|Year : 2015 | Volume
| Issue : 5 | Page : 74-79
The expression and prognostic significance of Mucin 13 and Mucin 20 in esophageal squamous cell carcinoma
Hui Wang, Luyan Shen, Yao Lin, Qi Shi, Yongbo Yang, Keneng Chen
Department of Thoracic Surgery I, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, Beijing, P.R. China
|Date of Web Publication||31-Aug-2015|
Prof. Keneng Chen
Department of Thoracic Surgery I, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Haidian, Beijing 100142
Source of Support: None, Conflict of Interest: None
Background: Mucin 13 (MUC13) and Mucin 20 (MUC20) are high molecular weight transmembrane O-linked glycoprotein secreted by epithelium and are widely overexpressed in epithelial tumor cells. Previously, it has been reported that MUC13 and MUC20 were prognostic molecular biomarkers of some epithelial tumors, but few has studied the relations between MUC13 or MUC20 expression and the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Thus, the aim of our study was to explore this issue.
Materials and Methods: Immunohistochemistry was used to detect the expressions of MUC13 and MUC20 in 186 patients with ESCC who received neoadjuvant chemotherapy followed by surgery. We analyzed the correlations between MUC13 or MUC20 expression and long-term survival of these patients.
Results: The expression of MUC13 and MUC20 in ESCC mainly occurred in the cytoplasm. Among the 186 subjects, 53.8% had high MUC13 expression and 38.2% had high MUC20 expression. Univariate analysis showed that neither MUC13 nor MUC20 was significantly associated with long-term survival. When combining expression of MUC13 with MUC20, we found that the median survival time of patients with low MUC13/high MUC20 expression was significantly shorter than that of patients with high MUC13/low MUC20 expression (27.7 months vs. 59.5 months, P = 0.021). Multivariate analysis showed that combination of MUC13/MUC20 expression was an independent prognostic factor (hazard ratios = 0.531, 95% confidence interval: 0.299-0.944, P = 0.031).
Conclusion: A combination of MUC13/MUC20 expression was a potential prognostic marker for patients with ESCC, who received neoadjuvant chemotherapy followed by surgery.
Keywords: Esophageal squamous cell carcinoma, mucin 13, mucin 20, prognosis
|How to cite this article:|
Wang H, Shen L, Lin Y, Shi Q, Yang Y, Chen K. The expression and prognostic significance of Mucin 13 and Mucin 20 in esophageal squamous cell carcinoma. J Can Res Ther 2015;11, Suppl S1:74-9
|How to cite this URL:|
Wang H, Shen L, Lin Y, Shi Q, Yang Y, Chen K. The expression and prognostic significance of Mucin 13 and Mucin 20 in esophageal squamous cell carcinoma. J Can Res Ther [serial online] 2015 [cited 2020 Feb 19];11:74-9. Available from: http://www.cancerjournal.net/text.asp?2015/11/5/74/163846
| > Introduction|| |
Esophageal carcinoma is one of the most common malignancies in China. In recent years, multi-modality treatment dominated by surgery has improved the life quality and prognosis of the patients to a certain degree. However, the long-term survival remains far from satisfactory, with a 5-year survival rate rarely exceeds 40%.  So far, the evaluation of the prognosis of esophageal carcinoma patients mainly depends on tumor node metastasis (TNM) staging system. But in clinical practice, different outcomes are often observed among patients with the same stage, thus, to identify prognostic biomarkers other than TNM staging system becomes the hotspots in cancer research field.
Mucins are high molecular weight (ranges from 0.5 to 20 MDa) transmembrane O-linked glycoprotein secreted by epithelium and are widely expressed in normal mucous of respiratory tract, digestive tract, and urogenital tract, etc. Mucins have various functions including lubricating tracts, regulating cell-cell and cell-matrix adhesion, and acting as a barrier to protect from invading of pathogen and toxicant. , Human mucin gene family consists of 19 members, which could be divided into membrane-bound and secreted types in terms of structure and function, whereas Mucin 13 (MUC13) and Mucin 20 (MUC20) belong to the former type.  All of the membrane-bound mucins have epidermal growth factor (EGF)-like domain, which is rich in cysteine residues and highly homologous with the ligand of epidermal growth factor receptor (EGFR).  The binding of EGF with its receptors like EGFR and HER2 could activate a series of signaling pathways involving in cell proliferation, cell differentiation, cell adhesion, and migration. In physiological status, the expression of mucin shows tissue-specific and temporal-specific features, which are disturbed in diseases including carcinoma. Recently, it has been reported that MUC13 and MUC20 were overexpressed in many cancers including gastric cancer, , ovarian cancer,  colorectal cancer,  endometrial cancer,  etc. Expression of MUC13 was increased successively in normal mucosa, intestinal metaplasia, and gastric cancer, indicating that it could be a biomarker for intestinal metaplasia and high expression of MUC13 is an independent prognostic factor of early-staged gastric cancer.  Although MUC13 is not an independent prognostic factor for colorectal cancer, patients with high MUC13 expression have a poorer prognosis than that with low MUC13 expression.  Patients with overexpressed MUC20 had a shorter disease-free survival time and overall survival time than that with low-level expressed MUC20, which is an independent prognostic factor other than TNM staging for patients with colorectal cancer.  Chen et al.  reported the similar results in endometrial cancer that patients with high MUC20 expression survived shorter than those with low MUC20 expression. Furthermore, MUC20 was an independent prognostic factor for endometrial cancer. However, few reported the expression of MUC13 and MUC20 in esophageal carcinoma and their correlations with prognosis of patients. Previously, we found that the expressions of MUC13 and MUC20 were associated with patients' response to neoadjuvant chemotherapy. In this study, we aimed to explore the correlation between expressions of MUC13 and MUC20 and the prognosis of esophageal squamous cell carcinoma (ESCC) patients.
| > Materials and Methods|| |
Patients in the study
A total of 712 patients with ESCC from January 2000 to December 2012 underwent esophagectomy by a single surgeon team in our department, among which 259 patients received neoadjuvant chemotherapy. One hundred and eighty-six cases were finally enrolled in the analysis after excluding nine cases with exploratory surgery, four cases with malignancy history, three cases underwent salvage surgery, 12 cases received radiation before surgery, two cases died within perioperative period, and 43 cases with incomplete tissue blocks.
Platinum-based two-drug combination, mainly paclitaxel and cisplatin with the proportion of 95%, was used in perioperative chemotherapy. On day 1, paclitaxel at a dose of 175 mg/m 2 of body surface area was administered intravenously. On day 1-3, cisplatin at a dose of 25 mg/m 2 of body surface area was administered intravenously, a single course of treatment lasted 21-28 days. Enhanced chest computed tomography (CT) and esophagography were used to evaluate the curative effects of the treatment. Approximately 1-4 cycles of neoadjuvant chemotherapy was administered before surgery. Among them, 66.1% received 2 cycles (123/186), 29 cases 1 cycle, 18 cases 3 cycles, and 16 cases 4 cycles.
All of the 186 subjects underwent radical surgery for ESCC, 166 subjects had R0 resection, and 20 subjects had R 1/2 resection, with a complete resection rate of 89.2%. A total of 155 cases were treated using the right transthoracic procedure (83.3%, 13 cases with Ivor-Lewis and 142 cases with McKeown). Six cases (3.2%) were treated using the left transthoracic procedure and 25 cases (13.4%) with transhiatal esophagectomy. Surgery was conducted 3-6 weeks following neoadjuvant chemotherapy, with 129 cases (69.4%) underwent surgery for 3-4 weeks after neoadjuvant chemotherapy and 57 cases (30.6%) for 4-6 weeks. Adjuvant chemotherapy was depended on pathological stage, patients' postoperative recovery, and the response to neoadjuvant chemotherapy.
A total of 83.1% of the visits were outpatient follow-ups, whereas 16.9% were telephone or letter follow-ups. After surgery, outpatient follow-up visits were conducted once in every 3 months in the first 2 years, once in every 6 months from 2 to 5 years, and once in every year after 5 years. Outpatient follow-up visits included recording of symptoms and findings of body examinations such as CT, upper esophagography, ultrasound, and gastroscopy, if necessary. After 2010, some subjects underwent positron emission tomography-CT examinations. Overall survival was defined as the time from operation to death or the last follow-up. The last follow-up checkpoint was October 2014.
After routine deparaffinization and hydration, tissue sections were treated with 3% hydrogen peroxide and then heated in sodium citrate for antigen retrieval. After antigen retrieval, the sections were incubated with 10% normal goat serum to block any nonspecific reaction. Then, the sections were incubated with a rabbit polyclonal antibody against human MUC13 (dilution, 1:400, Abcam, Cambridge, UK) or MUC20 (dilution, 1:200, Sigma, California, USA), at 4°C overnight, followed by incubation with a horseradish peroxidase (HRP)-ligated goat antibody against rabbit immunoglobulin (Beijing Zhongshan Jinqiao Corporation, Beijing, China) at room temperature for 15 min, and further incubated with HRP labeled streptavidin working solution for 15 min at room temperature. The sections were washed 3 times with 1 × phosphate-buffered saline (PBS) in each step. The staining was visualized by reaction with 3,3′-diaminobenzidine, then counter-stained with hematoxylin. Color separation was conducted using hydrochloric-alcohol solution. Running water was used to return blue after dehydrating by gradient ethyl alcohol and hyalinized by dimethylbenzene. Resin was used to seal the blocks. Simultaneously, each section was incubated with PBS instead of the primary antibody as an internal negative control. The already known positive slides were used as positive control.
Immunohistochemistry (IHC) signals were scored by two independent specialist pathologists. To evaluate MUC13 and MUC20 expressions, IHC staining was classified into the following four groups according to intensity and extent. The proportion of cells proteins expression was categorized as follows: (0) <10% immunopositive cells; (1) 10-30% positive cells; (2) 30-60% positive cells; and (3) >60% positive cells. The staining intensity was categorized by relative intensity as follows: (0) Negative; (1) pale yellow; (2) brown yellow; and (3) dark brown. The proportion and intensity scores were then multiplied to obtain a total score, scores <6 was considered as low-level expression, whereas scores >6 were considered as high-level expression.
SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied to conduct statistical analysis. Chi-square test was used to analyze the relations between expressions of MUC13 or MUC20 and clinicopathological characteristics. Kaplan-Meier survival analysis and log-rank test were used to evaluate the prognostic significance of MUC13 and MUC20 expression in ESCC patients. Cox proportional hazard model was used to explore the independent prognostic factors for ESCC patients. P <0.05 was considered statistically significant.
| > Results|| |
Relations between Mucin 13 or Mucin 20 expression and clinicopathological characteristics and their correlation
The expressions of MUC13 and MUC20 mainly occur in the cytoplasm. Among 186 ESCC patients, high expressions of MUC13 and MUC20 account for 53.8% (100/186) and 38.2% (71/186), respectively [Figure 1]. MUC13 and MUC20 expressions were not significantly correlated with each other (r = -0.070, P = 0.340). The expression of MUC13 was not associated with clinicopathological characteristics. However, the expression of MUC20 was significantly positively associated with tumor differentiation, the depth of tumor invasion, lymph node metastasis, pathological tumor node metastasis (pTNM) staging, tumor regression grade (TRG), lymphatic vessel invasion (LVI), and downstaging [Table 1].
|Figure 1: Expressions of Mucin 13 and Mucin 20 protein in esophageal squamous cell carcinoma tissues. (a) High-level expression of Mucin 13 in esophageal squamous cell carcinoma, ×200. (b) High-level expression of Mucin 20 in esophageal squamous cell carcinoma, ×200|
Click here to view
|Table 1: Association between MUC13 or MUC20 expression and clinicopathological characteristics of patients with ESCC (n=186) |
Click here to view
Association between expression of Mucin 13 or Mucin 20 and prognosis of esophageal squamous cell carcinoma patients
Univariate analysis showed that there was no significant difference in long-term survival between patients with high and low MUC13 or MUC20 expression [P = 0.132 and P = 0.060, respectively; [Table 2]. However, patients with high MUC13 expression tended to have a better survival than those with low expression, whereas patients with low MUC20 expression tended to have a better survival than those with high expression. Then, we further stratified the patients into two subgroups: high MUC13/low MUC20 and low MUC13/high MUC20, including 101 patients. Univariate survival analysis showed that the median survival time of patients with low MUC13/high MUC20 expression was significantly shorter than that of patients with high MUC13/low MUC20 expression [27.7 months vs. 59.5 months, P = 0.021; [Table 3] and [Figure 2], and the depth of tumor invasion, lymph node metastasis, pTNM staging, LVI, R 0 resection, TRG, and tumor downstage were significantly correlated with the survival of patients. After putting these factors which identified from univariate analysis into COX proportional hazard model, we observed that the combination of MUC13/MUC20 expression was an independent prognostic factor for ESCC patients who received neoadjuvant chemotherapy followed by surgery (hazard ratios = 0.531, 95% confidence interval: 0.299-0.944, P = 0.031).
|Figure 2: Survival analysis of the combination of Mucin 13/Mucin 20 expression in a subgroup of 101 esophageal squamous cell carcinoma patients. The overall survival of patients with low Mucin 13/high Mucin 20 expression was significantly shorter than that of patients with high Mucin 13/low Mucin 20 expression (P = 0.021)|
Click here to view
|Table 2: MUC13 and MUC20 expression were not associated with overall survival time (n=186) |
Click here to view
|Table 3: The association between MUC13/MUC20 expression and overall survival time (n=101) |
Click here to view
| > Discussion|| |
It is common knowledge that neoadjuvant chemotherapy could improve the R 0 resection rate for resectable ESCC patients and prolong their survival time. Nevertheless, there do exist some patients that are resistant to neoadjuvant chemotherapy and thus could not benefit from it, therefore, to identify biomarkers to predict response to neoadjuvant chemotherapy may serve as a method to improve the efficacy of neoadjuvant chemotherapy and prevent overtreatment. Our previous study found that the expressions of MUC13 and MUC20, which were members of the human mucin gene family, were correlated with chemotherapy sensitivity to platinum. Patients with high MUC13 and low MUC20 expression benefited more from neoadjuvant chemotherapy. Our present study aimed to explore the relations between expression of MUC13 or MUC20 and prognosis of ESCC patients who received neoadjuvant chemotherapy followed by surgery.
The function of MUC13 and MUC20 in tumorigenesis requires their extracellular EGF-like domain, which binds with EGFR and induces the dimerization of receptor and conformational change, activating the tyrosine kinase of EGFR, which results in phosphorylation of tyrosine residues. The phosphorylated receptor then binds with SH2 domain of Grb-2, which further activates Ras through binding with SOS (guanine nucleotide exchange factor). The activated Ras then induces expression of extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase signal pathway, enhances the transcription activity of certain transcription factor and promotes cell proliferation. ,, Likewise, the binding of EGF-like domain of MUC20 with EGFR activates the phosphorylation of STAT3, which results in upregulation of CCND1 and downregulation of interferon beta, interferon gamma, interleukin 8, and further promotes cell proliferation.  The massiveness and complex conformation of these high molecular weight transmembrane glycoproteins could prevent external substances from adhering to cell surface, playing a role in cell-cell adhesion.  Normally, MUC could protect mucosa from the invading of external microorganisms, whereas in tumors, MUC could protect tumor cells from the exposure to immune cells, or even specifically binds to antibody on the surface of the immune cells and consequently avoids immunologic cytotoxicity.  On the other hand, MUC is usually located at the upper surface of polarized epithelial cells whereas in depolarized tumor cells, MUC could be redistributed in the whole surface of cells, which leads to destruction of adhesion between tumor cells, enabling tumor cells to escape from their original sites, which is a key step in infiltration and metastasis.  Therefore, in the view of function and the underlying mechanism, we can infer that MUC13 and MUC20 may promote tumorigenesis.
Increasing reports showed MUC13 and MUC20 were frequently and aberrantly expressed in several cancers including gastric, colorectal, and endometrial cancer. Overexpression of these two proteins was significantly correlated with poor prognosis. Our study showed that patients with high MUC13/low MUC20 expression obtained a better survival than those with low MUC13/high MUC20 expression, which is due to the patients with a high MUC13/low MUC20 expression could benefit more from neoadjuvant chemotherapy. However, to be noticed is that the result in this study is inconsistent with the result from other studies. In other studies, the survival time of patients with high MUC13 expression was shorter than those with low MUC13 expression, whereas our result was just on the contrary. The potential reasons are as follows: (1) In our study we examined the expression of MUC13 and MUC20 in the postoperative specimens of patients who received neoadjuvant chemotherapy, which may change the expression pattern; (2) our study explored the prognostic significance of MUC20 and MUC13 expression in patients who received neoadjuvant chemotherapy. We previously demonstrated that patients with high MUC13/low MUC20 expression showed a better chemotherapy sensitivity, so their survival time was correspondingly longer. To further explicit the prognostic value of MUC13 and MUC20 in ESCC patients, we will examine their expression in ESCC patients who underwent surgery alone in the future.
| > Acknowledgments|| |
This work was supported by National Basic Research Program of China (Grant No. 2011CB504300), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20130001110108), National Natural Science Foundation for Young Scholars (Grant No. 81301748), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (Grant No. IRT13003), Beijing Star of Science and Technology Plan (Grant No. xxhz201405).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
van Hagen P, Hulshof MC, van Lanschot JJ, Steyerberg EW, van Berge Henegouwen MI, Wijnhoven BP, et al.
Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med 2012;366:2074-84.
Gendler SJ, Spicer AP. Epithelial mucin genes. Annu Rev Physiol 1995;57:607-34.
Hollingsworth MA, Swanson BJ. Mucins in cancer: Protection and control of the cell surface. Nat Rev Cancer 2004;4:45-60.
Williams SJ, Wreschner DH, Tran M, Eyre HJ, Sutherland GR, McGuckin MA. Muc13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells. J Biol Chem 2001;276:18327-36.
Carr JC, Sherman SK, Wang D, Dahdaleh FS, Bellizzi AM, O′Dorisio MS, et al.
Overexpression of membrane proteins in primary and metastatic gastrointestinal neuroendocrine tumors. Ann Surg Oncol 2013;20 Suppl 3:S739-46.
Shimamura T, Ito H, Shibahara J, Watanabe A, Hippo Y, Taniguchi H, et al.
Overexpression of MUC13 is associated with intestinal-type gastric cancer. Cancer Sci 2005;96:265-73.
Chauhan SC, Vannatta K, Ebeling MC, Vinayek N, Watanabe A, Pandey KK, et al.
Expression and functions of transmembrane mucin MUC13 in ovarian cancer. Cancer Res 2009;69:765-74.
Xiao X, Wang L, Wei P, Chi Y, Li D, Wang Q, et al.
Role of MUC20 overexpression as a predictor of recurrence and poor outcome in colorectal cancer. J Transl Med 2013;11:151.
Chen CH, Wang SW, Chen CW, Huang MR, Hung JS, Huang HC, et al.
MUC20 overexpression predicts poor prognosis and enhances EGF-induced malignant phenotypes via activation of the EGFR-STAT3 pathway in endometrial cancer. Gynecol Oncol 2013;128:560-7.
Lee HJ, Nam KT, Park HS, Kim MA, Lafleur BJ, Aburatani H, et al.
Gene expression profiling of metaplastic lineages identifies CDH17 as a prognostic marker in early stage gastric cancer. Gastroenterology 2010;139:213-25.e3.
Walsh MD, Young JP, Leggett BA, Williams SH, Jass JR, McGuckin MA. The MUC13 cell surface mucin is highly expressed by human colorectal carcinomas. Hum Pathol 2007;38:883-92.
Komatsu M, Jepson S, Arango ME, Carothers Carraway CA, Carraway KL. Muc4/sialomucin complex, an intramembrane modulator of ErbB2/HER2/Neu, potentiates primary tumor growth and suppresses apoptosis in a xenotransplanted tumor. Oncogene 2001;20:461-70.
Hirai S, Izumi Y, Higa K, Kaibuchi K, Mizuno K, Osada S, et al.
Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. EMBO J 1994;13:2331-40.
Galang CK, Der CJ, Hauser CA. Oncogenic Ras can induce transcriptional activation through a variety of promoter elements, including tandem c-Ets-2 binding sites. Oncogene 1994;9:2913-21.
Komatsu M, Carraway CA, Fregien NL, Carraway KL. Reversible disruption of cell-matrix and cell-cell interactions by overexpression of sialomucin complex. J Biol Chem 1997;272:33245-54.
Carraway KL, Perez A, Idris N, Jepson S, Arango M, Komatsu M, et al.
Muc4/sialomucin complex, the intramembrane ErbB2 ligand, in cancer and epithelia: To protect and to survive. Prog Nucleic Acid Res Mol Biol 2002;71:149-85.
Pino V, Ramsauer VP, Salas P, Carothers Carraway CA, Carraway KL. Membrane mucin Muc4 induces density-dependent changes in ERK activation in mammary epithelial and tumor cells: Role in reversal of contact inhibition. J Biol Chem 2006;281:29411-20.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]