Detection And Expression Of FAS Correlated Signals In Human Breast Cancer Tissues

Breast cancer is one of the leading causes of death among women. The most lethal "invasive carcinoma" represents 65-80% of all breast cancers. The chances of developing invasive breast cancer during a woman's lifetime are approximately 1 in 7 (13.4%). So far, the major thrust of all efforts to cure this disease has remained confined to conventional drugs and radiation therapies, which has the well-known side effects. Before possibilities for some genetic therapies could be explored, identification of important genes associated with breast cancer is the logical first step. Fatty acid synthase (FAS) is a complex multifunctional enzyme that plays a central r01e in endogenous lipogenesis in mammals. In well-nourished adults, FAS is responsible primarily for energy storage by converting excess carbohydrate to fatty acids that are then sterified to store triacylglycerols. However, after numerous clinical and basic research studies, it now appears that human cancers constitutively express high levels of FAS and undergo significant endogenous fatty acid biosynthesis. Importantly, tumor-associated FAS over-expression and hyperactivity seem to be independent of the regulatory signals that down-regulate fatty acid synthesis in normal cells. So it's necessary to clarify the signals regulate the expression of FAS in human breast cancer.Objectives:1,To detect the expression variation of different members of ErbBs, PI-3Ks and FABPs that are expected to associated with FAS signal pathway in breast cancer.2,Try to find the upstream and downstream molecules related to the FAS signal pathway.3,To discussed the molecular mechanism of the FAS signaling in ductal infiltrating carcinomas (DICs).4,To find potential markers and therapeutic targets for human breast cancer.Methods:1. Collected Data (the expression windows where the genes are located and the length of the genes)of the target genes (ErbB and PI-3K family members) in database (http://www.qbio-gene.com/displayfit/)supplying by Qbio-gene Inc.MP Biomedicals, Inc. Amplified the genes with corresponding primers of the expression windows by RFDD-PCR technique.2. Separated the obtained fragments of the target genes by electrophoresis on 7% urea denaturing polyacrylamide gel and scanned with Typhoon9200 Image system. Analyzed Image of the gels by the software of ImageQuant TL, Image Tool, Fragment Analysis.3. Compared the expression differences of target genes (ErbBs and PI-3Ks) between DICs tissue and adjacent normal tissue by RFDD-PCR and RT-PCR. Compared expression differences of these genes between positive and negative groups of FAS by RT-PCR.4. Amplified FAS and FABPs genes by RT-PCR (because they were not available in the gene database) in 76 DICs and 16 fibroadenoma of breast.5. Detected the spatial and temporal expression differences of the FAS associated proteins such as ErbB-1, ErbB-2, E-FABP, H-FABP, L-FABP and VEGF by immunohistochemical staining in different breast tissues and in different grades of 76 DICs.6. Surveyed the quantitive differences of the proteins expressions by Western blot analysis.7. Compared the means by T test. Analyzed the correlation between two samples by x² test. P＜0.05 was regarded as statistical significance.8. Results:1. The expressions of all other members of ErbBs were up-regulated significantly in DICs comparing with those in adjacent normal tissue except for ErbB-4. The levels of EGFR (ErbB-1) and ErbB-2 were elevated significantly in FAS positive group comparing with those in FAS negative group. Furthermore, we found significant correlation between the protein expressions ofErbB-1/ErbB-2 and those of FAS.2. The expressions of PIK3CA, PIK3CB, PIK3C2A, PIK3C2B and PIK3CG were up-regulated significantly in DICs comparing with those in adjacent normal tissues, whereas, PIK3R1, PIK3R3 and PIK3R4 were down-regulated significantly. Moreover, expression levels of PIK3CA and PIK3CB were increased significantly in FAS positive group comparing with those in FAS negative group.3. The expressions of E-FABP, H-FABP and L-FABP were up-regulated significantly in DICs comparing with those in benign tissues, however, A-FABP, B-FABP, I-FABP and G-FABP showed no expression differences between these two kinds of breast tissues.4. The expressions ofFAS, E-FABP, H-FABP and L-FABP were associated with the grades of DICs, but were not correlated with the age, lymph nodes metastasis, ER and PR status.5. The expression levels of FAS, E-FABP and H-FABP were down-regulated significantly in gradeâ…¢comparing with those in gradeâ… and gradeâ…¡DICs, furthermore, the protein expressions of E-FABP and H-FABP were correlated with that of FAS.6. The expressions of L-FABP, VEGF were up-regulated significantly in gradeâ…¢comparing with those in gradeâ… and gradeâ…¡DICs, moreover, the protein expression of L-FABP was correlated with that of FAS, but was not correlated with the expression of FAS.Conclusions:1. It is suggested that amplification or over-expression of FAS, ErbB-1,ErbB-2,ErbB-3 may related to the neopiasia and development of DICs. Moreover, as the upstream of FAS signal, ErbB-1 and ErbB-2 regulate the expression of FAS.2. It is implied that amplification of PIK3CA, PIK3CB, PIK3C2A, PIK3C2B, PIK3CG and down-regulation of PIK3R1, PIK3R3 and PIK3R4 genes are related to the proliferation, survival, adherence, migration and metastasis of DICs cells.3. The closed relation of PIK3CA, PIK3CB with ErbB-1, ErbB-2, FAS suggests that PIK3CA, PIK3CB conduct the signal from the ErbB-1,ErbB-2 to FAS through the phosph0rylation of PI-3K substrate.4. The intimate association of FAS expression and E-FABE H-FABP in gradeâ… andâ…¡DICs suggests that E-FABP and H-FABP might act as the downstream of FAS to combine and transport the long chain fatty acids synthesized by FAS.5. Accompanying with the down-regulation of FAS in gradeâ…¢DICs, the up-regulation of L-FABP and VEGF may compensate the requirement for O₂ and fatty acids of DICs cells..