Expreesion Of GCS And Its Methylation Status In Invasive Ductal Breast Cancer

Background and objectivesBreast cancer is the most frequent cancer to threaten women’s health. Besides surgical methods, chemotherapy, endocrine and molecular targeted therapy are fundamentals of breast cancer treatment. MDR to chemotherapeutic agents is a major cause for the treatment failure of breast cancer. MDR refers to the resistance of tumors not only to individual cytotoxic drugs used in chemotherapy, but also to cross-resistance to a range of drugs with different structures and cellular targets. MDR1/P-gp, a product of multidrug resistance gene-1(mdr-1), belongs to ABC (adenosine triphosphate-binding cassette) transporter family, acts as a drug efflux pump, lowers the intracellular drug levels to sublethal concentrations and helps cells to escape from death. P-gp is expressed in almost50%of breast MDR cancers. Recently, many studies have indicated that mdr-1is up-regulated by glucosylceramide synthase (GCS), which is a pivotal enzyme in the regulation of cellular ceramide. GCS transfers UDP-glucose to ceramide to form glucosylceramide (GlcCer) and allows cells to escape from ceramide-induced cellular apoptosis. Recently, accumulating evidence has pointed towards an important role for GCS in MDR. GlcCer is modified by a series of glycosyltransferase and synthethizes higher forms of glycosphingolipids. GlcCer is a characteristic of some MDR cancer cells. Neoadjuvant chemotherapy (NC) is the chemotherapy before local therapy of cancers. It is demonstrated that NC could up-regulate MDR associated protein expression such as P-gp, MRP, LRP and induce MDR of cancers. However, there are currently few reports describing the expression of GCS or whether chemotherapeutic agents could modulate the expression of GCS.In part one of this research, we examined the expression of GCS in invasive ductal breast carcinoma tissue samples, and investigated its associations with the clinical characteristics, molecular subtypes and prognosis. We also analyzed the relationship between NC of locally advanced breast cancer samples and GCS expression.Even though genetic aberrant plays critical roles, it’s not sufficient to explain the mechanism of carcinogenesis. Epigenetics is becoming one important field of cancer research. DNA methylation is the predominant epigenetic gene expression modification. DNA methylation plays a key role in gene expression, cell differentiation and carcinogenesis. Moreover, increasing evidence indicates that aberrant methylation of gene may involve in chemoresistance. Demethylation of CpG islands in the mdr-1promotor region is a mechanism of chemoresistance, which induces the expression of MDRl/P-gp. GCS gene promotor region is full of G+C sequences. However, there is no relevant research on GCS methylation in breast cancer. In part two of this research we randomly collected40GCS positive and40GCS negative samples from200cases of invasive ductal breast carcinoma samples. We reported the GCS methylation status to explore whether GCS methylation was associated with the clinicopathological features and GCS expression. We also investigated the GCS methylation status, GCS mRNA and protein expression in breast cell lines MCF-7(ER positive), MDA-MB-231(ER negative), T47D (PR positive) and MCF-7/ADM (GCS overexpression). Then we observed whether demethylation intervention5-aza-2’-deoxy-cytidine (5-Aza-dc) could change the GCS promotor methylation status and regulate the GCS mRNA and protein expression in order to further investigate the mechanism of GCS expression. If methylation of GCS promotor could be used as a target for resistance reversal, it could provide guidance for clinical use.Methods and resultsChapter I Expression of MDR associated gene GCS in breast cancerMethods:1. We collected200cases of breast invasive ductal carcinoma surgery samples and their clinical data from June2007to January2009in Jinan Central Hospital Affiliated to Shandong University and Yuhuangding Hospital Affiliated to Qingdao University. They did not receive radiation therapy or chemotherapy before biopsy or surgical resection. Immunohistochem istry (IHC) analyses were conducted to determine the expression of GCS.2. All calculations were performed with the SPSS17.0for windows statistical software package (SPSS, Chicago, IL, USA). Chi-square test was used to analyze the relationship between the expression of GCS and each histopathological variable. All patients were followed up. The median follow-up period was57.9months (range38to60months). Five year disease free survival (5-y-DFS) and five year overall survival (5-y-OS) curves were plotted using the Kaplan-Meier method. Given P-values less than0.05, the result was considered statistically significant.3. We collected60cases of locally advanced breast excision biopsies from patients who had undergone preoperative chemotherapy from January2012to January2014in Yuhuangding Hospital Affiliated to Qingdao University. IHC was used to analyze the expression of GCS before and after NC.Results:1. In the invasive cancers, a lower positive rate of GCS expression was observed in younger patients compared with that in older patients (p=0.033). We also found that the positive expression of GCS was much higher in grade I than that in grade II-III (p=0.038). And there was a significant correlation between the GCS upregulation and ER positivity (p=0.030) or HER-2negativity (p=0.002), but there was no statistical significance in the relationship between GCS expression and other clinicopathological parameters, including tumor size, nodal stage, Ki67.2.200invasive breast carcinomas were grouped into102luminar A subtype cases,39luminar B subtype cases,40basal-like subtype cases and19HER-2enrich subtype cases. The positive rate of GCS was highest in luminar A tumors and was lowest in basal-like tumors. The GCS expression levels were different in luminar A and basal-like tumors (p=0.038). And there were no difference among luminar A and other two groups (p>0.05).3. Kaplan-Meier survival curves for patients with invasive ductal breast carcinoma were calculated according to GCS protein expression status. There was no relationship between GCS positive expression and5y-DFS or5y-OS.4. Overall,15%(9/60) of all locally advanced breast cancer samples were positive for GCS. After NC, the percentage of GCS was41.7%(25/60),(p=0.002). Chapter ⅡGCS methylation status in breast cancerMethods:1. We randomly collected40GCS positive and40GCS negative samples from200invasive ductal breast carcinoma patients. Methylation-specific PCR (MSP) was used to measure the methylation status of GCS in breast cancer patients. Correlation analysis was performed between the methylation status and clinical characteristics or GCS expression with SPSS17.0. Given P-values less than0.05, the result was considered statistically significant.2. In breast cancer cell lines, MSP analysis was performed to measure the GCS methylation status. GCS mRNA and protein expression were detected by reverse transcription real-time quantitative PCR (qRT-PCR) and westernblot (WB). To further test the association between GCS expression and GCS methylation, DNMTs inhibitor5-Aza-dc was used to treat different cell lines and compare the different expression of GCS before and after treatment. Changes of methylation status, mRNA and protein expression of GCS were detected by MSP, qRT-PCR and WB.Results:1. In breast cancer tissues, the frequency of GCS CpG islands mehtylation status in GCS positive group and negative group is25%(10/40) and87.5%(35/40), respectively. There was significant statistical difference between these two groups (r=-0.63,p<0.01).35.2%(19/54) of the estrogen receptor positive group exhibited lower methylation levels of GCS CpG island compared to61.5%(16/26) of the estrogen receptor negative group (r=-0.249, p=0.026).45.3%(24/53) of the HER-2receptor negative group exhibited lower methylation levels of GCS CpG island compared to77.8%(21/27) of the HER-2receptor positive group (r=0.31,p=0.006). There was a negative correlation between the GCS methylation status and GCS expression or ER positivity, but positive correlation with HER-2positive. And there was no statistical significance in the relationship between the GCS methylation and other clinicopathological parameters, including age, histological differentiation, tumor size, nodal stage or Ki67.2. No methylation of GCS gene was detected in MDA-MB-231cell line. Complete methylation of GCS gene was detected in MDA-MB-231cell line. Partial methylation was detected in MCF-7and T47D cell lines. The mRNA and protein levels of GCS gene in MCF-7/ADM cells were the highest among four different cell lines. The mRNA and protein levels of GCS gene in MDA-MB-231cells were the lowest among four different cell lines.3. Complete methylation of GCS gene was detected in MDA-MB-231cell line and was completely reversed by5-Aza-dc. Consistently, GCS mRNA and protein expression were increased. Methylation was detected in MCF-7and T47D cell lines and was partially reversed by5-Aza-dc. No change was found in MCF-7/ADM cell line.Conclusion1. In invasive ductal breast carcinoma, the expression of GCS is related to estrogen receptor (ER) and HER-2status.2. The GCS expression levels were different in luminar A and basal-like tumors.3. There was no statistical significance in the relationship between GCS expression and5y-DFS or5y-OS.4. The expression of GCS was significantly increased after NC.5. There was a negative correlation between the GCS CpG island methylation status and GCS expression or ER positive. And there was a positive correlation between the GCS CpG island methylation status and HER-2positive.6. Demethylation agent5-Aza-dc can lead to demethylation of GCS gene promotor CpG island, thus up-regulate the expression level of GCS. GCS promotor methylation can modulate the expression of GCS and may play an important role in MDR.