| BACKGROUNDAs the most common malignancy in females worldwide, breast cancer with its ever increasing incidence in recent decade, severely threatens the health of women. It is a highly heterogeneous disease and can be categorized into ER positive and ER negative subgroups according to the estrogen receptor status or into luminal A, luminal B, basal-like, HER-2enriched and Normal-like subgroups based on genomic test. Although there is a variety of therapeutic choices for these patients, the fundamental importance of chemotherapy cannot be surpassed, especially with the advent of neoadjuvant chemotherapy which can downgrade the patients in T2or T3to increase their chance of complete resection of tumor or receiving breast conserving surgery and in turn having better life quality. Recent researches revealed that the overall chemotherapy response of ER positive breast cancer patients is not ideal except a proportion of chemosensitive populations in this subgroup despite the lack of understanding of its underlying mechanisms. Therefore evidence based medicine(EBM) weakly recommends its use in these ER positive patients and support the application of neoadjuvant endocrine therapy which is proven to have better efficacy in this subgroup. However, part of the ER positive patients have drug resisrence, relapse or metastasis during treatment with pre-or postoperative endocrine therapy anyway. Both the EBM and clinical routines highly recommend the use of chemotherapy in this situation since these endocrine resistence patients could possibly respond to chemotherapy. Due to the fact that there is no molecular biomarker or clincal pathological surrogate to predict the efficacy of chemotherapy in ER positive breast cancer patients effectively, it is usually difficult to choose the appropriate regimen either postoperative or after recurrence for them. Considering the large amount of ER positive patients, the number of the patients who could benefit from certain chemotherapy regimen is conceivable. Therefore, it is important to find an effective biological marker to predict the therapeutic efficacy of chemotherapy and prognosis of ER positive breast cancer patients, and screen out the sensitive ones who could benefit from effective chemotherapy. FOXA1, also named HNF3a(hepatic nuclear factor3a), is a member of Forkhead box winged-helix transcription factor family. It is found to be expressed higher in ER positive breast cancer cells than in normal breast tissues and can serve as a pioneer factor for ER to influence its combination with DNA. In this thesis, we plan to explore the possible predictive value of FOXA1for therapeutic efficacy of chemotherapy in ER positive breast cancer patients. Firstly, our study wants to explore the correlation between expression of FOXA1and chemotherapeutic efficacy in neoadjuvant chemotherapy settings since it is considered as an effective drug sensitive model to screen out the sensitive patients and their effective regimens. Secondly, we plan to explore the preliminary underlying mechanisms through in vitro tests for this association if it truly exists. Thirdly, we plan to clarify the prognostic relevance of FOXA1in ER positve patients who had received neoadjuvant chemotherapy in past.METHODS AND RESULTS1. Correlation analysis between FOXA1and the pathological response of ER positive breast cancer following neoadjuvant chemotherapy In total, there are123cases of ER positive breast cancer patients who had complete data record and core needle biopsy before neoadjuvant chemotherapy in our hospital from August2002to December2012. At a total of123cases, pathological response(no more than10%tumor residual) was observed in25.2%(31/123) patient while pathological nonresponse(more than10%tumor residual) in74.8%(92/123) of patients. The expression of FOXA1in ER positive breast cancer patients was correlated with pathological response by chi-square statistical analysis (P=0.002), and high FOXA1expression levels were more frequently found in nonresponding patients. Besides that, FOXA1expression was not associated with ER by the same statistical approach(p>0.05), suggesting the possible predictive value of FOXA1in chemotherapeutic efficacy of ER positive breast cancer patients independent of ER expression. However, the mechanism of whether and how FOXA1can lead to chemoresistance is still unknown.2. The characteristic of chemoresistance and its underlying mechanism of FOXA1in ER positive breast cancer cell linesMTT was performed in ER positive breast cancer cell lines MCF-7, T47D and Adriamycin induced chemoresistent cell line MCF-7/ADR using the most widely applied regimen in breast cancer treatment, adriamycin and paclitaxel. The Western blot assay were performed to evaluate FOXA1expression in the above cell lines at the protein level. The results showed that chemosensitivity from high to low is MCF-7, T47D, MCF-7/ADR respectively whereas the FOXA1expression level from low to high is also MCF-7, T47D, MCF-7/ADR respectively, indicating a correlation between FOXA1expression and chemosensitivity in ER positive breast cancer cell lines.Then we used lentiviral expression system to transfect MCF-7cells with FOXA1plasmid (pMD2G、pSPAX2、pSI-FOXA1) and vector plasmid (pMD2G、pSPAX2、 pSI-NEG) with subsequent Puromycin selection and confirmation by Western blot Choose MCF-7/FOXA1-15(MF15) and MCF-7/FOXA1-20(MF20) as overexpression clones, MCF-7and MCF-7/vector as negative control, and T47D, MCF-7/ADR as positive control. In the above six cell lines, MTT assay were performed to evaluate the drug sensitivity and growth curve, flow cytometry to detect any changes in cell cycle and apoptosis, colony formation assay to test the clonal formation, Transwell to test migration and invasion ability. The results showed that the chemosensitivity in negative control group was better than positive control and overexpression clones. Overexpression or high level of FOXA1can promote the cell cycle progression from G1phase to S/G2phase, inhibit apoptosis, promote proliferation, increase clonal formation and ability of migration and invasion.We then used siRNA interference technology to knockdown FOXA1in MCF-7, T47D and MCF-7/ADR followed by confirmation through RT-qPCR and Western blot assay. Then we tested the chemorensitivity and functional changes in the above cell lines by using the same approaches as mentioned above. The results showed that after FOXA1knockdown, the chemosensitivity of MCF-7, T47D and MCF-7/ADR were increased, cell cycle was blocked at G1phase, apoptosis was increased, proliferation was slowed down, the abilities of colony formation, migration and invasion were impaired.We next examined the preliminary mechanism of FOXA1in mediating the above changes related to chemosensitivity using Western blot and RT-qPCR assay. The results showed that overexpression or high levels of FOXA1could lead to upregulation of GST-pi, cyclinE1and Bcl-xl whereas downregulation of p53, p27, p21and cyclinD1both at mRNA and protein levels. Knockdown of FOXA1caused the opposite changes in these genes. Additionally, the change in expression of FOXA1can influence the protein level of TGF-β and phosphorylated AKT and MAPK(p38).Therefore we assumed that FOXA1may influence the possible signaling pathways including TGF-β, AKT and MAPK(p38), the expression level of p53、p27、p21、 cyclinE1、Bcl-x1、GST-pi, and their downstream biological functions such as cell cycle control, apoptosis and proliferation. And the above changes may all contribute to the chemosensitivity of ER positive breast cancer cell lines to chemotherapy including but not limited to adriamycin and paclitaxel.3. The expression variation of FOXA1around neoadjuvant chemotherapy in ER positive patients and relevant prognostic analysisSelect110cases who had operative tissue sections out of123patients being studied in the first part of this thesis. Use the same approach as the first part to stain and score FOXA1in these operative samples. Statistical analysis revealed no significant changes neither in positive rate nor in expression levels before and after neoadjuvant chemotherapy whereas the staining position changed obviously from mainly nuclear to both cytoplama and nuclear. However, this change did not correlate with chemotherapeutic efficacy or prognosis of these ER positive patients.The Chi-square test found that FOXA1expression defined by Histoscore more than3is not correlated with any clinicopathological variables including age, menstrual status, histological categories, lymphovascular embolus, nerve invasion, cancer nodular formation, pathological stages, IHC staining of ER, PR, HER-2, Ki67, E-cadherin, p53and luminal subtypes. Kaplan-Meier survival analysis also revealed no prognostic relevance of FOXA1expression in ER positive, luminal A or luminal B breast cancer patients.By increasing the criteria of FOXA1expression, we found that FOXA1histoscore no less than20and less than20can separate ER positive and luminal A breast cancer patients into groups with different prognosis, and serve as an independent prognostic marker. Although this grouping did not correlate with chemoresponse, it was associated with age and menstrual status.CONCLUSIONOur study revealed that FOXA1expression correlated with chemosensitivity of ER positive breast cancer patients, and could possibly serve as a predicative marker for these patients. In vitro test indicated that FOXA1may influence the possible signaling pathways including TGF-β,AKT and MAPK(p38), the expression level of p53、p27、 p21、cyclinE1、Bcl-xl、GST-pi, and their downstream biological functions such as cell cycle control, apoptosis and proliferation. And the above changes may all contribute to the chemosensitivity of ER positive breast cancer cell lines to chemotherapy including but not limited to adriamycin and paclitaxel. Retrospective study showed that the staining position of FOXA1changed after neoadjuvant chemotherapy without other clinical relevance. FOXA1histoscore no less than20and less than20can separate ER positive and luminal A breast cancer patients into groups with different prognosis, and serve as an independent prognostic marker. |
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