3593 DJ-1 Could Predict Worse Prognosis in Esophageal Squamous Cell Carcinoma Hiu-Fung Yuen,1,3 Yuen-Piu Chan,1 Simon Law,2 Gopesh Srivastava,1 Mohamed El-tanani,3 Tak-Wah Mak,4,5 and Kwok-Wah Chan1 Departments of 1Pathology and 2Surgery, The University of Hong Kong, Hong Kong, People’s Republicof China; 3Centre for Cancer Research and Cell Biology, Queen’s University of Belfast, Belfast, United Kingdom; and 4Advanced Medical Discovery Institute, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute; 5Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Abstract Recent studies have revealed an oncogenic role of DJ-1 promotes ESCC progression through phosphatidylino- through its ability to transform normal cells, prevent sitol3-kinase pathway and modulationof apoptosis, oxidative damage, and inhibit apoptosis. However, we performed immunohistochemistry of pAkt and its role in esophageal squamous cell carcinoma (ESCC) Daxx. We found that DJ-1 expression was significantly is unknown. In this study, by immunohistochemistry, associated with pAkt, whereas nuclear DJ-1 expression we analyzed the expression of DJ-1 in 81 ESCC tumors, was significantly correlated with nuclear expression 31 paired nonneoplastic esophageal epithelia, and 19 of Daxx. These results suggest that phosphatidyli- paired ESCC lymph node metastases. We found nositol3-kinase pathway and Daxx-regulatedapoptosis that cytoplasmic DJ-1 expression was significantly might be important in DJ-1-mediated ESCC progres- higher in ESCC and ESCC lymph node metastases sion. By using multivariate Cox regression, we fur- P than in nonneoplastic esophageal epithelium. ESCC ther showed that T4 stage ( =0.003)andDJ-1 specimens with high distant metastatic potentialalso (P = 0.034) are independent predictors of patient had a significantly higher level of nuclear DJ-1 survival. In conclusion, our results suggest that DJ-1 expression (P = 0.018). By Kaplan-Meier analysis, plays a very important role in transformation and we found that a high level of nuclear DJ-1 was sig- progression of ESCC and may be used as a prognostic nificantly associated with poorer patient survival in marker in ESCC. (Cancer Epidemiol Biomarkers Prev our cohort (P = 0.028). To investigate whether DJ-1 2008;17(12):3593–602) Introduction Esophageal squamous cell carcinoma (ESCC) is common for its antiapoptoticand transformation ability (5), among Asian populations. Despite recent advances in whereas DJ-1 also interacts with and inhibits the func- the detection of premalignant lesions and the develop- tion of Daxx, which binds to apoptosis signal-regulating ment of combination therapies, its incidence is increas- kinase 1 to promote apoptosis induced by oxidative ing and its outcome remains poor (1). Proper clinical stress (6). The antioxidative activity of DJ-1 also links to management should be given to patients with aggres- the ability of DJ-1 to induce Nrf2 stability, which in turn sive ESCC, which might improve survival. Identification activates the transcription of antioxidant and detoxifi- of prognosticmarkers for ESCC might help to make the cation enzymes (7). In another study, DJ-1 has been decision on which treatment regimen is the most suitable shown to up-regulate glutathione synthesis, which is a for a patient in terms of quality of living. downstream target of Nrf2, to protect cells from oxida- DJ-1 has been identified as an oncogene that can tran- tive stress-induced apoptosis (8). More recently, DJ-1 sform mouse NIH3T3 cells together with ras (2). Its has been shown to decrease the expression of Bax and oncogenic effect is partly attributed to its antiapoptotic inhibit caspase activation, suggesting a direct role of ability. DJ-1 is an important factor governing apoptotic DJ-1 in inhibiting the apoptotic pathway (9). In cancer response in neuronal cells. It has been shown to pro- cells, decreased expression of DJ-1 enhances apoptosis tect neuronal apoptosis, whereas decreased expression in prostate (10) and lung cancer cells (9, 11). These lines of DJ-1 increases apoptosis of neuronal cells to various of evidence suggest that DJ-1 is important in regulat- stresses (3, 4). Recent functional studies on DJ-1 revealed ing apoptosis and hence plays an important role in several important mechanisms on how it inhibits apop- carcinogenesis. tosis. Sumoylation of the K130 residue of DJ-1 is essential The roles and prognostic significance of DJ-1 have been studied in certain types of cancer. In breast cancer patients, serum level of DJ-1 is increased when compared Received 3/10/08; revised 8/4/08; accepted 9/5/08. with healthy subjects (12). DJ-1 negatively correlates with Note: Supplementary data for this article are available at Cancer Epidemiology the expression of a potent tumor suppressor, PTEN, in Biomarkers and Prevention Online (http://cebp.aacrjournals.org/). human breast cancer specimens (13). DJ-1 is increased in Requests for reprints: Kwok-Wah Chan, Department of Pathology, Queen Mary primary lung cancer (11) and a high level of DJ-1 is Hospital, Hong Kong, People’s Republicof China. Phone: 852-28554874; Fax: 852-28725197. E-mail: [email protected] correlated with poor prognosis of lung cancer patients Copyright D 2008 American Association for Cancer Research. (13). Reduced DJ-1 sensitizes prostate cancer cells toward doi:10.1158/1055-9965.EPI-08-0214 apoptotic-inducing agents (10). DJ-1 has been shown to Cancer Epidemiol Biomarkers Prev 2008;17(12). December 2008 Downloaded from cebp.aacrjournals.org on October 5, 2021. © 2008 American Association for Cancer Research. 3594 DJ-1 in Esophageal Squamous Cell Carcinoma be an androgen receptor coactivator (14), which interacts Table 1. Patient clinical and pathologic features with androgen receptor, and is up-regulated in human No. (%) Median prostate cancer on androgen deprivation therapy (15). cases (range) These results strongly suggest that DJ-1 promotes prostate cancer progression. Recently, DJ-1 has also been Age (y) 81 67 (41-87) suggested to confer prognostic value to ovarian carcino- Sex ma with effusions (16). Male 62 (77) Female 19 (24) There has not been any report concerning the T stage significance of DJ-1 in ESCC. In this study, we analyzed, T1 2 (3) by immunohistochemistry, the expression of DJ-1 in 81 T2 13 (16) ESCC specimens with 31 paired nonneoplastic esopha- T3 49 (61) geal epithelium and 19 paired ESCC lymph node T4 17 (21) N stage metastasis. The expression of DJ-1 was then correlated N 30 (37) with the clinicopathologic data of the patients. In 0 N1 51 (63) addition, as DJ-1 has been shown to promote cell sur- M stage vival through the phosphatidylinositol 3-kinase (PI3K) M0 68 (84) pathway by modulating PTEN and Akt activity (13, 17) M1 13 (16) and apoptoticresponse through Daxx (6), we further pTNM staging Stage II 32 (40) investigated the association between DJ-1 and pAkt or Stage III 36 (44) Daxx in the ESCC tumor specimens. Stage IV 13 (16) Histologicgrade Well-differentiated 13 (16) Materials and Methods Moderately differentiated 48 (59) Poorly differentiated 20 (25) Patients and Specimens. Our cohort consists of 81 Metastaticstatus Nonmetastatic55 (68) ESCC Chinese patients who underwent total esophagec- Metastatic(within 1 y) 26 (32) tomy for ESCC in Queen Mary Hospital from 1998 to 2005. The esophagectomy specimens were formalin-fixed and paraffin-embedded for routine histopathologicdi- night at 4jC. pAkt (Ser473) antibody from Cell Signaling agnosis. The specimens were collected consecutively. and Daxx antibody from Upstate were applied at a Patients who had received prior treatment directed dilution of 1:50 and 1:100, respectively, for overnight against ESCC or specimens with insufficient tumor tis- at 4jC. The primary antibody was omitted in the nega- sue for microarray incorporation or subsequent immu- tive control. nohistochemical study were excluded from this study. The extent of tumor at the time of operation was class- Evaluation of Immunohistochemical Staining ified according to the tumor-node-metastasis (TNM) Results. The stained sections were reviewed by two staging system, whereas tumor grades were classified independent observers (K.W.C. and Y.P.C.) who had no based on the WHO classification (18). The clinical data prior knowledge of the clinicopathologic data of the were retrieved and are summarized in Table 1. Adjuvant patients. All three cores of each specimen were scored therapies were given to the patient in a ‘‘curative intent’’ and the ‘‘hot spot’’ core was selected for later statistical manner despite the heterogeneity of the treatment. analysis. Cytoplasmicstaining was graded by an Paired nonneoplasticesophageal epithelium was selected arbitrary scale based on the extent and the intensity of from the upper resection margin of esophagectomy for the whole tissue core. A cytoplasmic staining score was comparison with tumor. The ESCC tumors were given, ranging from 0 to 3, which represent negative, regarded as having a high distant metastaticpotential if weak, moderate, and strong staining, respectively. For distant metastasis could be detected within 1 year of nuclear staining, nuclei from the whole tissue core esophagectomy, else they were considered to have a low were counted and only those cores with >100 nuclei of distant metastaticpotential. The mean follow-up time of target cells were submitted for statistical analysis. The our patients is 14.5 months (range, 0.7-65.2 months). DJ-1 nuclear score was then divided into four groups according to the percentage of cells positively stained as Tissue Microarray Construction. Tissue microarray negative (V5% nuclear stained positive), weak (>5% and (TMA) was constructed with a Beecher Instruments V20%), moderate (20% and V50%), and strong (z50%). tissue microarrayer as described previously (19). Briefly, For pAkt and Daxx, their nuclear score was respectively three representative areas of each specimen were selected graded into four groups as negative (V5% nuclear stained by a pathologist (K.W.C.) and transferred to a TMA block positive), weak (>5% and V20%), moderate (20% and using a 0.6 mm core diameter needle.