| Tamoxifen, a selective estrogen receptor modulator(SERM), is the first-line adjuvant endocrine therapy for breast cancer. However, primary and acquired tamoxifen resistance induced breast cancer metastasis and death, which was severely restricted the survival estimation of patients. Currently, the core mechanism in tamoxifen-resistant breast cancer has been recognized as abnormal estrogen receptor and its associated pathways. The changes in ERα66 which are associated with tamoxifen resistance include ERα66 expression absent, mutation or post-translational modification. Estrogen receptor α36(ERα36) is known as an ERα66 truncated variant, but the mechanism of ERα36 participated in tamoxifen resistance during breast cancer treatment is still poorly understood.With the gradually further research of breast cancer stem cells(CSCs), a new perspective was provided to study the progression of breast cancer. Currently, breast cancer stem cell markers are CD44+/CD24- / low/ESA+ or ALDH1 high. Moreover, estradiol and tamoxifen treatment increase the number and percentage of the normal mammary epithelial stem cells(MaSCs) and breast CSCs. However, no expression of ERα66 was detected in breast Ma SCs and CSCs. So it is of great value to study the molecular mechanisms of hormone signaling in tamoxifen-induced breast CSCs maintain.ALDH1high breast CSCs were detected with high ERα36 expression. ERα36 were correlated with estrogen genomic and non-genomic signaling pathway, via PI3 K / Akt and MAPK / ERK pathway. The signaling activates the downstream kinases, such as c-Jun N- terminal kinase(JNK) and the mobilization of intracellular Ca2+ activity, which participates in the proliferation and invasion of breast cancer cells. Therefore, ERα36 should have broader pathophysiology activity. It is also needs further study for the regulation of malignant behavior of breast cancer, especially the mechanism of stemness maintain of breast CSCs.ERα66 is the unique biomarker for clinical treatment with tamoxifen. Clinical trials showed that tamoxifen may induce CSC phenotype aggressive tumors. Therefore, further molecular analysis is also required for patients with endocrine therapy in breast cancer treatment. Therefore, in this study, based on membrane localization of ERα36, we have sorted ERα36+ cells from heterogeneous cell populations by fluorescence-activated cell sorting(FACS) technology. In addition, we have established a database of breast cancer specimens, combined with detailed clinical information. We performed a thorough study of the expression of its clinical significance. The main resluts and conclusions are summarized as follows:I. The large multi-cohort study of human breast cancer samples showed that ERα36 high expression predicted poor prognosis. More importantly, tamoxifen therapy increased the metastasis of ERα36 positive breast cancer.1. High ERα36 expression is a clinical marker for breast cancer metastasis and poor prognosis.We use the unique peptides of ERα36 to construct specific ERα36 monoclonal antibodies. The ERα36 expression of four separate cohorts, totally 1537 cases breast cancer was studied with immunohistochemical assays. All the information of the breast cancer research was collected for the database establishment(http://swcc.xnyy.cn/clinical,). We observed that ERα36+ tumors tend to metastases in breast cancer patients, and ERα36 expression is a risk factor for poor prognosis in breast cancer.2. Tamoxifen therapy increases the metastasis of ERα36+ breast cancer with reduced survival estimation.We explored the correlation between ERα36 expression in breast cancer and endocrine treatment-related clinical information. We found that compared to ERα36- cancer patients, ERα36 + breast cancer patients who receiving tamoxifen therapy were significantly suffere d from reduced metastasis-free survival(MFS). Furthermore, in the terms of ERα36+ patients, the patients received tamoxifen therapy suffered from significantly reduced MFS than those did not receive tamoxifen therapy. With the multiple regression analysis of 342 cases who were ERα36 and ERα66 double-positive breast cancer patients, we observed that tamoxifen therapy in these patients had a negative impact on both DFS and MFS. By analyzing the three other independent Cohorts, we further found that the risk ratio of receiving tamoxifen treatment for the patients with ERα36- or ERα36+ was 13.67(95% CI, 3.31-56.45). For the ERα36 + cancer patients, the risk ratio of receiving tamoxifen treatment compared to other therapies is 3.21(95% CI, 1.38~7.44). Further evidence confirmed ERα36 was involved in tamoxifen-induced breast cancer metastasis.3. Aromatase inhibitors treatment benifits ERα36 + breast cancer patients.We found that AIs treatment was significantly prolonged the metastasis-free survival than tamoxifen treatment group, with the data of ERα36/ERα66 double-positive breast cancer patients. Also, no significant difference of MFS was observed in AIs treated patients, compared between ERα36- and ERα36 + breast cancer patients. Regression analysis showed that tamoxifen reduced DFS of ERα66+/ ERα36+ breast cancer post-menopausal patients. And aromatase inhibitors can improve the prognosis of breast cancer patients with ERα36 + tumors.II. Tamoxifen activates the malignant phenotype of ERα36 + breast cancer cells.1. Tamoxifen activates ERα36 to increase the proliferation and metastasis of breast cancer cells.We constructed breast cancer cells with different ERα36 expression levels. The in vitro and in vivo treatment with tamoxifen, ERα36+ breast cancer cells showed significantly enhanced proliferation, invasion and metastasis, which suggests that tamoxifen acts as an agonist to activate ERα36 to enhance malignant phenotype of breast cancer cells.2. ERα36 is involved in breast cancer stem cell properties maintain.The stem cell characteristics were examined in ERα36 expressing breast cancer cells, we found that ERα36 positive breast cancer cells exhibit a significant self-renewal capacity, tumorigenicity, invasion and metastasis ability. Moreover, by further analysis of clinical samples, we found that ERα36 expression was corealated with breast cancer stem cell markers ALDH1A11. A significant co-expression phenomenon was observed, which suggests ERα36 may play a crucial role in breast CSCs phenotype maintenance.3. Tamoxifen enhanced stem cell-like properties of ERα36 + cells, and increased the expression of ALDH1A1.We examined the breast CSCs characteristics of ERα36+ cells with tamoxifen treatment. In vitro and in vivo experiments showed that tamoxifen significantly improved the self-renewal capacity of ERα36+ cells, and significantly enriched of breast ALDH1 high subgroup cells. Significant increases of ALDH1A1 expression were observed in metastatic clinical samples.III. Tamoxifen activates ERα36 to transcriptionally upregulate ALDH1A1 mRNA and protein expression.1. Tamoxifen activates ERα36 to participate estrogen genomic signaling pathway regulation.The gene expression analysis was performed with 4-OHT treated MCF-7-ERα36+/- cells. We found that cell adhesion molecules(CAMs) family was expeienced significant regulation, and significantly higher expression of stem-like cell-related genes was observed in ERα36+ cells. More importantly, although ERα36+ cells was of low ERα66 gene expression, the target gene of ERα66 genomic signaling pathway was observed significantly higher expression after tamoxifen treatment in ERα36 + cells.2. Tamoxifen /ERα36 transcriptionally regulates the expression ALDH1A1.Previous studies on whether the ligand binding ERα36 was remain controversial. We used computer simulation methods and ligand binding experiments to prove the combination of tamoxifen or 17-β estradiol on ERα36. In 20-40 minutes after tamoxifen treatment, we observed the translocation of ERα36 from the cytoplasm or membrane to the nucleus. We further demonstrated that the phosphorylation of ERα36-Ser132 played an important role in the nuclear translocation of ERα36. Nuclear translocated ERα36 may combine to ERE sites of the genome, which served as nuclear transcription factor to directly participate in transcriptional regulation of ALDH1A1.IV. Targeting ALDH1A1 or ERα36 to estimate tamoxifen-induced proliferation and metastasis of breast cancer.We use diethylamino benzene(DEAB) or disulfiram(DS) to inhibit ALDH1A1 activity, which can significantly reduce the self-renewal ability of breast cancer cells. We also observed obviously reduction of the tumor invasion and metastasis ability of breast cancer cells. In addition, we also found that ERα36 monoclonal antibodies significantly inhibit ERα36 mediated breast cancer proliferation and metastasis. Our findings support the potential clinical significance of targeting ALDH1A1 or ERα36.In summary, the main conclusions of this study are:I, Basic mechanism study: we firstly discovered that tamoxifen binded to and activate ERα36, and regulate estrogen genomic signaling pathway, and elevate the expression of ALDH1A1, the breast CSCs marker.1. ERα36 combines ligands(estradiol and tamoxifen), and tamoxifen works as activator.2. ERα36 activates genomic signaling pathway after phosphorylation and nuclear translocation in a ligand-dependent manner, which works as a nuclear transcription factor;3. ERα36 directly regulates the expression of ALDH1A1 to maintain the self-renewal ability of CSCs in breast cancer.II, Pathological diagnosis: we discovered that ERα36 expression in breast cancer is valuable for the molecular classification of breast cancer, and endocrine therapy medicine selection.1. ERα36 is a novel molecular marker for breast cancer metastasis, suggesting ERα36 +breast cancer is an important molecular classification;2. Tamoxifen therapy facilitates the metastasis of ERα36+ breast cancer, suggesting that tamoxifen should be removed from the therapy for ERα36 + breast cancer patients.III, Clinical therapy strategies: we discovered that ERα36 and ALDH1A1 are promising targets for breast cancer stem cells attenuation.1. Aromatase inhibitors are reasonable endocrine therapy choice for ERα36 positive breast cancer treatment, which are sensitive for ERα36+ breast cancer cells;2. Targeting ALDH1A1 and ERα36 can significantly attenuate the metastasis of breast cancer, which are potential treatment targets for metastatic breast cancer therapy.The study not only revealled the molecular mechanisms of ERα36 in tamoxifen promoted breast cancer metastasis, but also provided an important proves for breast cancer molecular classification and individualized treatment for breast cancer. |
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