Expression Of Sonic Hedgehog In Breast Cancer And Its Regulation Mechanisms

IntroductionBreast cancer is one of the most common types of malignant tumor.In fact,breast cancer is the leading cause of morbidity and mortality in women.The development and progression of breast cancer are related to the alterations in genetic and epigenetic events such as mutation of oncogene,loss of tumor suppressor gene,and DNA methylation.It is well known that hedgehog(Hh) signaling is crucial for tissue development during the embryogenesis,such as the left-right asymmetry decision, anterior-posterior axis decision in limb pattern determination,and patterning of various organs,including brain,spinal cord,craniofacial structures,lung,teeth,eye,and hair. Recent data have shown an association between the Hh pathway activation and the initiation of human tumors.Constitutive activation of the Hh pathway has been found in several types of tumors,including breast cancer.Three Hh homologues have been identified in mammals,including Sonic hedgehog(Shh),Indian hedgehog,and Desert hedgehog.Among them,Shh is the hot point of research.This pathway is a highly coordinated and orchestrated network involving in inhibition of the twelve transmembrane protein Pathedl(Ptch1) by binding Hh protein,activation of the seven transmembrane protein Smoothened(Smo), release of a five-zinc finger transcription factor Gli from a large protein complex, nuclear translocation of Gli,and transcription of target genes.In the presence of Shh, Smo is activated and results in Gli released from the large protein complex which is transported into the nucleus to activate Hh target genes.A previous study indicated that the expression of Shh was up-regulated in breast cancer,suggesting Shh is involved in the initiation of breast cancer.However,the up-regulation mechanisms of Shh in breast cancer are still not known.In the present study,we investigated the expression of Shh in normal breast tissues and breast cancer tissues;analyzed the relationship between Shh expression and clinical pathology parameters;and explored the underlying mechanisms of Shh up regulation in breast cancer.Materials and Methods1.The selected paraffin embedding sections,including 119 cases of breast carcinomas and 22 cases of normal breast tissues,were obtained from 2004 to 2007 at the First affiliated hospital of China medical university.Using S-P immunohistochemis try method,we detected the expression of Shh and analyzed the score.In addition,we investigated the relationship between Shh expression and clinical pathology parameters. Furthermore,we measured the expression of Shh in three breast cancer cell lines (MDA-MB-435,MDA-MB-231,and MCF-7) and one normal breast cell line (MCF-10A) by Western blotting.2.We extracted genomic DNA from 61 cases of breast cancer tissues and 15 cases of normal breast tissues by resin.Using methylation specific PCR(MS-PCR) method, we studied methylation status of Shh promoter region in breast cancer tissues and normal breast tissues.In addition,we analyzed the relationship between methylation status of Shh promoter region and the level of Shh.Furthermore,we also detected the methylation status of four breast cell lines and the expression level of Shh in the presence or absence of 5-azacytidine,a demethylation agent,by MS-PCR and Western blotting.3.We detected the expression of NF-κB in 95 cases of breast cancer tissues and 11 cases of normal breast tissues by S-P immunohistochemistry.In addition,we investigated the relationship between the expression of Shh and NF-κB.4.We investigated the effects of Cyclopamine,a inhibitor of Shh signaling pathway, on the growth and apoptotic ability of MDA-MB-435 and MCF-7 cell lines by MTT and flow cytometry. Results1.Our data showed that Shh staining was mainly localized in cytoplasma.In normal breast tissues and breast cancer tissues,the mean score of Shh expression was 2.95+1.91 and 5.25+2.88,respectively.There was significant difference in Shh expression between normal breast tissues and breast cancer tissues(P＜0.001).There were 18 cases(81.8%) of negative Shh expression(Immunostaining Score,IS＜5) and 4 cases(18.2%) of positive Shh expression(IS＞5) in 22 cases of normal breast tissues.Whereas,there were 47 cases(48.5%) of negative Shh expression and 50 cases (51.5%) of positive Shh expression in 97 cases of breast carcinomas.Statistical analysis indicated that the expression of Shh was higher in breast carcinomas and in breast cancer cell lines.In addition,we found that the expression of Shh was positively associated with the expression of p53,but was negatively related to the progression of clinical stage.2.In an attempt to clarify the regulatory mechanism of Shh expression,we invest igated the methylation status of its promoter region in breast carcinomas and normal breast lesions.The methylation status of Shh was determined by methylation-specific PCR(MS-PCR).We found that methylation of the Shh promoter region was frequent in normal breast cells(19/33,57.6%),but very rare in carcinoma(4/28,14.3%),and significantly correlated with Shh expression(P＜0.05).Additionally,after treatment with 5-azacytidine,the methylation was reversed in MDA-MB-435 and MCF-10A cells along with the expression of Shh was increased.3.The expression of NF-κB was positively associated with the expression of Shh in breast tissues.Among 56 cases with negative expression of Shh,there were 37 (66.1%) cases with downregulation of NF-κB.On the contrary,of 49 cases with positive expression of Shh,there were 35(71.4%) cases with upregulation of NF-κB.4.MTT results indicated that Cyclopamine could inhibit the growth of MCF-7 and MDA-MB-435 cells at concentration-dependent manner after treatment for 72 h.In order to underly the mechanism of growth inhibition of Cyclopamine,we detected the cell cycle and apoptosis.We found that Cyclopamine at 20μmol/L had no effects on cell cycle and apoptosis in MCF-7 cell line.However,after treatment with Cyclopamine, the apoptosis was induced in MDA-MB-435 cells.In addition,our data showed that mitochondrial membrane potential was decreased to 37%in MDA-MB-435 cells after treatment with Cyclopamine for 72 h.Conculsion1.The expression of Shh in breast carcinoma was significantly higher than that of normal breast tissues;the expression of Shh was positively related to the expression of p53,but negatively associated with the progression of clinical stage.2.The expression of Shh was negatively related to the methylation status of Shh promoter region in breast tissues,and 5-azacytidine,a demethylation agent,could reverse the methylation status and increase the expression of Shh,suggesting promoter methylation may be an important regulatory mechanism of Shh expression in breast cancer.3.The expression of NF-κB was positively related to the expression of Shh in breast tissues,suggesting NF-κB may be another important regulatory mechanism of Shh expression in breast cancer.4.Cyclopamine can significantly inhibit the proliferation of MCF-7 and MDA-MB -435 cells at concentration-dependent manner;Cyclopamine can induce apoptosis of MDA-MB-435 cells through decreasing the mitochondrial membrane potential;Cyclo pamine has not effect on cell cycle of MCF-7 and MDA-MB-435 cells.