The Expressions Of Hif-2a And ABCG2in Breast Cancer And The Relationship Between Them And Multidrug Resistance

[Background and objective]Breast cancer is the most common cancer in women worldwide. In China, the morbidity of the disease has increased by30%in the last10years. Therefore, how to estimate precisely the prognosis of the patients and how to choose the appropriate strategy of treatments are the key points in the breast cancer research.At present, the well acknowledged prognostic factors in breast cancer include the age of the patient, the condition of axillar lymph nodes, the size of the tumor, histological type, histological grade, clinical stage, condition of hormonal receptors (ER, PR), Ki67and HER2. In the matter of treatment, surgery is the mainstay of the treatment of breast cancer. Many patients receive adjuvant (post-operative) therapy, which reduces the risk of loco-regional and distant disease recurrence. Adjuvant treatment options include chemotherapy, radiotherapy, endocrine therapy and targeted therapy, aiming to provide maximum survival benefit with minimum toxicity. Chemotherapy plays an important role in breast cancer treatment, but researches show that about90%of chemotherapy failures attribute to multidrug resistance (MDR), and the MDR becomes the greatest obstacle of breast cancer therapy. The MDR is induced by many factors, and resistance phenotype is associated with increased expression of drug resistance related proteins that mediate energy-dependent transport of substrate drugs out of the cell against a concentration gradient. All the drug resistance related proteins are membrane proteins, which belong to ATP-binding cassette (ABC) transporters super-family. Hypoxia is an important factor involved in the progression of solid tumors and has been associated with various indicators of tumor metabolism, angiogenesis and metastasis. The presence of widespread hypoxia within tumors has been associated with reduced survival after radiotherapy or chemotherapy. Hypoxia has also been linked to poor outcome in a number of tumors regardless of the treatment modalities used. The transcriptional response to hypoxia is primarily mediated by two hypoxia-inducible factors-Hif-la and Hif-2a. While these proteins are highly homologous, increasing evidence suggests they have unique transcriptional targets and differential impact on tumor growth. Karolina Helczynska et al. found a significant association between Hif-2a protein and adverse prognosis of breast cancer, and no such association was found for Hif-la.ABCG2(ATP-binding cassette sub-family G member2), or breast cancer resistance protein (BCRP), is the second member of the G subfamily of the ABC efflux transporter superfamily that has been extensively studied, which play an important role in MDR. Two functional elements in the ABCG2promoter, the estrogen response elements (ERE) and the hypoxia response elements (HRE), and a peroxisome proliferator-activated receptor g (PPARg) response element upstream of the ABCG2gene have been shown to control ABCG2expression. It indicates that ABCG2could be associated with or regulated by Hif-2a or clinicopathological characteristics of breast cancer, such as ER et al.In this study, we firstly examined the expression of ABCG2and Hif-2a by immunohistochemistry on the tissue microarray paraffin sections of surgically removed samples from196cases of primary invasive ductal carcinoma patients with clinicopathological data, and analyzed respectively the correlations between the expressions of ABCG2/Hif-2a and the above clinicopathological characteristics, and the association between the expressions of ABCG2and Hif-2a as well by statistics. Furthermore, according to the results of correlations, in order to discuss the possible regulation relationship between Hif-2a and ABCG2, we silenced the expression of Hif-2a by small interference RNA in human breast cancer cell lines MCF-7and T47D respectively, and observed the changes of the mRNA and proteins of ABCG2and Hif-2a, and assess the changes of chemosensitivity of Hif-2a silencing cells to anticancer drug mitoxantrone. Based on the results of above study, we can draw a thesis that ABCG2could possibly be a direct downstream target of Hif-2a in breast cancer cells.[Methods]1. Tissue samples and tissue microarrayFormalin-fixed and paraffin-embedded tissue specimens of primary breast invasive ductal carcinoma from196patients in the Department of Pathology of Qilu Hospital of Shandong University were selected, in which every case’s clinicopathological data was available. For each case, two representative areas on the paraffin-embedded block were selected and punched out to make tissue microarray.2. Immunohistochemistry and analysis of corralationWe examined the expression of ABCG2and Hif-2a in the tissue microarray slices which contain196breast cancer samples by IHC2-step Detection System (PV-9000). After setting up the judge criterion of the staining, we analyzed the relationship between ABCG2/Hif-2a and the clinical pathological characters respectively, as well as the correlation between ABCG2and Hif-2a in invasive ductal carcinoma.3. Plasmids and cloningSmall hairpin small interference RNA (siRNA) sequences specifically targeted at Hif-2a and non-targeting siRNAs (as negative controls) sequences were designed and synthesized as64oligonucleotides. Then they were inserted into the pSUPER. neo+GFP expression vector respectively. After confirmed by enzyme digestion and DNA sequencing, the recombinant plasmids (pSUPER-siHif-2a and pSUPER-siN) were extracted.4. The detection of human breast cancer cell linesWe detected the level of mRNA and protein of ABCG2and Hif-2a in MCF-7、 T47D cells by RT-PCR, qRT-PCR and Western Blotting.5. Cell lines culture and transfectionThe plasmids were transfected into MCF-7and T47D respectively. After transfection, the level of mRNA and protein of ABCG2and Hif-2a were detected by RT-PCR, q RT-PCR and Western blotting. MTT assay was used to assess the effect of Hif-2a silencing on the chemosensitivity of cells to anticancer drug mitoxantrone.[Results]1. The results of immunohistochemistryA positive stain for ABCG2was defined as a brown stain observed in the cytoplasm and cytomembrane of the cells of the breast invasive ductal carcinoma, and ABCG2expression was present in different intensities and different cell distributions. Following the staging criteria of stain intensity,15cases (7.7%) were identified as completely negative,80cases (40.8%) were identified as "+",77cases (39.2%) were identified as "++", and24cases (12.3%) were identified as "+++". According to the assessment methods and evaluation criterion, combining the proportion of positively stained tumor cells, the high level, low level and negative expression of ABCG2was observed in73cases (37.2%),97cases (49.5%) and26cases (13.3%), respectively.A positive stain for Hif-2a was defined as a brown stain observed mainly in the nucleus of the cells of the breast invasive ductal carcinoma, Hif-2a expression was present in different intensities and different cell distributions. Following the staging criteria of stain intensity,12cases (6.12%) were identified as completely negative,78cases (39.80%) were identified as "+",69cases (35.20%) were identified as "++", and37cases (18.88%) were identified as "+++". According to the assessment methods and evaluation criterion, combining the proportion of positively stained tumor cells, the negative, low level and high level expression of Hif-2a was observed in25cases (12.76%),114cases (58.16%) and57cases (29.08%), respectively.2. The relationship between ABCG2/Hif-2a and the clinical pathological characters respectively, as well as the correlation between ABCG2and Hif-2a in invasive ductal carcinoma.There was no significant correlation between the expression level of ABCG2and biological factors such as patients’ age, histology type, histology-grade, tumour size, ER, PR and Ki67. In contrast, statistical analyses indicated that ABCG2expression was positively related with HER-2expression and the correlation was statistically significant. Meanwhile, the correlation between ABCG2expression and lymph node metastasis/clinical stage was significant. There was no significant correlation between the expression level of Hif-2a and biological factors such as patients’ age, histology type, tumour size, lymph node metastasis, ER, PR, HER-2expression and clinical stage. In contrast, statistical analyses indicated that Hif-2a expression was positively related with ABCG2expression and the correlation was statistically significant; meanwhile, the correlation between Hif-2a expression and histology-grade/Ki67was significant.3. The experiment results after the interference of Hif-2a by the transfection of the plasmid of pSUPER-siHif-2aRT-PCR and Western blot analyses were performed to verify the effects of Hif-2a interference. The expressions of Hif-2a mRNA and Hif-2a protein were down-regulated remarkably in comparison to the control in the cells of MCF-7/pSUPER-siHif-2a and T47D/pSUPER-siHif-2a, which certificated the effect of the interference. The expressions of ABCG2mRNA and ABCG2protein were also down-regulated remarkably, which means there is possible some kind of regulation relationship between Hif-2a and ABCG2. MTT assay showed that IC50values of the anti-cancer drug mitoxantrone decreased remarkably after Hif-2a interference. The drug resistance of the transfectted cells to mitoxantrone was significantly reversed.[Conclusion]1. The expression of ABCG2found in the majority tumor cells of invasive ductal carcinoma of breast. Its expression is correlated significantly with that of HER2, lymph node metastasis, and clinical stage in invasive ductal breast cancer. These findings suggest that ABCG2not only is one of the drug-resistance genes, but also is a novel potential bio-marker which can predict biological behavior, clinical progression, prognosis and chemotherapy effectiveness.2. The expression of Hif-2a found in the majority tumor cells of invasive ductal carcinoma of breast. Its expression is correlated significantly with that of histology-grade and Ki67in invasive ductal breast cancer. And the Hif-2a expression was also correlated with ABCG2expression significantly. These findings suggest that Hif-2a is not only one of the hypoxia inducible factors, but also a novel potential bio-marker which could predict biological behavior and worse chemotherapy effectiveness in invasive ductal carcinoma of breast.3. In the breast cancer cells, the expressions of ABCG2mRNA and ABCG2protein are changed along with the expressions of Hif-2a, combined with Hif-2a expression was correlated with ABCG2expression significantly, which suggests Hif-2a be a potent transcriptional regulator of the Abcg2gene and Abcg2be a direct downstream target of Hif-2a in the cells of invasive ductal breast cancer. Furthermore, Hif-2a participating in the MDR of breast cancer cells mediated by ABCG2, could possibly be a new mechanism. The silencing of Hif-2a in breast cancer cells can suppress the expression of ABCG2and reverse partly the MDR of the tumor cells, which hints that Hif-2a is possible to be identified as a new molecular marker to predict multidrug resistance and the target for reversing MDR mediated by ABCG2in breast cancer cells.