FAM83A/阻断肽调控Wnt/β-catenin信号通路及其对胰腺癌发展进程的作用机制研究

Wnt/β-catenin通路对胰腺癌的发展进程起着重要作用。我们前期发现，干扰FAM83A后肿瘤增殖及裸鼠荷瘤能力均显著降低，且FAM83A明显上调Wnt/β-catenin通路靶基因并与β-catenin降解复合体作用而抑制其组装。当Wnt3a刺激后，FAM83A被诱导入核而导致HDAC1/2与TCF4复合体解离。据此推测，FAM83A极有可能抑制β-catenin降解复合体组装而影响核内TCF4的转录活性。本课题拟研究：①FAM83A通过β-catenin降解复合体调控Wnt通路的分子机制；②FAM83A联合β-catenin/TCF4调控基因转录的分子机制；③FAM83A-β-catenin阻断肽的筛选及在斑马鱼模型胰腺癌转移进程的作用；④小鼠模型研究FAM83A调控Wnt/β-catenin通路对胰腺癌进程的作用。本项目的研究为临床上胰腺癌的有效治疗及创新药物研发提供全新的思路。

Pancreatic cancer is one of the most lethal malignant tumors with an extremely low five-year survival rate and known as the king of cancer. Functional evidence is accumulating that implicates a supporting role for Wnt/β-catenin signal pathway in PDAC maintenance and progression. In our study of 61 commercial pancreatic cancer tissue chips, we found that the expression level of FAM83A protein was significantly higher than that of normal tissues, and was correlated with the malignancy of pancreatic cancer stage. After subcutaneous injection of FAM83A shRNA expressing pancreatic cancer PANC-1 cells into a 4-week-old nude female mouse, we found that the growth of FAM83A-interfering cells was significantly lower than normal cells and the tumors were smaller. The volume and weight of subcutaneous tumorigenesis after 35d were significantly lower than that of the normal control group. We also found that the expressions of Wnt/β-catenin target proteins CyclinD1, C-myc and Axin2 were decreased after FAM83A depletion, and there was a dose-dependent relationship with the level of FAM83A. We further found by Co-IP and GST pull-down assays that FAM83A interacts with Axin1, GSK3β and β-catenin. There is a significant decrease in ubiquitinated β-catenin after overexpression of FAM83A, and a dose-dependent decreased β-catenin in GSK3β drop-down content after overexpression of FAM83A. This indicated that FAM83A could affect the stability of the β-catenin protein and the assembly of the β-catenin degradation complex. Moreover, in the PNAC-1 cells overexpressing FAM83A, after nucleo-cytoplasmic separation of cell lysates, we found that with the stimulation of Wnt3a, the protein of FAM83A was significantly increased in the nucleus, at the same time, FAM83A was found interacting with transcription factors TCF4 and Pygo2. Additionally, the interaction between HDAC1/2 and TCF4 was much more enhanced after FAM83A depletion, indicating that FAM83A was likely involved in transcriptional regulation of the Wnt signaling pathway. Based on these preliminary results, in this proposal, we will use cultured cell, zebrafish and transgenic mouse models of pancreatic cancer, in combination with molecular biology, domain mapping, mutagenesis, CRISPR-Cas9 knockout, and over-expression to determine whether and how FAM83A regulates the development of pancreatic cancer through affecting the assembly of β-catenin degradation complex and the transcriptional activity of TCF4 in the nucleus, and its role in pancreatic cancer progression. For this purpose we will perform the following experiments in three aspects: .1) We propose to investigate whether the regulation of FAM83A on the level of β-catenin in the cytoplasm through the stability of β-catenin protein and the assembly of the β-catenin degradation complex. Further, we propose to investigate whether FAM83A influences the activity of TCF4 transcriptional factor and the acetylation modification level in the promoter region of Wnt target genes at the cellular level;.2) Screen the blocking peptide inhibiting the interaction between FAM83A and β-catenin and verify the effect of the blocking peptide on cell migration in zebrafish transplanted tumor model; .3) Examine whether the FAM83A-β-catenin interaction mediates the regulation of Wnt/β-catenin pathway and pancreatic cancer progression in FAM83A+/- knockout mice, and verify the effect of the above blocking peptides on the occurrence and development of pancreatic cancer in mouse model. Successful completion of experiments outlined in this proposal will enhance our current understanding of the mechanism of how FAM83A promoting the activity of Wnt/β-catenin in pancreatic cancer and its important role in the occurrence and development of pancreatic cancer, and to provide a new idea for the effective treatment of pancreatic cancer in clinical practice and the development of innovative drugs.