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- Open Access
Nuclear expression of TCF4/TCF7L2 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma
© The Author(s) 2016
- Received: 30 October 2015
- Accepted: 7 December 2015
- Published: 28 July 2016
The prognosis for patients with esophageal cancer remains poor. Therefore, the identification of novel target molecules for the treatment of esophageal cancer is necessary. Here, we investigated the clinicopathological significance of transcription factor 4/transcription factor 7-like 2 (TCF4/TCF7L2) in resectable esophageal squamous cell carcinoma (ESCC), because TCF4/TCF7L2 expression has not been studied in esophageal cancer previously.
This study included 79 patients with esophageal cancer who underwent surgery between 1998 and 2005. The expression of the TCF4/TCF7L2 protein in the nucleus of esophageal cancer cells was analyzed using immunohistochemistry. We examined the correlation between TCF4/TCF7L2 expression, clinicopathological factors, and prognosis in patients with ESCC.
TCF4/TCF7L2 was expressed in 57 % (45/79) of patients. TCF4/TCF7L2 expression was correlated with T factor (T1 vs. T2-4, p = 0.001), stage (I vs. II-IV, p =0.0058), Ly factor (p =0.038), and V factor (p =0.038) and did not correlate with age, gender or N factor. Furthermore, patients who were positive for TCF4/TCF7L2 had a significantly lower survival rate than those who were negative for TCF4/TCF7L2 (log-rank test, p = 0.0040). TCF4/TCF7L2 expression significantly affected the survival of patients with ESCC. Positive expression of TCF4/TCF7L2 was correlated with a poor prognosis after a curative operation in patients with ESCC.
- Esophageal cancer
- Wnt signal
- Clinicopathological factor
The prognosis of patients with esophageal cancer remains poor, emphasizing the need for the development of new treatment strategies. Today, the overall 5-year survival rate is less than 50 %, despite the use of multimodal therapies. Even in early-stage disease, many patients develop a local recurrence of tumors or distant metastasis within a short period of time after operation. To develop novel treatment strategies, it is important to understand the biological behavior of esophageal cancer. Recent studies identified several genes and molecules involved in the origin and/or progression of esophageal cancer, including TP53 , deleted in esophageal cancer 1 (DEC1) , deleted in colorectal cancer (DCC) , deleted in lung cancer 1 (DLC1) , cyclin D1 , adenomatous polyposis coli (APC) , and survivin . However, the precise mechanisms that underlie the development and progression of esophageal squamous cell carcinoma (ESCC) remain unclear.
The Wnt signaling pathway regulates important cellular processes, including development and differentiation, apoptosis, immunologic and inflammatory responses, cell-cycle progression and cellular division [8, 9]. Transcription factor 4/transcription factor 7-like 2 (TCF4/TCF7L2) is a key molecule of the Wnt signaling pathway, which acts as a transcriptional factor in the nucleus [8, 10]. Downstream genes of the Wnt signaling pathway include cyclin D1 and c-myc. To the best of our knowledge, no reports have described the clinicopathological significance of TCF4/TCF7L2 protein expression in the progression of various malignancies.
In this study, we investigated the clinicopathological significance of TCF4/TCF7L2 protein expression in 79 patients with resectable ESCC.
Samples were obtained from 79 patients with ESCC who underwent operation at the Department of Gastroenterological Surgery, Nagoya City University Medical School between 1998 and 2005 without pre-operative chemotherapy or radiation. The tumors were classified according to the guidelines for clinical and pathological studies on carcinoma of the esophagus. The samples were used after obtaining written consent from the patients.
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded primary human ESCC tissues using the monoclonal anti-TCF4 antibody (Cell Signaling, NY) at 1:200. Paraffin-embedded tumor sections were deparaffinized, rehydrated, heat-treated by microwaving in 10 mM citrate buffer for 15 min for antigen retrieval, and cooled to room temperature. The sections were then treated with 0.3 % H2O2 in methanol for 30 min to neutralize the endogenous peroxidases, blocked with nonspecific goat serum for 10 min, and incubated with the H-100 antibody overnight at room temperature in a humid chamber. The immunoreactive protein was detected with a DAKO Envision System, HRP (DAB), and sections were counterstained with hematoxylin. Two independent investigators subjectively assessed the immunostaining of TCF4, and discordant results were resolved by consultation with a third investigator. For the evaluation of TCF4 expression, immunostaining was considered positive only when unequivocally strong nuclear staining was present in more than 50 % of the tumor cells, as analyzed using a light microscope. Cases with faint staining only were considered negative.
The chi-squared test was used to analyze the correlations between the clinicopathological factors and the expression of TCF4/TCF7L2. The survival rates were calculated according to the Kaplan-Meier method. Multivariate analysis of Cox’s proportional hazard risk model was used to obtain the conditional risk of death due to ESCC. Differences were considered statistically significant for P values less than 0.05.
Expression of TCF4/TCF7L2 in ESCC
Correlation of TCF4 IHC in esophageal cancer with clinicopathological factors, including patient and tumor characteristics
No. of patients (n = 79)
Age at surgery
T1 vs T2-4
Lymph node status
N0 vs N1
I vs II-IV
Blood vessel invasion
Survival curves and expression of TCF4/TCF7L2
Univariate analysis showed that, among the clinicopathological factors examined in this study, the extent of primary tumor (risk ratio, 4.184; p < 0.0001), lymph node metastasis (risk ratio, 4.149; p < 0.0001), lymphatic invasion (risk ratio, 6.622; p = 0.003), vein invasion (risk ratio, 2.816; p = 0.0003), and immunostaining for TCF4/TCF7L2 (risk ratio, 2.506; p = 0.0049) were statistically significant prognostic factors. Multivariate analysis revealed that TCF4/TCF7L2 expression was not an independent prognostic factor (data not shown).
The Wnt signaling pathway plays important roles in axis formation during early vertebrate development . Upon Wnt signaling, the phosphorylation of beta-catenin is suppressed through undefined mechanisms, and beta-catenin functions as a transcriptional regulator in the nucleus together with TCF4/LEF1 [12, 13]. A number of downstream genes, such as c-myc , cyclin D1 [14, 15], c-jun, fra-1, uPAR, ZO-1 , and novel protein band 4.1 like 4 (NBL4)  have been reported; however, the precise regulatory mechanisms remain to be resolved. Alterations of APC, AXIN, or beta-catenin itself, lead to the accumulation of beta-catenin in the cytoplasm and/or nucleus, resulting in the unregulated transcription of downstream genes [12–14, 16–18]. However, in esophageal cancer cells, the frequency of beta-catenin accumulation in the nucleus is lower than in colon and liver cancer 
TCF4/TCF7L2 is a major component of the Wnt signaling pathway. However, because few reports have described the mechanisms mediating Wnt signaling activation in ESCC, the factors that regulate TCF4/TCF7L2 expression in this type of cancer are not known.
In many cancer cells, TCF4/TCF7L2 is localized to the nucleus . Consistent with this observation, our current experiments show that TCF4/TCF7L2 is also expressed in the nucleus of ESCC cells (Fig. 1). In colon cancer cells, TCF4/TCF7L2 is located in the nucleus with beta-catenin . By contrast, for esophageal squamous cancer cells, our data suggest that TCF4/TCF7L2 alone is located in the nucleus without beta-catenin, because beta-catenin is not detected in the nucleus in ESCC . However, the mechanisms that regulate TCF4/TCF7L2 expression in ESCC remain unclear.
There are a few reports that the Wnt signaling pathway is activated in ESCC. Cyclin D1, a downstream gene of the Wnt signaling pathway, is highly expressed in ESCC [21, 22]. Other mechanisms of translocation to the nucleus for TCF4/TCF7L2 may exist. Downstream genes of the Wnt signal pathway in esophageal cancer may be activated by TCF4/TCF7L2 activation.
Because TCF4/TCF7L2 plays a role in cancer proliferation, additional studies are necessary to determine whether TCF4/TCF7L2 contributes to the growth of esophageal cancers. In our study, TCF4/TCF7L2 was correlated with the T factor of patients with ESCC (Table 1).
Interestingly, we found that TCF4/TCF7L2 predicted the prognosis of patients with ESCC. Therefore, our data suggest that TCF4/TCF7L2 is involved in the cell proliferation of esophageal carcinoma and that TCF4/TCF7L2 is a useful biomarker for predicting prognosis in patients with ESCC.
Several clinical studies have reported that TCF4/TCF7L2 is an indicator of poor prognosis or malignant potential in hepatocellular carcinomas  and colon cancer . The current study may be the first report demonstrating that TCF4/TCF7L2 is correlated with the prognosis of patients with esophageal squamous cell carcinoma.
95 % CI
Age at surgery
Lymph node metastasis
Immunostaining for TCF4
Additionally, whether TCF4/TCF7L2 expression is mediated by other mechanisms will be the focus of future studies.
In patients with esophageal cancer, many prognostic markers, including cyclin D1, E-cadherin, and MDM2, have been reported [21, 23]. Furthermore, we also reported that pituitary tumor transforming gene 1 (PTTG1) , DNA fragmentation factor 45 (DFF45) , NOTCH1 , VEGF-C , and DROSHA  may be prognostic markers of ESCC. Therefore, TCF4/TCF7L2 represents an additional potential prognostic indicator for patients with ESCC.
Although the precise molecular mechanisms through which TCF4/TCF7L2 is activated must be clarified, our data clearly indicate that TCF4/TCF7L2 may be a molecular target for the development of effective therapeutic agents for patients with esophageal cancer.
APC, adenomatous polyposis coli, DCC, deleted in colorectal cancer, DEC1, deleted in esophageal cancer 1, DFF45, DNA fragmentation factor 45, DLC1, deleted in lung cancer 1, ESCC, esophageal squamous cell carcinoma, NBL4, novel protein band 4.1 like 4, PTTG1, pituitary tumor transforming gene 1, TCF4/TCF7L2, transcription factor 4/transcription factor 7-like 2
The authors would like to thank Ms. Seiko Inumaru for her excellent technical assistance.
Availability of data and materials
The authors state that the data and materials can be available.
HI; leader of this research, immunostainig, statistics. TW; Patients’ characterization. YT; Patients’ characterization. NS; Obserber of immunostainig. TT; Obserber of immunostainig. HS; Patients’ characterization. TO; immunostaing. HT; Proofreading of this paper. All authors read and approved the final manuscript.
The authors have no proprietary or commercial interest in any product mentioned or concept discussed in this article.
Ethics approval and consent to participate
The authors declare that they have the approval codes, No.71 obtained from the corresponding ethical committee on human research of our institute.
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- Robert V, Michel P, Flaman JM, Chiron A, Martin C, Charbonnier F, Paillot B, Frebourg T. High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis. Carcinogenesis. 2000;21(4):563–5.View ArticlePubMedGoogle Scholar
- Nishiwaki T, Daigo Y, Kawasoe T, Nakamura Y. Isolation and mutational analysis of a novel human cDNA, DEC1 (deleted in esophageal cancer 1), derived from the tumor suppressor locus in 9q32. Genes Chromosomes Cancer. 2000;27(2):169–76.View ArticlePubMedGoogle Scholar
- Miyake S, Nagai K, Yoshino K, Oto M, Endo M, Yuasa Y. Point mutations and allelic deletion of tumor suppressor gene DCC in human esophageal squamous cell carcinomas and their relation to metastasis. Cancer Res. 1994;54(11):3007–10.PubMedGoogle Scholar
- Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E, Nakamura Y. Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3. Cancer Res. 1999;59(8):1966–72.PubMedGoogle Scholar
- Jiang W, Zhang YJ, Kahn SM, Hollstein MC, Santella RM, Lu SH, Harris CC, Montesano R, Weinstein IB. Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer. Proc Natl Acad Sci U S A. 1993;90(19):9026–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Boynton RF, Blount PL, Yin J, Brown VL, Huang Y, Tong Y, McDaniel T, Newkirk C, Resau JH, Raskind WH, et al. Loss of heterozygosity involving the APC and MCC genetic loci occurs in the majority of human esophageal cancers. Proc Natl Acad Sci U S A. 1992;89(8):3385–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Kato J, Kuwabara Y, Mitani M, Shinoda N, Sato A, Toyama T, Mitsui A, Nishiwaki T, Moriyama S, Kudo J, et al. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer. 2001;95(2):92–5.Google Scholar
- Duval A, Busson-Leconiat M, Berger R, Hamelin R. Assignment of the TCF-4 gene (TCF7L2) to human chromosome band 10q25.3. Cytogenet Cell Genet. 2000;88(3–4):264–5.View ArticlePubMedGoogle Scholar
- Ishiguro H, Tsunoda T, Tanaka T, Fujii Y, Nakamura Y, Furukawa Y. Identification of AXUD1, a novel human gene induced by AXIN1 and its reduced expression in human carcinomas of the lung, liver, colon and kidney. Oncogene. 2001;20(36):5062–6.View ArticlePubMedGoogle Scholar
- Saadeddin A, Babaei-Jadidi R, Spencer-Dene B, Nateri AS. The links between transcription, beta-catenin/JNK signaling, and carcinogenesis. Mol Cancer Res. 2009;7(8):1189–96.View ArticlePubMedGoogle Scholar
- McMahon AP, Moon RT. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis. Cell. 1989;58(6):1075–84.View ArticlePubMedGoogle Scholar
- He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science. 1998;281(5382):1509–12.View ArticlePubMedGoogle Scholar
- Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275(5307):1787–90.View ArticlePubMedGoogle Scholar
- Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, Ben-Ze'ev A. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999;96(10):5522–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Tetsu O, McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999;398(6726):422–6.View ArticlePubMedGoogle Scholar
- Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, et al. Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci U S A. 1999;96(4):1603–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Ishiguro H, Furukawa Y, Daigo Y, Miyoshi Y, Nagasawa Y, Nishiwaki T, Kawasoe T, Fujita M, Satoh S, Miwa N, et al. Isolation and characterization of human NBL4, a gene involved in the beta-catenin/tcf signaling pathway. Jpn J Cancer Res. 2000;91(6):597–603.View ArticlePubMedGoogle Scholar
- Brabletz T, Jung A, Dag S, Hlubek F, Kirchner T. beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol. 1999;155(4):1033–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Kudo J, Nishiwaki T, Haruki N, Ishiguro H, Shibata Y, Terashita Y, Sugiura H, Shinoda N, Kimura M, Kuwabara Y, et al. Aberrant nuclear localization of beta-catenin without genetic alterations in beta-catenin or Axin genes in esophageal cancer. World J Surg Oncol. 2007;5:21.View ArticlePubMedPubMed CentralGoogle Scholar
- Mulholland DJ, Read JT, Rennie PS, Cox ME, Nelson CC. Functional localization and competition between the androgen receptor and T-cell factor for nuclear beta-catenin: a means for inhibition of the Tcf signaling axis. Oncogene. 2003;22(36):5602–13.View ArticlePubMedGoogle Scholar
- Itami A, Shimada Y, Watanabe G, Imamura M. Prognostic value of p27(Kip1) and CyclinD1 expression in esophageal cancer. Oncology. 1999;57(4):311–7.View ArticlePubMedGoogle Scholar
- Wu MY, Zhuang CX, Yang HX, Liang YR. Expression of Egr-1, c-fos and cyclin D1 in esophageal cancer and its precursors: An immunohistochemical and in situ hybridization study. World J Gastroenterol. 2004;10(4):476–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Shimada Y, Imamura M, Shibagaki I, Tanaka H, Miyahara T, Kato M, Ishizaki K. Genetic alterations in patients with esophageal cancer with short- and long-term survival rates after curative esophagectomy. Ann Surg. 1997;226(2):162–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Shibata Y, Haruki N, Kuwabara Y, Nishiwaki T, Kato J, Shinoda N, Sato A, Kimura M, Koyama H, Toyama T, et al. Expression of PTTG (pituitary tumor transforming gene) in esophageal cancer. Jpn J Clin Oncol. 2002;32(7):233–7.View ArticlePubMedGoogle Scholar
- Konishi S, Ishiguro H, Shibata Y, Kudo J, Terashita Y, Sugiura H, Koyama H, Kimura M, Sato A, Shinoda N, et al. Decreased expression of DFF45/ICAD is correlated with a poor prognosis in patients with esophageal carcinoma. Cancer. 2002;95(12):2473–8.View ArticlePubMedGoogle Scholar
- Sugito N, Ishiguro H, Kuwabara Y, Kimura M, Mitsui A, Kurehara H, Ando T, Mori R, Takashima N, Ogawa R, et al. RNASEN regulates cell proliferation and affects survival in esophageal cancer patients. Clin Cancer Res. 2006;12(24):7322–8.View ArticlePubMedGoogle Scholar