The Function And Molecular Mechanisms Of DAB2IP In Invasion And Metastasis Of Colorectal Cancer

Background and PurposeColorectal cancer (CRC) is the second highest cause of cancer-related deaths in the world. In China, CRC occupies the fifth position in the mortalities caused by cancer, and its incidence rate still continues to increase.Many patients have occurred micro-metastasis before the operation, and metastasis is the direct cause of recurrence and death.So it is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.DAB2IP, also known as ASK1-interacting protein-1(AIP1), a novel member of the Ras GTPase-activating protein family.It is located on chromosome9q33.1-q33.3. DAB2IP directly interacts with the N-terminal domain of DAB2(also known as DOC-2) protein, then forms a unique protein complex and has a negative regulatory activity to the Ras-mediated signal pathway.Recently it was reported DAB2IP is down-regulated in various human cancers mainly because of altered epigenetic regulation of its promoter, such as by DNA hypermethylation and/or histone modification In addition, DAB2IP involved in regulation of tumor growth and metastasis. DAB2IP by EZH2functions as a signaling scaffold that coordinately regulates Ras and nuclear factor-kB (NF-kB) through distinct domains to promote tumor growth and metastasis in prostate cancer. Most importantly, DAB2IP leads to MET through recruitment of PP2A and GSK-3b resulting in β-catenin degradation in prostate epithelia. DAB2IP mediates vascular endothelial cell growth factor receptor-3-dependent angiogenic and lymphangiogenic responses.Low expression of DAB2IP contributes to malignant development and poor prognosis in hepatocellular carcinoma. The function and molecular mechanisms of DAB2IP in invasion and metastasis of colorectal cancer is unclear.In this study, we aim to clarify the possible role of DAB2IP gene in the proliferation, invasion and metastasis of CRC. It will be helpful to understand the molecular basis of CRC, and establish Snail/DAB2IP as a new target for early metastatic diagnostic markers and novel therapeutic strategies.Methods1. Investigating the effect of DAB2IP silence on the biological behaviors of human CRC cell lines in vivo and in vitro.1) The expressions of DAB2IP gene in eight colorectal carcinoma cell lines were detected by reverse Real-Time PCR and Western blot.2) Stable cell lines were established by lentiviral expression system. DAB2IP silenced cell lines(SW480/SiDAB2IP-1/-2, HCT116/SiDAB2IP-1/-2) and negative control groups(SW480/NC、HCT116/NC). The efficiency of the interference were detected by Q-PCR and Western blot.3) The biological fuction of DAB2IP in cell proliferation, cell cycle,cell apoptosis, adhesion ability was investigated by MTT assay, coloy formation assay, flow cytometry,heterogernous adhesion. Cell migrate ability and cell invasion ability were tested by intro migrate assay and intro invasion assay. The expression of makers in EMT was detected by Western blot and immunofluorescensse staining.4) The effects in vivo tumorigenesis, invasive and metastasis ability were measured by tumorigenesis assay,tail vein injection of nude mice.2. Predicting and confirming the transcription factor of DAB2IP1) Prediction of the sites in DAB2IP-promoter bonded with Snail were by software and identification of the sites and activity were by Luciferase activity assay and Chromatin Immunoprecipitation (CHIP).2) We transfected pEGFP-Snail or SiSnail into cells and detected the expression of Snail and DAB2IP by Western blot.3. EZH2, HDAC1/HDAC2and Snail form a co-repressor complex to silence DBA2IP1) pcDNA3.1-EZH2and pcDNA3.1-EZH2Δset expression vector was constructed.2) We transfected SiSnail and EZH2/SiSnail into cells, respectively, and detected the expression of EZH2, Snail and DAB2IP by Western blot.3) The expression of EZH2, H3, H3K27me3, DAB2IP was deteced in SW480transfected with pcDNA3.1-EZH2、pcDNA3.1-EZH2Δset or treated with TSA (Trichostatin A).4) Co-immunoprecipitate assay and Immuno-colocalization assay was used to analysis the interactive relationship between EZH2, HDAC1/HDAC2and Snail.5) The expression of EZH2, Snail, DAB2IP was detected by western blot in SW480/MOCK and SW480/EZH2treated with SiSnail.6) Luciferase activity of the DAB2IP promoter in SW480/MOCK and SW480/EZH2was measured by Lucifere activity assay after the depletion of endogenous Snail, HDAC1/HDAC2.7) CoIP were used to analysis the interactive relationship between EZH2and Snail after treating cells with TS A or SiHD AC1/2.8) CoIP were used to analysis the interactive relationship between EZH2, HDAC1/HDAC1and Snail after treating cells with T S A.9) Luciferase activity of the DAB2IP promoter in SW480/MOCK and S W480/EZH2was measured after treating cells with DMAO or T S A.4. The positive feedback loop exists between snail and DAB2IP1) The expression of p-PI3K、p-AKT、p-GSK-3β、Snail、β-catenin was detected by Western blot after treating cells with SiSnail. Subcelluar localization of Snail and β-catenin was detected by immunofluorescensse staining2) Luciferase activity of the DAB2IP promoter was measured after treating SW480with SiDAB2IP. Wnt pathway was detected by TOP FLASH/FOP FLASH luciferase activity assay after treating SW480with SiDAB2IP.3) The expression of DAB2IP, Snail, GSK-3β, p-GSK-3p, p-catenin was detected after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132.4) Luciferase activity of the DAB2IP promoter was measured after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132.5) Wnt pathway was detected by TOP FLASH/FOP FLASH luciferase activity assay after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132.5. Investigating the effect of SiSnail and SiSnail/SiDAB2IP on the biological behaviors of human CRC cell lines in vivo and in vitro.1) The biological effect of SiSnail and SiSnail/SiDAB2IP in cell proliferation was investigated by MTT assay,coloy formation assay.Cell migrate ability and cell invasion ability were tested by intro migrate assay and intro invasion assay.The expression of makers in EMT were detected by Western blot.2) The biological effect of SiSnail and SiSnail/SiDAB2IP in vivo tumorigenesis and metastasis ability was measured by tumorigenesis assay,tail vein injection of nude mice.6.Expression of DAB2IP in CRC cell, CRC samples and paraffin section1) The expressions of DAB2IP gene in20CRC samples and their paird normal colorectal mucosa were detected by Real-Time PCR.2) The expressions of Snail and DAB2IP in8CRC samples and their paird normal colorectal mucosa were detected by Western blot. The relationship of DAB2IP and Snail was analyzed by SPSS13.0software3) The expression of DAB2IP and EZH2was examined in specimens of CRC with follow-up visit information by immunohistochemistry(IHC) method.The relationship of DAB2IP expression and clinical pathological factors was analyzed by SPSS13.0softwareResults 1.Investigating the effect of DAB2IP silence on the biological behaviors of human CRC cell lines in vivo and in vitro.1) The Real-Time PCR and Western blot result of expression of DAB2IP in the CRC cell lines showed that there is significant difference among them(F=6.664, P=0.001). The expression of DAB2IP in SW620and Lovo cell line was lowerer than other cell lines.2) Western Blot results showed that the expression of DAB2IP was significantly down-regulated after transfecting lentivirus of SiDAB2IP-1/-2into SW480and HCT116cells compared to NC group (Real-Time-PCR SW480:F=404.920, P=0.000; HCT116:F=1756.099, P=0.000).3) DAB2IP silenced cells showed a significantly increased potency of proliferation(SW480:F=12.487, P=0.000; HCT116:F=34.931, P=0.000) and clonies formation(SW480:F=52.000, P=0.000; HCT116:F=37.186, P=0.000).4) DAB2IP silenced cells showed a significantly enhanced the potency of cells invasion(SW480:F=413.872, P=0.000; HCT116:F=234.832, P=0.000) and migration(SW480:F=49.306, P=0.000; HCT116:F=240.050, P=0.000) compared with NC cells as determined by in vitro invasion assay and in vitro migration assay。5) The cell cycle results indicated that DAB2IP silence accelerated the G1to G2or S-phase transition in CRC cell lines(SW480:G1F=409.814,P=0.000, G2F=8.493, P=0.000, SF=63.672,P=0.000; HCT116:G1F=179.890, P=0.000, G2F=66.639, P=0.000,SF=41.639, P=0.000).6) The cell apoptosis results indicated that DAB2IP silence did not affect cell apoptosis (SW480:F=0.007, P=0.993; HCT116:F=0.063,P=0.940).7) DAB2IP silenced cells showed the same adhesion ability compared with NC cells as determined by heterogeneity adhesion assay (SW480:F=0.820, P=0.456; HCT116:F=1.223, P=0.318).8) DAB2IP silenced cells showed the same stem cell propertie compared with NC cells as determined by stem cell ball assay(SW480:F=0.916, P=0.450; HCT116: F=0.124, P=0.886). 9) SW480/SiDAB2IP-1/-2cell and HCT116/SiDAB2IP-1/-2cell exhibited the downregulation of epithelial marker E-Cadherin and a-Catenin, mealwhile, the upregulation of mesenchymal makers Vimentin as compared with NC cells,respectively.10) DAB2IP silenced cells showed a significantly enhanced tumour growth and invasion in nude mice subcutaneous (SW480:F=2.480, P=0.019; HCT116:F=2.700, P=0.011) and metastasis ability(SW480::F=6.762, P=0.008)) compared with NC cells as determined by tumorigenesis assay and tail vein injection of nude mice.Conclusion:DAB2IP silenced cells showed a significantly enhanced potency of proliferation, invasion, migration, EMT and accelerated the G1to G2or S-phase transition in vitro. DAB2IP silenced cells showed a significantly enhanced tumour growth in nude mice subcutaneous and metastasis ability.2. Predicting and confirming the transcription factor of DAB2IP1) Finding the promotor sequence of DAB2IP in Ensembl. And then by using the online Mapper2、TRED and Consite, we predictited and considered transcription factor Snail as a putative upstream regulator of DAB2IP.2) In HEK293A, SW480and HCT116cell lines, Luciferase reporter system showed that the luciferase activity was decreased significantly respectively in PGL3-Basic-DAB2IP-promoter group compared to the other group (P<0.001; P<.001).3) The result of ChIP assay indicated there are1binding sites (CHIP1:151bp-156bp) between the DAB2IP promoter and Snail.4) By Western blot, expression of DAB2IP were decreased markedly in480cell line and HCT116cell lines after transfected with pEGFP-Snail. While expression of DAB2IP protein were increased after transfected with SiSnail.Conclusion:Transcription factor Snail is a putative upstream regulator of DAB2IP.3. EZH2, HDAC1/HDAC2and Snail form a co-repressor complex to silence DBA2IP1) By Western blot, expression of DAB2IP were decreased markedly in480cell line and HCT116cell lines after transfected with pcDNA3.1-EZH2. While expression of DAB2IP protein were increased after transfected with SiEZH2.2) When ectopic overexpression of EZH2in SW480was observed, the levels of DAB2IP were reduced and accompanied by an increased expression of H3K27me3. Furthermore, the treatment of TSA partially restored the reduced levels of DAB2IP in pcDNA3.0-EZH2/SW480cells, but the levels of H3K27me3were riot decreased.3) By co-IP, we finded EZH2, HDAC1/HDAC2and Snail could form a co-repressor complex.4) The repressive activity of EZH2toward DAB2IP expression was prevented in Snail depleted.5) The repressive activity of ectopic EZH2in the DAB2IP promoter of SW480cells(t=11.565, P=0.000) was dramatically inhibited after the depletion of endogenous Snail(t=1.494, P=0.209), but was only partially inhibited after the depletion of either HDAC1(t=2.625, P=0.058)or HDAC2(t=2.191, P=0.094).6) Either siHDAC1/HDAC2or TSA inhibits the interaction between EZH2and Snail.7) We found that TSA significantly decreased the interaction between HDAC1/2and Snail, but did not affect the association of HDAC1/2and EZH2.8) The luciferase reporter assay supported these results, that is, the repressive activity of ectopic EZH2on the DAB2IP promoter was blocked in the SW480cells pretreated with TSA(t=22.972, P=0.000).Conclusion:EZH2, HDAC1/HDAC2and Snail form a co-repressor complex to silence DBA2IP.4. The positive feedback loop exists between snail and DAB2IP1) SW480/SiDAB2IP-1/-2cell exhibited the downregulation of Snail, mealwhile, the upregulation of p-GSK-3β as compared with NC cells. DAB2IP silence did not affect the expression of PI3K、AKT、p-PI3K、p-AKT、GSK、P-catenin but promote P-catenin protein into nuclei.2) DAB2IP silence inhibited luciferase activity of the DAB2IP promoter and upregulated luciferase activity of Wnt pathway(SW480TOP/FOP:F=46.966, P=0.000).3) When SW480was transfected with SiSnail, the level of Snail was reduced and accompanied by an increased expression of DAB2IP. Furthermore, the transfected of SiSnail/SiDAB2IP partially restored the reduced levels of Snail, while the level of DAB2IP was decreased.4)The expression of DAB2IP was upregulated and the expressin of Snail, p-GSK-3β,β-catenin was downregulated in SW480/SiSnail compared with SW480/SiNC, while, the expression of DAB2IP and Snail, p-GSK-3β,β-catenin was partly reverted after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132. SW480/SiDAB2IP.5) Luciferase activity of the DAB2IP promoter was upregulated in SW480/SiSnail compared with SW480/SiNC, while, the luciferase activity was partly reverted after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132(SW480DAB2IP-promoter:F=52.169, P=0.000).6) Luciferase activity of Wnt pathway was downregulated in SW480/SiSnail compared with SW480/SiNC, while, the luciferase activity was partly reverted after treating SW480/SiSnail with SiDAB2IP, LiCL or MG132(SW480TOP/FOP: F=57.994, P=0.000).Conclusion:The positive feedback loop exists between snail and DAB2IP.5..Investigating the effect of SiSnail and SiSnail/SiDAB2IP on the biological behaviors of human CRC cell lines in vivo and in vitro.1) Snail silenced cells showed a significantly decreased potency of proliferation (SW480:F=20.523, P=0.000; HCT116:F=14.610, P=0.000) and forming plate colonies(SW480:F=197.303, P=0.000; HCT116:F=32.042, P=0.001) compared with NC cells, meanwhile the effects was decreased in SiSnail/DAB2IP groups, as determined by in vitro MTT assay and plate colony formation assay.2) Snail silenced cells showed a significantly decreased the the potency of cells invasion(SW480:F=180.863, P=0.000; HCT116:F=385.370, P=0.000) and migration (SW480:F=20.523,P=0.000; HCT116:F=14.610, P=0.000) compared with NC cells, meanwhile, the effects was decreased in SiSnail/DAB2IP groups, as determined by in vitro invasion assay and in vitro migration assay3) Snail silenced cells showed a significantly inhibited tumour growth in nude mice subcutaneous (SW480:F=5.047, P=0.000; HCT116:F=2.182, P=0.038) and metastasis ability(SW480:F=5.316, P=0.018; HCT116:F=6.951, P=0.007) compared with NC cells, meanwhile, the effects was decreased in SiSnail/DAB2IP groups, as determined by tumorigenesis assay and tail vein injection of nude mice.4) SW480/SiSnail cell and HCT116/SiSnail cell exhibited the upregulation of epithelial marker E-Cadherin, mealwhile, the downregulation of mesenchymal makers Vimentin as compared with NC cells, respectively. Meanwhile, the effects was partly rescused in SiSnail/DAB2IP groups.Conclusion:Snail silenced cells showed a significantly decreased the potency of proliferation, invasion, migration and metastasis compared with NC cells, meanwhile, the effects was decreased in SiSnail/DAB2IP groups.6.Expression of DAB2IP in CRC fresh samples and paraffin section1) The Real-Time PCR result in the fresh tissues showed that the expression of DAB2IP at mRNA (t=2.555, P=0.012) in20CRC samples is lower than their paird normal colorectal mucosa.2) Western blot result in the fresh tissues showed that the expression of DAB2IP in8CRC samples is lower than their paird normal colorectal mucosa, meanwhile, the expression of Snail is higher than their paird normal colorectal mucosa. The expressions of DAB2IP and EZH2had significant negative correlation(r=-0.503, P=0.047).3) Expression of DAB2IP in CRC paraffin section by IHC. DAB2IP expression was lower in200CRC tissues than in adjacent normal tissues (P<0.001,z=-9.236) Its expression was positively correlated with patients ages(P=0.009), tumor size(P=0.044), lymph metastasis(P=0.039), Ducks stage(P=0.045), remote metastasis(P=0.042). Patients with lower DAB2IP expression had shorter overall survival time (P=0.000) especially in DucksC/D stage.4) Expression of EZH2in CRC paraffin section by IHC. DAB2IP expression was higher in120CRC tissues than in adjacent normal tissues.The expression of DAB2IP and EZH2had significant negative correlation(χ2=4.585, P=0.032)。Conclusion:The expression of DAB2IP is lower than their paird normal colorectal mucosa. The expression of DAB2IP and EZH2/Snail had significant negative correlation.Conclusion:1) DAB2IP, as a tumor suppressor, inhibited proliferation, invasion, migration and metastasis of CRC.2) Transcription factor Snail is a putative upstream regulator of DAB2IP. EZH2, HDAC1/HDAC2and Snail form a co-repressor complex to silence DBA2IP.3) The positive feedback loop existing between Snail and DAB2IP enhanced proliferation, invasion, migration and metastasis of CRC.4) The expression of DAB2IP is lower than their paird normal colorectal mucosa. DAB2IP may be a valuable marker for evaluating the progression and prognosis of CRC.Innovation points of this paper1) We have present the mechanism of of Snail/DAB2IP positive feedback loop in the CRC metastasis and invasion.2)The data will be helpful to elucidate a new target of early warming, progression and prognosis evaluation of CRC.