Epithelial Mesenchymal Transition and its Roles on Chemoresistance in Non—small Cell Lung Cancer

Abstract Objective：Previous reported have demonstrated that an intricate link between epithelial-mesenchymal transition （EMT） and anticancer drug resistance in cell culture and animal model. The aim of this study is to further investigate the relationship between chemoresistance and EMT in non-small cell lung cancer （NSCLC） through observing the expression status of EMT markers and resistance protein in histological level. Methods： The resistance protein， excision repair cross-complementing 1 （ERCCl） and EMT markers， including E-cadherin and vimentin， were detected by immunohistochemistry in 100 cases of NSCLC， half of that were treated with pre-operative neoadjuvant chemotherapy （neoadjuvant chemotherapy group）， and the other underwent surgery alone （simple surgery group）. Results： There were significant positive correlations between the expression of ERCCl and vimentin in neoadjuvant chemotherapy group （r = 0.471，P = 0.01） and simple surgery group （ r = 0.380，P = 0.01）， and significant negative correlations between the ERCCl and E-cadherin in neoadjuvant chemotherapy group（r = -0.401，P = 0.01） and simple surgery group （r = -0.295，P = 0.03. In neoadjuvant chemotherapy group， EMT status （p = 0.04） and drug resistance （p = 0.03） were more apparent than simple surgery group. The expression levels of ERCCl， vimentin and E-cadherin were all related to differentiated degree and lymph node metastasis in both groups（P<0.05）. Conclusion： This study indicated that chemoresistance is correlated with the occurrence of EMT in NSCLC at tissue level， suggesting that selective targeting of EMT-phenotypic cells for declining chemoresistance may be a plausible therapeutic strategy.

Key words Non-small cell lung cancer； Epithelial-mesenchymal transition； Chemoresistance

中圖分類號：R734.2 文獻標識碼：A

0 Introduction

Among lung cancer， non-small cell lung cancer （NSCLC） accounts for 75%-80%， with 5-year survival rates less than 20%[1]. Primarily due to local invasion and distant metastasis of cancer cells， NSCLC shows a poor prognosis. Surgery combined with chemotherapy has been the mainstay and cisplatin is still the most commonly used first-line chemotherapeutic agent in the treatment of NSCLC[2]. However， resistance to cisplatin is a major obstacle in potentially curative treatments for advanced NSCLC. Therefore， to improve the sensitivity to chemotherapy may be a key step to against cancer.

Excision repair cross-complementing 1 （ERCC1） is reported involving in the sensitivity of cancer cells to platinum-based chemotherapy， as it has a key role in evaluating tumor cisplatin-resistance clinically. A few recent studies have reported， that highly up-regulated expression of resistance protein leading to invasion and metastasis in lung cancer cells which were more chemoresistant and metastatic， as compared to their parent cells[3].

Some recent reports demonstrated that epithelial-mesenchymal transition （EMT） plays an important role not only in invasiveness and metastasis of cancer， but also in occurrence of chemoresistance of them[4-5]. With farther understanding in EMT， it is necessary to further observe the link between EMT and chemoresistance in vivo for the practical application of EMT theory in the treatment of tumor.

This study confirmed that the expression of resistance protein has a strong correlation with EMT status in respect of histology， consistent with EMT theory in cell culture study， indicated that targeting therapy through regulating EMT might be a potential strategy for the treatment of human NSCLC.

1 Materials and Methods

1.1 Primary tumor samples

One group of tumor specimens was collected from 50 patients in the Affiliated Hospital of Qingdao University Medical College， who underwent surgery following neoadjuvant chemotherapy in the last five years （neoadjuvant chemotherapy group）. Each patient received 2 cycles of cisplatin-based combined chemotherapy. Another group of 50 patients underwent surgery alone without neoadjuvant chemotherapy （simple surgery group）， which were taken from the Affiliated Hospital of Qingdao University Medical College between July 2011 and June 2012. NSCLC was definitively identified based on pathological diagnosis in every patient of both groups.

1.2 Immunohistochemistry

Serially continuous tissue sections of formalin-fixed， paraffin-embedded tumor tissues were used for immunohistochemical analysis. Sections were deparaffinized by xylene and rehydrated through an ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 minutes at room temperature. Source and dilution of primary antibody used were as follows： ERCC1（1：100），E-cadherin（1：200） and vimentin（1：100）. Primary antibodies were applied overnight at 4？€癈， and then with secondary antibodies for 30 minutes at 37？€癈. PBS instead of the primary antibody was used as negative controls. Three washes with PBS were applied between each step of antibody incubation. Consequently， all sections were stained with diaminobenzidine （DAB） and counterstained with haematoxylin. The above reagents used were purchased from ZSGB-BIO （China）.

2 Statistical Analysis

The date was statistically analyzed with SPSS19.0 software. The relationship between immune-markers expression and clinicopathological factors was analyzed with the chi-square test. The correlation between two variances was analyzed with Spearman rank correlation analysis. P values <0.05 were considered statistically significant.

3 Results

3.1 Expression of EMT markers and drug resistance protein in clinicopathological findings

The characteristics of 100 patients and immunohistochemical findings were shown in Table 1. The expressions of E-cadherin， vimentin and ERCC1 in neoadjuvant chemotherapy group and simple surgery group were related to both differentiated degree and lymph node metastasis （P<0.05）， but had no correlation with age， gender， histological types and therapeutic methods （P>0.05）.

3.2 Correlation between resistance protein and EMT makers

To explore whether the development of EMT is correlated with the occurrence of chemoresistance in vivo， we analyzed the expression of ERCC1， vimentin and E-cadherin in tumor tissues. Immunohistochemical findings showed that the expression of ERCC1， vimentin and E-cadherin were 76.0%（38/50）， 64.0%（32/50） and 30.0%（15/50） in neoadjuvant chemotherapy group， and 58.0%（29/50）， 52.0%（26/50） and 32.0%（16/50） in simple surgery group（Table 2）. There is a significant positive correlation between ERCC1and vimentin in neoadjuvant chemotherapy group（r = 0.471， P<0.05） and simple surgery group（r = 0.401， P<0.05）， and a significant negative correlation between ERCC1and E-cadherin in neoadjuvant chemotherapy group（r = -0.380， P<0.05） and simple surgery group（r = -0.295， P<0.05）. Meanwhile， compared with simple surgery group， EMT status （p = 0.04） and drug resistance （p = 0.03） were more apparent in neoadjuvant chemotherapy group.

Table 2 Expression of ERCC1、E-cadherin and vimentin

4 Discussion

In recent years， NSCLC has achieved considerable progress in therapeutic field. New therapeutic approaches such as multidisciplinary treatment and targeted therapy are applied， simultaneously traditional chemotherapy therapeutic is still essential. Cisplatin is a dominated chemotherapy drug in treatment of NSCLC， which binds to DNA and results in the formation of cross-links， consequently arresting DNA synthesis and replication in tumor cells[6]. Therefore resistance to cisplatin is a major influence to treatment for NSCLC. Excision repair cross-complementing 1 （ERCC1） is a structure-specific endonuclease， leading to the repair of DNA damage by the nucleotide excision repair pathway. Overexpression of ERCC1 indicates the enhancement of resistance to cisplatin in lung cancer cells. At present ERCC1 become an international generally acknowledged marker for evaluating resistance to cisplatin. However， the mechanism about cisplatin chemotherapy resistance is not clear.

Increasing evidences show that EMT closely correlated to chemotherapy resistance in cancer cells. Studies on mechanism of EMT may be the key step for investigating chemotherapy resistance[4]. The processes of EMT is a unique process by which cancer cells undergo remarkable morphologic change from their epithelial phenotype to an elongated fibroblastic phenotype （mesenchymal phenotype） accompanied by increased motility and invasion[7]. The most convincing evidence is that regulatory gene and signaling pathways of EMT widely take part in development and invasion of tumor cells[3]. Moreover， during the acquisition of EMT， molecular hallmarks of EMT will change. Study shows that cells decrease in the expression of epithelial markers such as E-cadherin and -catenin， and gain in the expression of mesenchymal markers such as vimentin， fibronectin， -smooth muscle actin （SMA） and so on[8].

The most outstanding character is down-regulation of E-cadherin in EMT and it becomes a hot spot in the research of tumor development， invasion and metastasis. In recent years， emphasis has been placed on the correlation studies of EMT and tumor drug resistance. The process of EMT has been shown to be important in conferring drug resistance to conventional chemotherapeutics including taxol， vincristine and oxaliplatin. For example， oxaliplatin-resistant colonic cancer cells showed phenotypic changes consistent with EMT with decreased expression of the epithelial adhesion molecules such as E-cadherin， and an increase in mesenchymal markers， vimentin. Rho established A549 cell line of NSCLC resistant to gefitinib （A549/GR）， and observed the phenomenon of EMT in cell line as mentioned above， moreover， invasive and exercise performance enhanced in transwell chambers. Clearly， these studies suggest a molecular and phenotypic association between chemoresistance and EMT of cancer cells， but all of the studies were based on cell culture level or transplant tumor model study， in view of that， we collected the specimens of NSCLC to divide into two groups： simple surgery group and neoadjuvant chemotherapy group.

We analyzed comparatively the expression levels of E-cadherin， vimentin and ERCC1 in continuously serial sections of tumor tissues. We observed there were significant positive correlations between ERCC1 and vimentin and negative correlations between ERCC1 and E-cadherin in the two groups. Our results indicate that drug resistance enhanced during the acquisition of EMT characteristics in the cancer cells， which is consistent with investigations above that chemoresistance is associated with EMT in cell culture. Moreover， we found cancer cells which expressed ERCC1 showed changes to markers of EMT， such as the downregulation of E-cadherin and the upregulation of vimentin， especially in the periphery area of tumors. Our results demonstrated in histological level that chemotherapy resistance is closely related to EMT. Moreover， we observed the phenomenon of EMT and drug resistance is more apparent in neoadjuvant chemotherapy group.

Conclusively， this study provides further evidence of link chemoresistance to EMT. On predicting sensitivity of chemotherapy and implementation of individualized treatment， further investigations on mechanism of EMT and chemoresistance is necessary.

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