Study Of β-catenin And APC Genes In Breast Cancer

The development and progression of breast cancer correlates with a number of genetic defects, including activation of proto - oncogenes and inactivation of tumor - suppressor gene. Such defects can initiate inappropriate intracellular signals that result in tumorigenesis. β— catenin plays two important roles in cells, as a adhesion protein associated with E - cadherin and a - catenin in cell — cell adhesion and maintenance of cellular shape, and as a transcriptional activator in Wnt signaling which is associated with occurrence of many tumor. In normal mature cell, most of cytosolic β- catenin integrate with E - cadherin and α-catenin to constitute cadherin — catenin complex, while the remnants of cytosolic β — catenin combined with destructive complex composed of tumor — suppressor protein APC, glycogen synthase kinase -3β ( GSK -3β) and Axin. The main function of the destructive complex is to phosphorylate β - catenin and thereby triggers its ubiquitination and subsequent degradation in proteasomes. In this way, cytosolic β- catenin is kept at a very low level. Wnt signal activation results in accumulation of cytosolic B - catenin and subsequent translocation of β-catenin to the nucleus, where it binds to T cell factor (TCF)/lymphoid enhance factor ( LEF) of transcriptional factors to activate downstream target gene, such as oncogene c - myc, cyclin D1, therefore results in abnormal proliferation and differentiation of cell and malignant tumorigenesis. In this study, we detected change of β- catenin and APC genes in normal breast tissues around tumor, breast cancer tissue, breast hyperplasia of usual type and atypical hyperplasia, and discussed the role of the two genes in breast tumorigenesis and development.MethodsExperiment 1. Expression of E - cadherin - catenin complex in breast cancerThe expression of E - cadherin, α- catenin and β- catenin in specimens of 42 breast cancers and normal breast tissues around tumor, 15 breast hyperplasia of usual type and 15 breast atypical hyperplasia was detected by immunohisto-chemical method.Experiment 2. mutation of p - catenin gene in breast cancer Mutation of exon 3 of β - catenin gene in 42 breast cancers, 15 breast hyperplasia of usual type and 15 breast atypical hyperplasia was detected by poly-merase chain reaction - single strand conformation polymorphism. Experiment 3. Research of APC gene in breast cancer Expression of APC protein in specimens of 42 breast carcinomas and normal brease tissues around tumor, 15 breast hyperplasia of usual type and 15 breast a-typical hyperplasia was detected by immunohistochemical method. Mutation of mutation cluster region in APC gene was detected by polymerase chain reaction - single strand conformation polymorphism. Loss of heterozygosity of APC gene and methylation status of APC gene promoter 1A were investigated by polymerase chain reaction - restriction fragment length polymorphism and methylation -specific PCR.ResultExperiment 1. Expression of E - cadherin - catenin complex in breast cancerIn normal breast tissue and breast hyperplasia of usual type, E - cadherin, α- catenin and β- catenin were expressed on cell membrane of ductal and acinic cells, showing cellular contour and border among cells. Abnormal expression of E - cadherin ( n = 1) , a - catenin ( n = 2) and |3 - catenin ( n =4) took place in breast atypical hyperplasia, the difference in abnormal expression rateof β- cateniii between hyperplasia of usual type and atypical hyperplasia was significant(P <0.05). In 42 cases of breast cancer, the abnormal expression rates of E - cadherin,α - catenin and β- catenin were 54. 8% , 64. 3% and 59. 5% , respectively. The difference in abnormal expression rate of three proteins between breast cancer and breast hyperplasia of usual type and atypical hyperplasia was significant (P < 0.05). Abnormal expression of a - catenin and β-catenin were significantly correlated with histological grade, abnormal expression rate of α - catenin in grade III was higher than that in grade I ( P < 0. 05 ) , and abnormal expression rate of β - catenin in grade II and III was higher than in grade I ( P < 0. 01). Abnormal expression of β - catenin in group of axillary lymph node metastasis was higher than that in group of negative axillary lymph node metastasis ( P < 0. 05 ). Abnormal expression rate of β -catenin increased with increase of TNM stage, the difference in abnormal expression rate of β- catenin between stage III and stage I was significant(P <0. 01). Abnormal expression of β- catenin was negatively correlated with expression of estrogen receptor(r = -0.308, P <0.05).Experiment 2. mutation of β - catenin gene in breast cancer Mutation of exon 3 of β- catenin gene was not identified in any of breast cancers, breast hyperplasia of usual type and atypical hyperplasia by PCR — SS-CP.Experiment 3. Research of APC gene in breast cancer Expression of APC protein was detected by antibody c -20 and N -15 respectively recognizing C - and N - terminus of APC protein. In normal breast tissue, breast hyperplasia of usual type and breast atypical hyperplasia, positive staining particles mostly focused on cytoplasm of ductal and acinic cells, positive expression rate being 100%. Negative expression of APC protein appeared in breast cancer. For antibody C -20 and N - 15, negative expression rate of APC protein in breast cancer was 47.6% (20/42) and 42.9% (18/42) , respectively. The difference in negative expression rate of APC protein between breast cancer and normal tissue, breast hyperplasia of usual type and atypical hyperplasia was significant (P<0.05). The results detected by two antibodies were identical in breast cancer( P <0.05). Expression of APC protein was positivelycorrelated with expression of β— catenin. Mutation of MCR of APC gene was not identified in any of breast cancers, breast hyperplasia of usual type and atypical hyperplasia by PCR - SSCP. Hypermethylation of CpG islands of APC gene promoter 1A was detected in 16 of 42 breast cancers. Unmethylated APC alleles were also present in 35 breast cancers. Only unmethylated APC alleles were detected in 42 normal brease tissues around tumor, 15 breast hyperplasia of usual type and 15 breast atypical hyperplasia. Difference in hypermethylation rate of APC promoter between breast cancer and normal tissue, breast hyperplasia of u-sual type and atypical hyperplasia was significant ( P < 0. 05 ). Hypermethylation of APC gene was negatively correlated with expression of APC protein in breast cancer. In 42 breast cancers, 24 cases were heterzygote, 8 cases being LOH. There was no correlation between hypermethylation of APC gene with ' LOH of APC gene. Hypermethylation of APC gene and expression of APC protein were not correlated with ages, tumor size, stage, histological grade, axillary lymph node metastasis, ER, PR and HER -2.Conclusion1. During breast tumorigenesis and tumor progression, E - cadherin - catenin complex is destroyed and abnormal expression of β - catenin play an important role. Abnormal expression of E — cadherin - catenin complex is correlated with differentiation disturbance and metastasis.2. Mutation of β - catenin gene in human breast cancer is very rare. Expression of β - catenin can be regulated by other mechanisms.3. Hypermethylation of CpG islands of APC gene promoter 1A is a cancer - specific change in the breast cancer, which may be common mechanism of in-activation of APC gene in primary breast cancer.4. In breast cancer, abnormal expression of β-catenin may be associated with negative expression of APC protein.5. β- catenin signal is pivot in Wnt signaling pathway, which is also regulated by other components, such as GSK -3β, Axin and disheveled. Therefore, relation between abnormal expression of β- catenin and other signaling compo-...