Role Of ASPP1 In The Regulation Of Epithelial-Mesenchymal Transition,Invasion And Metastasis In Colorectal Cancer

Part ? the association between ASPP1 expression and clinical characteristics in colorectal cancer(CRC)Objective: To analysis ASPP1 expression in paired human CRC and adjacent normal tissues and evaluate the relationship between expression of ASPP1 and clinical characteristics.Methods: A colorectal tissue microarray with 86 matched pairs of primary CRC samples and adjacent normal tissues were conducted IHC analysis.We assessed the correlation between ASPP1 expression and clinical characteristics.Results: We demonstrated that ASPP1 was expressed in the nucleus and cytoplasm,both in normal and CRC tissue.However,we determined that ASPP1 expression was significantly lower in both the nucleus and cytoplasm in CRC,compared to adjacent normal tissue.Next,we analyzed the correlation between expression of ASPP1 and clinical pathological factors by categorizing tissues into high and low expression groups according to both clinical stage and lymph node stage.Low nuclear,but not cytoplasmic,expression of ASPP1 was significantly correlated with both lymph node metastasis and higher clinical stage.Conclusion: ASPP1 expression was reduced in CRC and low ASPP1 expression was correlates with higher clinical stage,indicating that lower nuclear ASPP1 expression may promote invasion and migration in CRC.PART ? the function of ASPP1 in KRAS-mutation colorectal cancer in vivo and vitroObjective: To explore the role of ASPP1 in invasion and migration of colorectal cancer in vivo and vitro.Methods: We stably expressed control or ASPP1(PPP1R13B)sh RNA in HCT116 cells and then utilized transwell invasion and migration assays to investigate the variation of invasion and migration in colorectal cancer cells.We conducted 3D acini cultures of MCF10 A ER:HRAS V12 cells to detect RAS-induced invasion by immunofluorescence.Next,we built a mouse model to examine whether ASPP1-depleted cells have more metastatic potential by using tail vein injection of firefly luciferase tagged HCT116 cells with ASPP1 sh RNA or control sh RNA.Moreover,we knocked down ASPP1 in the HCT116 cell line to find target genes regulated by ASPP1 via Western Blot and PCR techniques.Last,we constructed HKe-3 ER: HRAS V12 cell line to confirm the target gene by immunofluorescence staining.Results: In both Transwell migration and invasion assays,depletion of ASPP1 significantly promoted invasion and migration in HCT116 cells.Similar results have been demonstrated in a MCF10 A derivative cell line,MCF10 A ER:HRASV12,which was engineered to express ER:HRAS.Addition of 4-hydroxytamoxifen(4-OHT)acutely activates the RASpathway in this cell line.Lung fluorescence signals of mice from ASPP1 sh RNA group were significantly more intense.The area of metastatic clusters present in the lungs of mice injected with ASPP2 sh RNA/HCT116 cells was significantly higher than the group injected with control sh RNA/HCT116 cells,as examined by hematoxylin and eosin(H&E)staining.We successfully knocked down ASPP1 in HCT116 and western Blot and PCR results showed that Snail2 was significantly up-regulated in down-regulating ASPP1 cell lines.Conclusion: ASPP1 inhibits RAS-induced invasion and ASPP1 may play a critical role in inhibiting invasion and metastasis of colorectal cancer cells.PART ? ASPP1 inhibition induces expression of Snail2 via NF-?B pathway to facilitates oncogenic RAS-induced invasionObjective: To investigate the target genes that ASPP1 regulates the invasion and metastasis of colon cancer and explore the pathways that ASPP1 regulates the target genes.Methods: The TCGA database was analyzed to find relevant pathway.Next,the reporter gene and co-immunoprecipitation method were used to confirm the molecular regulation mechanism of ASPP1.Results: We analyzed the TCGA database and found that ASPP1 is likely to regulate Snail2 expression through the NF-?B pathway.Next,we used NF-?B reporter gene detection to find that NF-?B activity in HCT116 transfected with ASPP1 si RNA was significantly increased compared to the control si RNA group.Finally,we knocked down ASPP1 in HCT116 cells and performed co-immunoprecipitation.The results showed that the binding between NF-?B1 p50 and p65 increased significantly after ASPP1 was depleted.At the same time,Snail2 or p65 was removed,and ASPP1 knockdown induced EMT in HCT116 cells,which showed that E-cadherin decreased and Snail2 protein level increased.Consumption of NF-?B p65 completely eliminated the increase in Snail2 and restored the expression of E-cadherin.Conclusion: Downregulation of ASPP1 induces EMT in CRC cells and this induction could be enhanced in the presence of active RAS.ASPP1 knockdown induces EMT via NF-?B pathway mediated regulation of Snail2.