B and MHCC-97H cells. (B) The anchorage-independent colony formation assay was performed in soft agar medium with DUSP21 silencing in PLC/PRF/5 cells with sh-NC as a negative control. The numbers of colonies were counted and statistically analyzed using a two-tailed t test. (C) The representative photographs of SA-b-gal staining are shown after DUSP21 knockdown in Focus cells, where SA-b-Gal staining-positive cells were counted in at least six random microscopic fields and then statistically analyzed using a twotailed t test. (D) Cell cycle analysis was performed after DUSP21 knockdown in Focus and PLC/PRF/5 cells, where the percentage of the cell population at different cell cycle phases is shown in the histograms and statistically analyzed using a two-tailed t test. *P < 0.05; **P < 0.01. (E) Some key molecules, including p53, p27, p16, cyclin D1, and cyclin E, which are associated with cell cycle and/or senescence, were assayed by western blotting after DUSP21 knockdown in Focus and PLC/PRF/5 cells. The signal intensities of western blots were measured quantitatively using ImageJ software, and b-actin was used as a normalization control. Data shown are representative of at least three independent experiments.

examination by immunofluorescence showed that DUSP21 did not colocalize with MitoTracker Red, a mitochondria-selective fluorescent probe (Supporting Fig. 6B). This result implied that the subcellular localization of ectopic DUSP21 in HCC cells was distinct from that in testis. DUSP21 Knockdown Plays an Antiproliferative Role in HCC Cells. To investigate the effect of DUSP21 on HCC cells, we examined growth and colony formation in HCC cell lines, including MHCC97L and Huh-7 cells with low DUSP21 expression, as well as Focus and PLC/PRF/5 cells with relatively high DUSP21 expression, after transfection of recombinant pcDNA3.1-DUSP21. The results showed that ectopic DUSP21 had no significant impact on proliferation and colony formation in these HCC cells (Supporting Fig. 7A,B).

To further confirm the inhibitory effect of DUSP21 knockdown on HCC cells, we used two additional siRNAs (si-3 and si-4) to silence endogenous DUSP21 in Focus and PLC/PRF/5 cells. The siRNAs also significantly inhibited the growth of Focus and PLC/PRF/5 cells (Supporting Fig. 7C). Furthermore, we performed the colony formation assay on additional HCC cell lines by transfecting recombinant pSUPER-shDUSP21 carrying shRNA against DUSP21 into these cells. The results demonstrated that the pSUPER-mediated DUSP21 knockdown significantly inhibited anchorage-dependent colony formation of the HCC cell lines Hep3B, MHCC-97H, Bel-7404, and QGY-7703 (Fig. 4A; Supporting Fig. 7D) and restrained anchorageindependent colony formation of PLC/PRF/5 cells cultured in soft agar medium (Fig. 4B). To explore


DENG, LI, ET AL.

525

Fig. 5. The effects of DUSP21 knockdown on tumorigenicity and tumor burden in a xenograft mouse model. (A,B) 5 3 106 Hep3B cells infected with Ad-shDUSP21 were subcutaneously injected into a single flank in nude mice (n 5 9), while cells infected with Ad-shNC as a control were subcutaneously injected into the opposite flanks of the same mice. Tumor size was examined by serial calibration, where mean tumor volume (6 SEM) (A), and the removed tumor are shown (upper). Tumor weights were statistically analyzed using two-tailed Mann-Whitney rank sum tests (B). (C) 5 3 106 PLC/PRF/5 cells infected with Ad-shDUSP21 were subcutaneously injected into a single flank in nude mice (n 5 10), and the same number of cells infected with Ad-shNC was used as a control. The tumor-free Kaplan-Meier survival curve of these mice was statistically analyzed using a log-rank test. (D,E) Intratumoral injection of Ad-DUSP21 into xenograft tumors derived from PLC/PRF/5 cells was performed. The means of the tumor size (D) and weight (E) with SEM are shown and were statistically analyzed using two-tailed Mann-Whitney rank sum tests, n 5 5. (F) siRNA against DUSP21 mixed with the atelocollagen-mediated synthetic siRNA delivery system was administered by intratumoral injection into nude mice with tumors derived from MHCC-97H cells. The mean with SEM of the tumor weight is shown, and statistical analysis was performed using two-tailed Mann-Whitney rank sum tests, n 5 5.

the cellular events involved in the inhibitory effects of DUSP21 knockdown on HCC cells, we next analyzed cell apoptosis, senescence, and cell cycle progression when DUSP21 was knocked down. Interestingly, upon DUSP21 knockdown, SA-b-gal staining-positive cells and the proportion of cell population in the G1 phase significantly increased (Fig. 4C,D). However, DUSP21 knockdown had no significant impact on apoptosis in Focus and PLC/PRF/5 cells (Supporting Fig. 7E,F). To further confirm the phenotypic changes, we examined some key molecules, including p53, p27, p16, cyclin D1, and cyclin E, which are known to be responsible for the regulation of the cell cycle and/or cell senescence. Interestingly, the resulting data showed that DUSP21 knockdown led to an increase in p53 and p16 as well as to a decrease in cyclin D1 and E (Fig. 4E), supporting the observation of DUSP21 knockdown-mediated G1 arrest and cell senescence.

DUSP21 Knockdown Reduces Tumorigenicity and Tumor Burden in a Xenograft Mouse Model. To investigate the effect of DUSP21 silencing on tumorigenicity in vivo, a recombinant adenoviral vector harboring an shRNA against DUSP21 (Ad-shDUSP21) was constructed and then transfected into Focus, PLC/PRF/ 5, and Hep3B cells (Supporting Fig. 8A). Subsequently, these Focus, PLC/PRF/5, and Hep3B cells were subcutaneously inoculated into the flanks of nude mice, whereas cells infected with the adenoviral vector carrying sh-NC (Ad-shNC) were inoculated into the opposite flank of the same mice as a control. Apart from Focus cells that lacked tumor formation, Ad-shDUSP21 significantly suppressed the tumorigenesis of Hep3B cells, with the size and weight of these xenograft tumors reduced compared to tumors formed from cells infected with Ad-shNC (Fig. 5A,B). More significant, AdshDUSP21 reduced the occurrence of visible tumors from PLC/PRF/5 cells over 2 months, with only 1 in

526

DENG, LI, ET AL.

10 mice bearing tumors from these cells, while the AdshNC infected cells produced tumors in 9 of the same 10 mice. Kaplan-Meier tumor-free survival analysis showed that DUSP21 silencing significantly reduced tumorigenesis (P 5 0.033, Fig. 5C). To further evaluate the antitumor action of AdshDUSP21, a xenograft mouse model with 50-60 mm3 visible tumors, established by the subcutaneous implantation of PLC/PRF/5 cells, was subjected to intratumoral injection with Ad-shDUSP21. Interestingly, AdshDUSP21 significantly reduced tumor volume in five mice compared with the control Ad-shNC (Fig. 5D,E). We sought to evaluate the therapeutic efficacy of siRNA against DUSP21 through an atelocollagenbased nonviral delivery method.25,26 A xenograft mouse model with visible tumors derived from MHCC-97H cells predisposed to metastasis was subjected to intratumoral injection every 3 days for a total of five injections using a mixture of atelocollagen and DUSP21 siRNA. We removed all tumors from the 10 tested mice to assess the relationship between DUSP21 silencing and therapeutic efficacy. The administration of siRNA significantly knocked down the endogenous DUSP21 levels in these xenograft tumors in 5 of the 10 mice tested (Supporting Fig. 8B). Interestingly, the sizes and weights of the tumors with lower DUSP21 levels were significantly reduced compared with those injected with control si-NC (P 5 0.006, Fig. 5F). These collective data suggest that DUSP21 plays a role in HCC cell survival, thereby serving as a potential therapeutic target for HCC treatment. Vanadate Inhibits DUSP21 Phosphatase Activity and HCC Cell Proliferation. To determine whether DUSP21 has phosphatase activity, DUSP21 was expressed and purified using a prokaryotic expression system, and its phosphatase activity was validated in vitro using pNPP as a substrate (Fig. 6A). We further evaluated the effects of 18 known phosphatase inhibitors, including 14 specific and four nonspecific phosphatase inhibitors, on DUSP21 phosphatase activity. The results showed that only vanadate (orthovanadate), a nonspecific phosphatase inhibitor, significantly inhibited DUSP21 phosphatase activity with an IC50 value of 62.5 lM (Fig. 6B,C; Supporting Fig. 9A), which is a relatively high concentration. This result suggested that high-affinity specific inhibitors for DUSP21 phosphatase activity should be further screened and validated. To evaluate whether vanadate also has an inhibitory effect on HCC cells similar to the antiproliferative result of DUSP21 silencing, we next examined the effects of vanadate on HCC cell proliferation. Expectedly, vanadate significantly inhibited cell viability and


colony formation in HCC cells in a dose-dependent fashion, whereas neither sodium fluoride nor sodium pyrophosphate (no more than 500 lM), which were shown to have no inhibitory effects on DUSP21 phosphatase activity (Fig. 6B), had significant effects on cell viability and colony formation of Focus, PLC/ PRF/5, and BEL-7404 cells (Fig. 6D,E; Supporting Fig. 9B,C). Collectively, these data suggested that vanadate could suppress HCC cell proliferation possibly through inhibiting DUSP21 phosphatase activity. DUSP21 Knockdown Plays an Antiproliferative Role Through Activation of p38 Mitogen-Activated Protein Kinase (MAPK) in HCC. Previous studies demonstrated that DUSPs are involved in MAPK signaling cascades.13 Here, we proposed that DUSP21 as an atypical DUSP could also regulate the MAPKs pathway in HCCs, based on data found in STRING, a database tool showing protein interactions (Supporting Fig. 10A). To validate this hypothesis, we first performed a coimmunoprecipitation (Co-IP) assay to determine whether DUSP21 binds to the three classical MAPKs, i.e., p38, JNK, and ERK, in Huh7 cells stably expressing FLAGtagged DUSP21. However, p38, JNK, and ERK were not immunoprecipitated by the FLAG-tagged DUSP21 (Supporting Fig. 10B). Subsequently, we examined the phosphorylation levels of the three MAPKs in HCC cells by western blot assay upon ectopic DUSP21 overexpression, endogenous DUSP21 knockdown, or vanadate treatment. Overexpression of DUSP21 had no obvious impact on the phosphorylation levels of the three MAPKs in Focus and PLC/PRF/5 cells (Supporting Fig. 10C), consistent with a previous report.23 However, upon the vanadate-mediated inhibition of phosphatase activity including DUSP21, the phosphorylation levels of p38, JNK, and ERK were markedly increased whereas the expression of cyclin D1 and cyclin E were significantly decreased in Focus cells in a dose-dependent manner (Supporting Fig. 10D). Intriguingly, upon DUSP21 knockdown, the phosphorylation level of p38, not of ERK or JNK, was observably increased in Focus and PLC/PRF/5 cells (Fig. 7A), and even in Focus cells treated with vanadate or epidermal growth factor (EGF) (Supporting Fig. 10E). Collectively, these observations suggest that DUSP21 knockdown may lead to the activation of the p38 MAPK pathway in HCC cells. To explore the molecular mechanism by which DUSP21 plays a role in the proliferation and survival of HCC cells, we investigated the role of p38 MAPK in HCC proliferation and survival. Our results demonstrated that p38a, a common member of the p38 MAPK family, significantly inhibited colony formation in Focus and PLC/PRF/5 cells (Fig. 7B), consistent


DENG, LI, ET AL.

527

Fig. 6. The effects of vanadate on DUSP21 phosphatase activity and HCC proliferation. (A) The in vitro phosphatase activity of purified DUSP21 was tested using p-nitrophenyl phosphate as a substrate as the volume of DUSP21 protein progressively increased. (B) The effects of three nonspecific phosphatase inhibitors, pyrophosphate, sodium fluoride, and vanadate, at exponentially increasing concentrations on DUSP21 phosphatase activity were analyzed. (C) The inhibitory effect of vanadate on DUSP21 phosphatase activity was evaluated at different concentrations. (D) Cell viability was evaluated in PLC/PRF/5 and Focus cells 3 days after treatment with vanadate, pyrophosphate, and sodium fluoride at a concentration gradient of 0-1,000 lM. (E) Representative photographs of colony formation in which PLC/PRF/5 and Focus cells were treated with different concentrations (10 lM, 25 lM, and 50 lM) of vanadate, pyrophosphate, and sodium fluoride for 3 weeks are shown.

with the previous report that p38a MAPK acts as a suppressor of cell proliferation and tumorigenesis.27,28 To further determine whether the activation of p38 MAPK contributes to the antiproliferative effect of DUSP21 knockdown in HCC cells, we evaluated cell viability and colony formation when both DUSP21 and p38 MAPK were simultaneously silenced by way of RNAi. Interestingly, RNAi against p38 abolished the inhibitory effect of DUSP21 knockdown on viability and colony formation in Focus, PLC/PRF/5, and MHCC-97H cells (Fig. 7C,D; Supporting Fig. 11A). To further confirm this observation, the chemical SB203580, a known p38 MAPK inhibitor, was employed in the same experiments. Expectedly, the inhibitory effect of DUSP21 knockdown on cell viability and colony formation was significantly blocked by the p38 inhibitor SB203580 in Hep3B, Focus, and MHCC-97H cells (Fig. 7E,F; Supporting Fig. 11B). These collective data suggest that the antiproliferative

role of DUSP21 knockdown in HCC is through the activation of the p38 pathway.

Discussion CT antigens have been considered therapeutic targets for many cancers. However, which CT antigens could serve as therapeutic targets remains obscure. It is reasonable that these CT antigens essential for cancer cell survival may serve as potential therapeutic targets. The cell-based RNAi screen has proven to be a powerful approach for characterizing biological features, including cell survival.12 In this study, we employed an RNAi screen against 179 potential CT-X genes to screen for CT genes required for sustaining cell survival. Significantly, we found nine candidates responsible for sustaining cell viability, implying that these genes and protein products could be considered potential therapeutic targets for HCC treatment.

528

DENG, LI, ET AL.


Fig. 7. The role for p38 MAPK in DUSP21-sustained proliferation in HCC. (A) The phosphorylation levels of p38, JNK, and ERK were examined by western blotting assay upon DUSP21 knockdown in Focus and PLC/PRF/5 cells. Data shown are representative of three independent experiments. (B) Overexpressed p38a suppressed colony formation in Focus and PLC/PRF/5 cells. p38a expression was evaluated by western blotting assay (upper), and the histograms (middle) represent the numbers of colonies. Columns, mean (n 5 3); bars, SD. Representative dishes showing colony formation in cells transfected with p38a and pcDNA3.1 (bottom). (C-F) The inhibitory effects of RNAi against DUSP21 on cell viability in Focus, PLC/PRF/5, Hep3B, and MHCC-97H cells (C,E) and colony formation in Focus and MHCC-97H cells (D,F) were neutralized by RNAi against p38 MAPK and the specific p38 inhibitor SB203580 (100 nM), respectively. Columns, mean (n 5 3); bars, SD. Statistical significance was calculated using a two-tailed t test. *P < 0.05; **P < 0.01.

Among these candidates, four are known CT genes. IL13RA1 encodes a subunit of the interleukin 13 receptor, which is a potent therapeutic target for tumors,29 and MAGEA9 and MAGEB6 are members

of the MAGE family and are potential biomarkers in HCC.30,31 CT45 is frequently expressed in lung cancer,21 Hodgkin’s lymphoma,20 myeloma,32 and breast cancers.33 In addition to the above four genes, the


remaining five genes are considered novel CT-X genes and potential biomarkers in HCC. Apart from DUSP21, ZCCHC13 contains six CCHC-type zinc fingers and functions as a transcription factor. PIH1D3, also known as NY-SAR-97, contains a PIH1 domain, which is involved in pre-rRNA processing.34 PNMA5 is a member of the paraneoplastic Ma antigen protein family, whose proteins are implicated in the development of paraneoplastic disorders.35 MPC1L is also termed the mitochondrial pyruvate carrier 1-like protein. Whether these genes could serve as potential therapeutic targets for HCC treatment should be investigated further. In this study, we focused on DUSP21, a member of the atypical DUSP family. The DUSPs, a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within one substrate, are major modulators of some critical signaling pathways that are deregulated in cancer, obesity, diabetes, inflammation, and Alzheimer’s disease.13,36 Thus, DUSPs have increasingly become promising therapeutic targets in drug discovery by way of high-throughput screening and compound-library development efforts. Here, our data indicated that the testis-restricted DUSP21 was upregulated in 30% of HCC specimens. Significantly, the DUSP21 reexpressed in HCC cells could be indispensable for sustaining cell proliferation and survival, possibly through nononcogene addiction, which is a promising therapeutic target for tumors.37 As for the molecular mechanisms involved in the functions of the atypical DUSP family, it has been reported that different atypical DUSP members have varied substrate specificity13. In some cases, the DUSPs (e.g., DUSP19, DUSP22, and DUSP23) may act as scaffold proteins that contribute to the interaction of signaling proteins.13,38 Our data revealed that DUSP21 knockdown resulted in the increased phosphorylation level of p38 in HCC cells, implying that DUSP21 knockdown may trigger p38 activity, even though we detected no interaction between p38 and DUSP21 in our Co-IP assay. However, it is possible that DUSP21 may act as scaffold in its role in the activation of the p38 MAPK pathway in HCC cells. Taken together, based on in vitro and in vivo experiments, we propose that DUSP21 could be a potential therapeutic target for HCC treatment. Acknowledgment: We thank Dr. Da-Li Zheng and Jian Huang for help with data analysis, and Dr. Angel R. Nebreda (Institute for Research in Biomedicine, Spain) for providing us with p38a plasmid.

DENG, LI, ET AL.

529

References 1. Hofmann O, Caballero OL, Stevenson BJ, Chen YT, Cohen T, Chua R, et al. Genome-wide analysis of cancer/testis gene expression. Proc Natl Acad Sci U S A 2008;105:20422-20427. 2. Old LJ. Cancer/testis (CT) antigens — a new link between gametogenesis and cancer. Cancer Immun 2001;1:1. 3. Scanlan MJ, Simpson AJ, Old LJ. The cancer/testis genes: review, standardization, and commentary. Cancer Immun 2004;4:1. 4. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. Cancer/ testis antigens, gametogenesis and cancer. Nat Rev Cancer 2005;5:615625. 5. Stevenson BJ, Iseli C, Panji S, Zahn-Zabal M, Hide W, Old LJ, et al. Rapid evolution of cancer/testis genes on the X chromosome. BMC Genomics 2007;8:129. 6. Ross MT, Grafham DV, Coffey AJ, Scherer S, McLay K, Muzny D, et al. The DNA sequence of the human X chromosome. Nature 2005; 434:325-337. 7. Mellstedt H, Vansteenkiste J, Thatcher N. Vaccines for the treatment of non-small cell lung cancer: investigational approaches and clinical experience. Lung Cancer 2011;73:11-17. 8. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917-924. 9. Han ZG. Functional genomic studies: insights into the pathogenesis of liver cancer. Annu Rev Genomics Hum Genet 2012;13:171-205. 10. Wu LQ, Lu Y, Wang XF, Lv ZH, Zhang B, Yang JY. Expression of cancer-testis antigen (CTA) in tumor tissues and peripheral blood of Chinese patients with hepatocellular carcinoma. Life Sci 2006;79:744-748. 11. Zhao L, Mou DC, Leng XS, Peng JR, Wang WX, Huang L, et al. Expression of cancer-testis antigens in hepatocellular carcinoma. World J Gastroenterol 2004;10:2034-2038. 12. Bai J, Binari R, Ni JQ, Vijayakanthan M, Li HS, Perrimon N. RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance. Development 2008;135:1439-1449. 13. Patterson KI, Brummer T, O’Brien PM, Daly RJ. Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochem J 2009;418:475-489. 14. Zhang ZY. Protein tyrosine phosphatases: structure and function, substrate specificity, and inhibitor development. Annu Rev Pharmacol Toxicol 2002;42:209-234. 15. Almeida LG, Sakabe NJ, deOliveira AR, Silva MC, Mundstein AS, Cohen T, et al. CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens. Nucleic Acids Res 2009;37: D816-819. 16. Jongeneel CV, Delorenzi M, Iseli C, Zhou D, Haudenschild CD, Khrebtukova I, et al. An atlas of human gene expression from massively parallel signature sequencing (MPSS). Genome Res 2005;15:1007-1014. 17. Boutros M, Bras LP, Huber W. Analysis of cell-based RNAi screens. Genome Biol 2006;7:R66. 18. Chang BD, Broude EV, Dokmanovic M, Zhu H, Ruth A, Xuan Y, et al. A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res 1999;59:3761-3767. 19. Wayne CM, MacLean JA, Cornwall G, Wilkinson MF. Two novel human X-linked homeobox genes, hPEPP1 and hPEPP2, selectively expressed in the testis. Gene 2002;301:1-11. 20. Heidebrecht HJ, Claviez A, Kruse ML, Pollmann M, Buck F, Harder S, et al. Characterization and expression of CT45 in Hodgkin’s lymphoma. Clin Cancer Res 2006;12:4804-4811. 21. Chen YT, Scanlan MJ, Venditti CA, Chua R, Theiler G, Stevenson BJ, et al. Identification of cancer/testis-antigen genes by massively parallel signature sequencing. Proc Natl Acad Sci U S A 2005;102:7940-7945. 22. Hahn Y, Lee B. Identification of nine human-specific frameshift mutations by comparative analysis of the human and the chimpanzee genome sequences. Bioinformatics 2005;21(Suppl 1):i186-i194.

530

DENG, LI, ET AL.

23. Hood KL, Tobin JF, Yoon C. Identification and characterization of two novel low-molecular-weight dual specificity phosphatases. Biochem Biophys Res Commun 2002;298:545-551. 24. Rardin MJ, Wiley SE, Murphy AN, Pagliarini DJ, Dixon JE. Dual specificity phosphatases 18 and 21 target to opposing sides of the mitochondrial inner membrane. J Biol Chem 2008;283:15440-15450. 25. Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, et al. Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo. Nucleic Acids Res 2004;32:e109. 26. Takei Y, Takigahira M, Mihara K, Tarumi Y, Yanagihara K. The metastasis-associated microRNA miR-516a-3p is a novel therapeutic target for inhibiting peritoneal dissemination of human scirrhous gastric cancer. Cancer Res 2011;71:1442-1453. 27. Tian K, Yang S, Ren Q, Han Z, Lu S, Ma F, et al. p38 MAPK contributes to the growth inhibition of leukemic tumor cells mediated by human umbilical cord mesenchymal stem cells. Cell Physiol Biochem 2010;26:799-808. 28. Campbell JS, Argast GM, Yuen SY, Hayes B, Fausto N. Inactivation of p38 MAPK during liver regeneration. Int J Biochem Cell Biol 2011; 43:180-188. 29. Kong S, Sengupta S, Tyler B, Bais AJ, Ma Q, Doucette S, et al. Suppression of Human Glioma Xenografts with Second-Generation IL13R-Specific Chimeric Antigen Receptor-Modified T Cells. Clin Cancer Res 2012;18:5949-5960. 30. Zhang Y, Li Q, Liu N, Song T, Liu Z, Guo R, et al. Detection of MAGE-1, MAGE-3 and AFP mRNA as multimarker by real-time quantitative PCR assay: a possible predictor of hematogenous microme-


31.

32.

33.

34.

35.

36.

37. 38.

tastasis of hepatocellular carcinoma. Hepatogastroenterology 2008;55: 2200-2206. Roch N, Kutup A, Vashist Y, Yekebas E, Kalinin V, Izbicki JR. Coexpression of MAGE-A peptides and HLA class I molecules in hepatocellular carcinoma. Anticancer Res 2010;30:1617-1623. Andrade VC, Vettore AL, Regis Silva MR, Felix RS, Almeida MS, de Carvalho F, et al. Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma. Exp Hematol 2009;37: 446-449. Chen YT, Ross DS, Chiu R, Zhou XK, Chen YY, Lee P, et al. Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers. PLoS One 2011;6:e17876. Gonzales FA, Zanchin NI, Luz JS, Oliveira CC. Characterization of Saccharomyces cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA processing. J Mol Biol 2005;346:437455. Schuller M, Jenne D, Voltz R. The human PNMA family: novel neuronal proteins implicated in paraneoplastic neurological disease. J Neuroimmunol 2005;169:172-176. Bakan A, Lazo JS, Wipf P, Brummond KM, Bahar I. Toward a molecular understanding of the interaction of dual specificity phosphatases with substrates: insights from structure-based modeling and high throughput screening. Curr Med Chem 2008;15:2536-2544. Luo J, Solimini NL, Elledge SJ. Principles of cancer therapy: oncogene and non-oncogene addiction. Cell 2009;136:823-837. Zama T, Aoki R, Kamimoto T, Inoue K, Ikeda Y, Hagiwara M. Scaffold role of a mitogen-activated protein kinase phosphatase, SKRP1, for the JNK signaling pathway. J Biol Chem 2002;277:23919-23926.