HOXA13 Promotes Cancer Cell Growth and Predicts Poor Survival of Patients with Esophageal Squamous Cell Carcinoma

Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-4546

Research Article

Zhen-Dong Gu,1 Lu-Yan Shen,1 Hua Wang,1 Xiao-Mei Chen,3 Yong Li,2 Tao Ning,2 and Ke-Neng Chen1

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Departments of 1Thoracic Surgery I, and 2Genetics, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, P.R. China; and 3Department of Gynecology and Obstetrics, Union Hospital of Fujian Medical University, Fuzhou, P.R. China

Abstract proved. Among the reasons are the advanced stage at diagnosis Homeobox genes are known to be classic examples of the in- and the lack of an efficacious system both in understanding its car- timate relationship between embryogenesis and tumorigene- cinogenic mechanism and clinical evaluation, especially the lack of sis. Here, we investigated whether inhibition of HOXA13,a sensitive and specific molecular markers for early detection. member of the homeobox genes, was sufficient to affect the It is known that the process of normal embryogenesis and tu- proliferation of esophageal cancer cells in vitro and in vivo, morigenesis share many of the same pathways, and tumorigenesis and studied the association between HOXA13 expression is an aberrant form of organogenesis (1, 2). Homeobox genes rep- and survival of patients with esophageal squamous cell carci- resent classic examples of the intimate relationship between em- HOX noma (ESCC). HOXA13 expression was permanently knocked bryogenesis and tumorigenesis. The mammalian genes, which HOXA HOXD down using an RNA interference technique, and cell strain are arranged in clusters ( through ) on four separate with stable knockdown of HOXA13 protein was established. chromosomes, encode a subset of transcription factors which reg- Colony formation assay showed that the number of colonies ulate axial regional specification during embryonic development in HOXA13 protein–deficient cells was significantly less than and have recently been shown to be aberrantly expressed in a va- – that of control cells (P < 0.01). Tumor growth in nude mice riety of solid tumors (3 5). HOX showed that the weight and volume of tumors from the genes are expressed with temporal and spatial colinearity – HOXA13 knockdown cells was significantly less than that from in development (6 8). Accordingly, Yahagi and colleagues (9) HOX the control cells (P < 0.01). Then, HOXA13 expression in ESCC showed that genes with high expression in the foregut had specimens and paired noncancerous mucosa was detected by a propensity for low expression in the hindgut, whereas those with immunohistochemistry, and overexpression of HOXA13 was a low expression in the foregut tended to have high expression in HOXA13, HOXB13 found to be more pronounced in ESCCs than paired noncan- the hindgut. Specifically, paralogue 13 ( ,and HOXD13 cerous mucosa (P <0.05).Furthermore,theassociationof ) had a tendency for high expression in the hindgut region HOXA13 expression and disease-free survival time was ana- and weak expression in the foregut including the esophagus. How- HOXA13 lyzed in 155 ESCC cases. The median survival time of patients ever, in our previous study, , a member of paralogue 13, expressing HOXA13 was significantly shorter than HOXA13- was found to be abnormally expressed in esophageal cancerous negative patients (P = 0.0006). Multivariate analysis indicated tissues but not in noncancerous tissues using reverse transcrip- HOXA13 that tumor-node-metastasis (TNM) stage and HOXA13 expres- tion-PCR, suggesting that might play a role in the carci- sion were independent predictors of disease-free survival time nogenesis of the esophageal epithelium (10). In the present study, of patients with ESCC. Our results showed that HOXA13 ex- we further supported the involvement of HOXA13 in esophageal a pression enhanced tumor growth in vitro and in vivo, and cancer growth because of the following evidences: ( ) the abnor- was a negative independent predictor of disease-free survival mal expression of HOXA13 was confirmed by the detection of its b of patients with ESCC. [Cancer Res 2009;69(12):4969–73] expression on protein level in an esophageal cancer cell line; ( ) the rate of colony formation and the capability of tumor growth in cells without HOXA13 expression were much lower than in cells Introduction expressing HOXA13; (c) the expression of HOXA13 protein was Esophageal cancer is the sixth most common cancer in the more abundantly found in ESCC tissues than its noncancerous world. The majority of esophageal squamous cell carcinoma counterparts; (d) further statistical analysis showed that the ex- (ESCC) cases occur in Asia, especially in north central China. Tre- pression of HOXA13 protein was a negative predictor of disease- mendous advances in diagnosis and treatment have been achieved free survival time of patients. recently; however, the survival rate has not been significantly im-

Materials and Methods Note: Supplementary data for this article are available at Cancer Research Online Cell culture and transfections. Human esophageal cancer cell line EC- (http://cancerres.aacrjournals.org/). 109 was cultured in DMEM (Life Technologies) supplemented with 10% Z-D. Gu and L-Y. Shen contributed equally to this work. Requests for reprints: Ke-Neng Chen, Key Laboratory of Carcinogenesis and heat-inactivated fetal bovine serum in a humidified atmosphere with 5% 5 Translational Research (Ministry of Education), Department of Thoracic Surgery I, CO2, at 37°C. EC-109 cells were plated at 70% confluence (∼2×10 cells) Peking University School of Oncology, Beijing Cancer Hospital and Institute. No. 52 in a 24-well plate and grown over night, and transfected with 0.8 μg of ei- Fucheng Road, Haidian District, Beijing 100036, P.R. China. Phone: 86-10-8819-6536; ther short hairpin (shRNA) targeting HOXA13 gene or scrambled shRNA, or Fax: 86-10-8819-6526; E-mail: [email protected]. ©2009 American Association for Cancer Research. empty vector using the Lipofectamine Reagent (Invitrogen). Stably trans- doi:10.1158/0008-5472.CAN-08-4546 fected cells were selected with 600 μg/mL of G418 (Promega). www.aacrjournals.org 4969 Cancer Res 2009; 69: (12). June 15, 2009

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Western blot analysis. Total cell extracts were prepared in 1× SDS load- was used as secondary antibody. Immunohistochemical signals were ing buffer, separated by SDS-PAGE, blotted onto polyvinylidene difluoride, scored by two independent observers. The scores were calculated as the then immunoreacted with goat anti-HOXA13 polyclonal antibody (Santa number of stained cells divided by the total number of cancer cells Cruz Biotechnology) as primary antibody. Rabbit anti-goat polyclonal counted. Four high-power fields (400×) per slide were calculated and the horseradish peroxidase–conjugated IgG was used as a secondary antibody. results were averaged. Unequivocal staining of the cytoplasm in >50% of Immunoreactivity was detected with an enhanced chemiluminescence cancer cells was considered positive. reaction kit (GE Healthcare). As a loading control, glyceraldehyde-3- Statistical analysis. SPSS 10.0 software was used to perform the statis- phosphate dehydrogenase was detected using a goat polyclonal antibody tical analyses. All in vitro experiments were performed at least thrice in (Santa Cruz Biotechnology). triplicates. When the data from different groups were compared, normal RNA interference. For down-regulation of HOXA13 expression in EC- analysis and homogeneity of variance were checked first, and then an un- 109, a plasmid-mediated shRNA was used. The candidate shRNA sequences paired two-tailed t test analysis was used. Bars and error bars on the graphs targeting HOXA13 mRNA were generated by Genechem Com, which were as well as data in the text represent the mean ± SD. The relationship be- cloned into plasmid pGCsi-U6NeoGFP-Blank. The oligonucleotide pairs tween the expression of HOXA13 protein and clinicopathologic character- used were listed in Supplementary Table S1. Empty vector and scrambled istics was tested by χ2 test. Univariate survival analysis was carried out by shRNA were used as controls. Kaplan-Meier method, and subjected to the log rank test. The Cox propor- Colony formation assay. Stable shRNA transfected and control cells tional hazards model with a stepwise procedure was used for multivariate (2 × 103) were cultured in 90-mm plate for 3 weeks in regular culture me- analysis. The variables in the multivariate analysis were age, sex, tumor dium with 600 μg/mL of G418. The formed colonies were then fixed, location, tumor cell differentiation, TNM stage, and HOXA13 expression. stained, and counted. P < 0.05 was considered significant. In vivo carcinogenesis. Stable shRNA transfected cells (1 × 106)in 100 μL serum-free DMEM (Life Technologies) were injected s.c. into the right flank of female nude mice at 18 to 20 g of weight. Scrambled shRNA- Results transfected cells were used as controls. Each group included 12 mice, and Expression of HOXA13 protein in EC-109. We have previously was housed in pathogen-free conditions. After 3 weeks of observation, the reported 11 of 39 HOX genes, including HOXA13, abnormally ex- mice were sacrificed. Tumors were excised, measured, and weighted. All pressed in esophageal cancerous tissues but not in noncancerous experiments were done in accordance with institutional standard guidelines tissues using reverse transcription-PCR (10). In this study, we ob- of Peking University School of Oncology for animal experiments. tained an esophageal cancer cell line EC-109 and further examined Patients and tissue samples. To investigate the difference of HOXA13 protein expression between cancerous and noncancerous tissue, nine ESCC the expression of HOXA13 protein by Western blotting. Endoge- patients were recruited, including eight men and one woman, ages 52 to 71 nous HOXA13 protein expression was detected in EC-109 cells years (median, 62 years), with stage IIa (n = 4), IIb (n = 2), and III (n =3) (Fig. 1A). diseases according to the criteria of the TNM classification system of ma- Down-regulation of HOXA13 protein in EC-109. We intro- lignant tumors (UICC, 1987). The patients underwent radical esophagect- duced plasmids expressing shRNA targeting three different regions omy at the Department of Thoracic Surgery, Peking University School of of HOXA13 into EC-109 cells to knock down the expression of Oncology from February to June, 2003. The samples of cancer tissues and HOXA13 protein. The efficacy of knockdown of HOXA13 protein paired noncancerous mucosa were obtained from the Department of Pa- was examined by Western blotting. Of the three plasmids used, thology of the hospital. None of these patients had received preoperative one worked well, whereas the other two did not. Meanwhile, one adjuvant therapy or suffered from severe postoperative complications, such plasmid with scramble shRNA was used as a control. After screen- as anastomotic leakage, hemorrhage, or chylothorax. ing by G418, we established a cell strain with stable knockdown of To study the association of HOXA13 protein expression and other clin- B icopathologic characteristics and disease-free survival time, 155 ESCC HOXA13 protein following three passages (Fig. 1 ). cases were included, which underwent radical esophagectomy from July Down-regulation of HOXA13 protein decreased cell prolifer- 1996 to November 2002 at the Department of Thoracic Surgery, Peking ation in vitro. To investigate if down-regulation of HOXA13 pro- University School of Oncology, and were selected using the same criteria tein could modulate cell proliferation, a colony formation assay described above. The selected cases involved 117 men and 38 women, ages was performed. The number of colonies formed (456 ± 7) in cells 39 to 78 years (median, 63 years), with stage I (n = 6), IIa (n = 62), IIb (n = with stable knockdown of HOXA13 protein was significantly re- n n 13), III ( = 72), and IV ( = 2) diseases. All the specimens had been rou- duced, as compared with that of the control (983 ± 78; Fig. 1C, μ tinely formalin-fixed, paraffin-embedded, and serially sectioned at 4 min P = 0.0003). The level of colony formation in parental cells (812 ± thickness. 100) showed no significant difference compared with that of the The clinical data were obtained from hospital records because all post- P operative patients were routinely scheduled for a regular physical examina- control ( = 0.495). tion (visiting our hospital) for life-long follow-up. The calculation of Down-regulation of HOXA13 protein decreased tumor disease-free survival time began at the date of surgery and ended when growth in vivo. After studying the effect of HOXA13 protein on any of the following events happened: recurrence, metastasis, or oncologi- cell proliferation in vitro, the involvement of HOXA13 protein in cal death. All patients in this study had recorded disease-free survival carcinogenesis in vivo was investigated. HOXA13 knockdown cells, times. This study was approved by both the Ethics and the Academic com- control cells, and parental cells were injected into nude mice. The mittees of Peking University School of Oncology, and informed consent was tumors were removed 3 weeks after injection and were measured obtained from all participants. and weighted. The weight and volume of tumors from the HOXA13 Immunohistochemistry. After routine deparaffinization and hydration, knockdown cells were 154.3 ± 71.1 mg and 0.249 ± 0.142 cm3, re- tissue sections were treated with 3% hydrogen peroxide and then heated in spectively, which were significantly less than those of the control EDTA (pH 8.0) for antigen retrieval. The HOXA13 antigen-antibody reaction 3 D P took place overnight at 4°C, following goat serum blocking. The streptavi- (482.2 ± 90.5 mg and 0.718 ± 0.209 cm , respectively; Fig. 1 ; < din/peroxidase amplification kit (Zymed) was applied to detect the signal 0.01). However, no difference was found between the weight and of the HOXA13 antigen-antibody reaction. Peroxidase activity was devel- volume of tumors from parental cells and those of the control. oped with diaminobenzidine. All sections were counterstained with hema- Expression of HOXA13 protein detected by immunohisto- toxylin. The purified rabbit polyclonal antibody against human HOXA13 chemistry in ESCC and paired noncancerous tissue. The above (Abcam) was used at 2 μg/mL and goat anti-rabbit biotin-conjugated IgG results suggested that HOXA13 protein might exert an effect on the

Cancer Res 2009; 69: (12). June 15, 2009 4970 www.aacrjournals.org

Univariate analysis using the log rank test showed that the expression of HOXA13 was significantly associated with disease-free survival time (P = 0.0006, Table 2; Fig. 3). The median survival time was 14 months for HOXA13-positive patients, which was significantly shorter than the 26 months for HOXA13-negative patients. Multivariate analysis showed that TNM stage and HOXA13 expression were independent poor predictors of disease-free survival time in this series of 155 patients (Table 3). In addition, we attempted to investigate the correlation between HOXA13 and its known downstream target genes, Enpp2 (Innis group) or Bmp2, Bmp7 (Stadler group), or Epha7 (Stadler or Zappavigna group). Seventy-eight of 155 cases were studied immunohisto- chemically. Twenty-seven of 78 cases were HOXA13-positive and 51 cases were HOXA13-negative. There is no concordant expression between HOXA13 and Bmp2, Bmp7, or Epha7 (Supplementary Table S2). The reason for this phenomenon is unknown. The expression of Enpp2 could not be determined because Enpp2 antibody is not commercially available.

Discussion Genes regulating normal cell proliferation and differentiation participate in tumorigenesis and/or tumor progression because of their deregulated function or interaction with deregulated target genes. Among the principal examples are homeobox genes, which encode transcriptional regulatory proteins. Those proteins play important roles during embryonic development and seize function in Figure 1. Down-regulated HOXA13 protein decreased cell proliferation in vitro mature organs. However, the aberrant expression is often found in and in vivo. A, expression of HOXA13 protein in EC-109. Lanes 1 and 2, lysates of EC-109 cell line; lanes 3 and 4, lysates of esophageal epithelial cells of mice. The molecular weight of HOXA13 is 43 kDa. B, down-regulation of HOXA13 protein in EC-109. Lanes 1 and 8, the parental cells of EC-109; lanes 2, 4, and 6, cell strains with stable knockdown of HOXA13 protein; lanes 3, 5, and 7, controls (for details, see full-length blot in Supplementary Fig. S1). C, down-regulation of HOXA13 protein decreased cell proliferation in vitro. Left, colonies formed by cell strains with a stable knockdown of HOXA13 protein; right, colonies formed by control cells. Cells with stable knockdown HOXA13 protein formed significantly fewer colonies, compared with that of the controls. D, down-regulation of HOXA13 protein decreased tumor growth in vivo. Top, tumors from the control cells; bottom, tumors from cells with stable knockdown of HOXA13 protein. The weight and volume of tumors from the HOXA13-knockdown cells were significantly lower than those from the control cells (P < 0.01).

cell proliferation of esophageal cancer cells. To further elucidate the expression of HOXA13 protein in esophageal cancer, we initial- ly used formalin-fixed, paraffin-embedded noncancerous colon and rectum mucosa specimens as positive controls. It was noted that HOXA13 protein was localized in the cytoplasm of colorectal epithelium (data not shown). As a transcription factor, HOXA13 was reported to show nuclear staining by Knosp and colleagues (11). However, we consistently detected it in the cytoplasm of esophageal cancer cells (Fig. 2). It is speculated that the cytoplasmic localization may be due to the modulation of nuclear localization signals, nuclear export sequences (12, 13), and interaction with cytoplasmic anchoring factor (12, 14–17). In the current study, HOXA13 protein showed increased expression in cancerous tissue in seven of nine patients (77.8%), as compared with paired noncancerous tissues in one of nine patients with ESCC (11.1%; Fig. 2; P = 0.015). Figure 2. Expression of HOXA13 protein in ESCC and paired noncancerous Association of HOXA13 protein expression with clinical tissues. A, positive control (×100), using established HOXA13-positive ESCC characteristics and disease-free survival. The expression of tissue. B, negative control (×100), with primary antibody replaced by PBS. C, high expression of HOXA13 in ESCC (case no. 68698). Left, ×40; right, HOXA13 protein was not significantly associated with clinical ×200. D, negative expression in paired noncancerous tissue (case no. 68698). characteristics (Table 1). Left, ×40; right, ×200. www.aacrjournals.org 4971 Cancer Res 2009; 69: (12). June 15, 2009

HOX Table 1. Association between HOXA13 expression and To elucidate these questions, further and detailed studies of clinical characteristics of patients with ESCC (n = 155) genes function are warranted. In our previous studies, we detected the expression of HOX gene Clinicopathologic HOXA13 P family members in esophageal cancer and paired noncancerous tis- characteristics expression no. (%) sue. Eight of 39 HOX genes were detected in cancerous tissue, but not in paired noncancerous tissue, which were HOXA10, HOXA13, Positive Negative HOXB7, HOXC4, HOXC8, HOXD9, HOXD10, and HOXD13 (10). Inter- estingly, according to the “spatial colinearity” of HOX genes which is Age (y) displayed in development, HOXA13 and HOXD13 are expressed spe- ≤ 60 12 (18.5) 53 (81.5) 0.913 cifically in the cloacal mesoderm and also uniquely in the hindgut >60 16 (17.8) 74 (82.2) and cloacal endoderm, but not in the esophagus (21, 22). Thus, the Sex HOXA13 Male 22 (18.8) 95 (81.2) 0.675 up-regulation of in esophageal cancer tissue implies that it Female 6 (15.8) 32 (84.2) might play a crucial role in carcinogenesis. Tumor location In order to further investigate the role of HOXA13 in esophageal Upper 3 (21.4) 11 (78.6) 0.656 carcinogenesis, we evaluated the effect of HOXA13 expression on Middle 19 (19.6) 78 (80.4) esophageal cancer cell proliferation. EC-109 is a HOXA13-expressing Lower 6 (13.6) 38 (86.4) esophageal cancer cell line. Using RNA interference technique, the Tumor cell differentiation expression of HOXA13 was down-regulated. A cell strain with stable Well 17 (19.8) 69 (80.2) 0.474 knockdown of HOXA13 protein was selected and used for the sub- Moderate 10 (18.5) 44 (81.5) sequent experiments. The colony formation ability of HOXA13 pro- Poor 1 (6.7) 14 (93.3) tein expression was examined in vitro. The results suggested that Tumor invasion (T) cells with a stable knockdown of HOXA13 proteins showed a signif- T1 0 (0) 7 (100.0) 0.445 icantly weaker colony formation ability, compared with the con- T2 4 (13.8) 25 (86.2) T3 21 (19.4) 87 (80.6) trols. In order to validate this observation, we injected the cells T4 3 (27.3) 8 (72.7) and control cells into nude mice to test their tumor formation abil- Lymph nodes metastasis (N) ity and growth in vivo. Consistently, the tumor formation ability of N0 10 (13.5) 64 (86.5) 0.159 the cells with HOXA13 protein knocked-down was significantly N1 18 (22.2) 63 (77.8) weaker than that of the controls. All of these results indicate that TNM stage the expression of HOXA13 protein is involved in carcinogenesis I 0 (0) 6 (100.0) 0.388 and growth of ESCC. This result is consistent with those reports IIa 9 (14.5) 53 (85.5) implying the role of HOX gene members in carcinoma. Whelan IIb 2 (15.4) 11 (84.6) III 16 (22.2) 56 (77.8) and colleagues (23) found that HOXA9 overexpression induced IV 1 (50.0) 1 (50.0) IGF-1R expression and subsequently promoted leukemia cell proliferation. Beslu and colleagues (24) reported that HOXB4 overexpression up-regulated the growth of hematopoietic stem cells. Transfection of HOXB7 into breast cancer cell line SkBr3 stimulated cancers (3). The products of homeobox genes are transcription fac- the amplification and colony formation ability of cancer cells (25), tors that regulate gene expression. The homeodomain is deemed as the functional motif of homeobox genes, which bind to specific DNA sequences, including promoters of other HOX genes, to en- hance or inhibit their transcription. It has been shown that HOX genes are abnormally expressed in many solid tumors and deriva- tive cell lines, as well as the corresponding embryonic tissues from which these tumor cells are derived (18–20). However, there is no detailed report thus far describing the function of HOX genes in cancer. Do HOX genes play a role in the carcinogenesis or growth of ESCC? Is the expression of HOX genes linked to the survival of patients, and does it have potential to be used as a clinical bio- marker of ESCC, and direct the patient-specific treatment strategy?

Table 2. HOXA13expressioninESCCtissueand association with disease-free survival time (n = 155)

Item No. (%) Median survival P time, mo (95% confidence interval)

HOXA13 + 28 (18.1) 14 (9–19) 0.0006 − 127 (81.9) 26 (19–33) Figure 3. Kaplan-Meier survival curves for 155 patients with or without HOXA13 expression. The survival of patients with HOXA13 expression was significantly shorter than that of patients without HOXA13 expression (P = 0.0006).

Cancer Res 2009; 69: (12). June 15, 2009 4972 www.aacrjournals.org

Table 3. Independent predictors of disease-free survival Because the final goal of these diagnosis and treatment strategies time in multivariate analysis (n = 155) is to improve the survival of patients, it is urgent to clarify if the promotion of carcinogenesis and growth from HOXA13 proteins ex- Variables Hazard ratio P erts any effect on the survival of patients. To investigate this, we (95% confidence interval) first confirmed that the overexpression of HOXA13 protein did exist in the cancer tissue of patients with ESCC, then we further testified TNM stage 0.006 that this overexpression did affect the survival; the survival of patients Stage I vs. stage IV 0.152 (0.021–1.089) 0.061 with HOXA13 overexpression was significantly shorter than that of Stage IIa vs. stage IV 0.445 (0.107–1.847) 0.265 patients without HOXA13 overexpression. HOXA13 expression, like Stage IIb vs. stage IV 0.296 (0.063–1.382) 0.121 TNM stage, was a negative, independent predictor of disease-free – Stage III vs. stage IV 0.741 (0.181 3.041) 0.677 survival time of patients with ESCC. Thus, combination of HOXA13 HOXA13 expression 0.003 expression and TNM stage classification might provide a more ac- Negative vs. positive 0.504 (0.322–0.787) 0.003 curate prediction of the postoperative outcome of patients with ESCC. Because HOXA13 is involved in the tumorigenesis of ESCC, it may provide a new aspect for drug development. and HOXB7 overexpression increased the proliferation ability of im- mortalized ovary epithelial cells (26). There are reports demonstrat- ing the role of HOXA13 in neoplasms other than solid tumors. Disclosure of Potential Conflicts of Interest NUP98-HOXA13 Taketani and colleagues (27) found the fusion gene No potential conflicts of interest were disclosed. in patients with acute myelogenous leukemia suggesting that HOXA13, like HOXA9, might play an important role in the pathogen- esis of leukemia. In addition, the deregulated expression of HOXA13 Acknowledgments was found in glioblastoma tissue (28), and melanoma (29). However, Received 12/2/08; revised 3/15/09; accepted 4/22/09; published OnlineFirst 6/2/09. to date, there is no report regarding the function of HOXA13 in solid Grant support: National Natural Science Foundation of China (grant no. tumors. Our results showed for the first time that HOXA13 overex- 30572130), and Capital Medical Development Research Fund (grant no. 2005-2020). The costs of publication of this article were defrayed in part by the payment of pression is involved in carcinogenesis and promoted tumor growth page charges. This article must therefore be hereby marked advertisement in accor- in ESCC. dance with 18 U.S.C. Section 1734 solely to indicate this fact.

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www.aacrjournals.org 4973 Cancer Res 2009; 69: (12). June 15, 2009

HOXA13 Promotes Cancer Cell Growth and Predicts Poor Survival of Patients with Esophageal Squamous Cell Carcinoma

Zhen-Dong Gu, Lu-Yan Shen, Hua Wang, et al.

Cancer Res 2009;69:4969-4973. Published OnlineFirst June 2, 2009.

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