| BACKGROUNDEstrogen receptor (ER) is regarded as an important molecular biological marker of breast cancer. There are two main subtypes of ER, ERαand ERβ.Due to the existence of splice variants,ERαhas three subtypes,namely ERα66,ERα46和ERα36,and ERα66 is simply called ER66.Clinical investigations indicated that ER66 was associated with the responsiveness of breast cancer to chemotherapy. Those breast tumors with positive ER66 expressions were less responsive to chemotherapy. A large amount of literatures proved that ER66 inhibited the apoptosis of breast cancer cells by up-regulating the expression of anti-apoptotic protein Bcl-2 to mediate the responsiveness of breast cancer to chemotherapy. However, some in vitro study found that antiestrogen drugs-tamoxifen was unable to reverse the responsiveness of positive ER66 breast cancer cells to chemotherapy. A large scale of studies indicated that the combination of tamoxifen and chemotherapy drugs was unable to enhance the responsiveness of breast cancer cells to chemotherapy. Therefore, the relationship between ER66 and breast cancer remains to be elucidated. METHODS AND RESULTS1. Correlation analysis between ER66, other drug-resistance markers and the pathological response of breast cancer following chemotherapyA total of 118 breast cancer cases with neoadjuvant chemotherapy were enrolled in this test. Prior to chemotherapy, biopsy specimens were subjected to histological staining for detecting estrogen receptora36 (ER66), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), P-glycoprotein (P-gp), multidrug resistance-related protein (MRP), glutathione S-transferase pi (GST-pi) and topoisomerase-Ⅱ(Topo-Ⅱ) expressions. After chemotherapy, the specimens were then subjected to H.E staining for pathological response and staging. All the cases were divided into 3 chemotherapy regimes, including DEC (docetaxel+epirubicin+ cyclophosphamide),VFC(vinorelbine/vincristine+5-fluorouracil+cyclophosphamide) and EFC(epirubicin+5-fluorouracil+cyclophosphamide).The expressions of molecular markers in breast cancer and pathological response post-chemotherapy were subjected to univariate and multivariate statistical analysis.Our studies found that ER66 was an independent predictor of pathological alterations post-chemotherapy in three different preoperative chemotherapy regimens including DEC, VFC and EFC.Breast cells with positive ER66 showed drug resistance to chemotherapy, whereas negative ER66 cells were sensitive to chemotherapy. HER2 was an independent predictor of pathological alterations post-chemotherapy in DEC group alone, positive HER2 breast cancer cells were susceptible to chemotherapy. PR and multidrug resistance markers P-gp,MRP, GST-pi和TopoⅡwere not independent predictors in all the three groups.2. In vitro study upon the relationship between ER66 and the resistance of breast cancer cells to chemotherapyIn the first section, we found that ER66 was associated with pathological alterations of breast cancer cells post-chemotherapy in all groups, suggesting that ER66 was a multidrug resistant predictor. To confirm the relationship between ER66 and multidrug resistance following chemotherapy, MTT was employed to investigate the IC50 of four drugs paclitaxol,epirubicin,5-fluorouracil,vinorelbine to ER66 negative Bcap37,MDA-MB-231 and positive MCF-7,T47D breast cancer cell lines, respectively. The results showed that the IC50 to ER66 positive cancers was significantly higher compared with that to ER66 negative breast cancer cell lines, indicating that ER66 positive breast cancer cells had multidrug resistance post chemotherapy.Both clinical and in vitro studies found that ER66 expression was correlated with the drug resistance of breast cancer cells to multiple drugs. So, did ER66 directly mediate the drug resistance of breast cancer to chemotherapy by regulating the expression of Bcl-2 as reported in previous findings? We used MTT and PI staining to the influence of ER66 expression inhibited by estradiol activation or fulvestrant inhibition upon the responsiveness of positive ER66 T47D breast cancer cells to chemotherapy. The pretreatment time of estradiol was set as 16 h and 12 d. Meanwhile, ER66 plasmid was transfected into negative ER66 Bcap37 breast cancer cells to establish BC-ER stably expressed ER66, and empty plasmid was transfected into cancer cells to construct BC-V.Our results showed that ER66 activation was able to increase the sensitivity of natural positive ER66 T47D breast cancer cells to multidrugs but inhibit its multidrugs resistance. By contrast, ER66 activation inhibited the drug resistance to chemotherapy for positive ER66 breast cancer cells transformed from negative ER66 Bcap37 cells. Compared with negative ER66 BC-V, positive ER66 BC-ER presented higher resistance to multiple chemotherapy drugs. We can not explain this phenomenon by stating that ER66 directly mediated the drug resistance of breast cancers to chemotherapy by regulating the expression of Bcl-2 and Bax (previous findings showed that ER66 mediated the process by regulating the expression of downstream proteins). Because, we found that ER66 activation upregulated the expression of Bcl-2 for positive ER66 breast cancer cells, whereas ER66 activation downregulated Bcl-2 expression and upegulated Bax expression for positive ER66 cancer cells transformed from negative ER66 breast cancer cells. We contributed this phenomenon to the influence of ER66 upon the growth of breast cancer cells:ER66 activation was able to enhance the growth of natural positive ER66 breast cancer cells, increase the amount of cells staged in G2+S phase, and finally increase the sensitivity to chemotherapy drugs. However, for Bcap37 cells transformed from negative ER66 breast cancer cells, ER66 activation inhibited the growth of cancer cells, lowered the percentage of G2+S cells, and increase the resistance of cancer cells to chemotherapy drugs.ER66 activation was unable to induce the drug resistance of natural ER66 positive T47D breast cancer cells. Although ER66 activation was able to increase the drug resistance of Bcap37 cells transformed from negative ER66 breast cancer cells, but it was only attributable to the inhibitory effect upon the growth of these treated Bcap37 cells, which was not applicable for common positive ER66 breast cancer cells. Taken together our clinical and in vitro findings, we indicated that ER66 was associated with drug resistance of breast cancer cells after chemotherapy, but ER66 itself did not directly mediate this process by regulating the expression of Bcl-2.3. Cell differentiation-the bridge connecting ER66 and drug resistance of breast cancer cellsBased upon the two sections, we knew that ER66 had intimate relationship with pathological alterations of breast cancer cells after chemotherapy, that is, positive ER66 increased its drug resistance. But in vitro study found that ER66 itself did not participate in the mediation of the drug resistance of breast cancer cells. So how does the relationship between ER66 and drug resistance of breast cancer cells form?The differentiation of cancer cells was an important predictor of breast cancers. It was postulated that the degree of cancer cells differentiation correlated with the responsiveness of breast cancer cells to chemotherapy. Previous findings showed that ER66 was not expressed in stem and progenitor breast cancer cells. Therefore, it was hypothesized that ER66 was expressed in cancer cells with certain differentiation and ER66 expression might be related with the differentiation of breast cancer cells. Differentiation stage of breast cancer cells was probably the bridge connecting ER66 and drug resistance of breast cancer cells. Two relationships below were the emphasis in the third section:the relationship between the differentiation of breast cancer cells and pathological changes after neoadjuvant chemotherapy; the relationship between ER66 expression and the differentiation of both normal breast epithelium and breast cancer cells. These findings were designed to explain that differentiation stage of breast cancer cells was probably the bridge connecting ER66 and drug resistance of breast cancer cells.Totally 142 breast cancer cases subjected to neoadjuvant chemotherapy were collected in this test. Prior to chemotherapy, H.E staining was employed to stage the histological grade and evaluate the differentiation stage of gland tubes. Post chemotherapy, biopsy specimens were subjected to grading of pathological changes. Chi-square analysis was used histological staging, gland tubes differentiation staging and pathological changes post-chemotherapy. The results above indicated that the differentiation stage of breast cancer cells was associated with pathological changes after chemotherapy, and the highly-differentiated cancer cells had resistance to chemotherapy drugs.In addition, we observed the distribution of positive ER66 cells in normal breast gland, and analyzed the correlation between ER66 expression and the differentiation grade of breast cancer cells, indicating that ER66 was probably a predictor for the differentiation of breast epithelium. Negative ER66 cells existed throughout the differentiation:process of normal breast epithelia, including breast epithelia both in immature and mature stages. However, positive ER66 cells only distributed in those epithelia at relative mature stage, suggesting that negative ER66 cells were contained in highly-, moderately, and low-differentiation stages, whereas positive ER66 cells were mainly distributed in breast cancers at relatively highly differentiation stages (including high-and moderate-differentiation stages, and relatively highly-differentiated parts in low-differentiation stage)The differentiation grade of breast cancer cells was related to responsiveness of cancer cells to chemotherapy. Highly-differentiated breast cancers had resistance to chemotherapy drugs, whereas positive ER66 breast cancer cells were mainly distributed in breast cancers at highly-differentiated stage. Therefore, it was postulated that the differentiation stage of breast cancer cells well explained that positive ER66 cancer cells showed chemotherapy drug resistance and negative ER66 cancer cells had sensitivity to chemotherapy. CONCLUSIONER66 is an independent predictor of pathological changes of breast cancer cells to chemotherapy. Positive ER66 breast cancer cells showed resistance to chemotherapy drugs, but ER66 activation does not directly mediate this process by mediating the expressions of Bcl-2 and Bax proteins. ER66 is probably a marker predicting the differentiation grade of breast cancer cells and breast cancers, positive ER66 breast cancer cells are highly differentiated, and had resistance to chemotherapy drugs. However, negative ER66 cancer cells are poorly differentiated, and showed sensitivity to chemotherapy drugs.ER is indirectly correlated with breast cancer chemoressitance through cell differentiation.
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