Molecular Mechanism Of The Formation Of Invadopodia In Colorectcal Cancer Induced By FMNL2

Background and PurposeColorectal cancer is a common malignant tumor. It has become one of the three largest cancers in our country, while second largest in the western developed countries inferior to lung cancer. The mortality of colorectal cancer is secondery in malignant tumors. There are30-40%patients undergoing radical surgery, radiation, chemotherapy and the comprehensive treatments result in metastases to liver and lung. Therefore, to clarify the molecular mechanism of colorectal cancer metastasis and the metastasis controling are the important directions of current research.At present, the loss of adhesion characteristics of metastasitic tumor cells, expression of proteolytic enzymes, enhancing athletic ability and forming new blood vessels in metastastic cells, et al. are required for the process of invasion and metastasis. When faced with a variety of cells stimulation signals both inside and outside, accurately changing the shape and movement behavior of tumor cells need reconstruction of tubulin and actin ceaselessly and constantly dynamic changing of filapodia, lobopodia,lamellipodia, invadopodia to regulate the movement of cells and to adapt to the invasion and metastasis process. Pseudopodia formation and cell morphology change is the first step in invasion and metastasis. Highly dynamic pseudopodia formation of Cell migration head structure depends on actin monomer polymerization and the extension of actin fibres. Filopodia and lamellipodia start tumor adhesion. Invadopodia is closely related with extracellular matrix degradation.In prophase work out, we select highly-expressed FMNL2gene in the high metastasis potential cell lines, an important actin nucleation factor, which induces epithelial-mesenchymal transition (EMT) to promote colorectal cancer cell invasion and metastasis. It is highly expressed in colorectal cancer tissues and associated with lymph node metastasis. In addition, the HMGA1/miR-137/FMNL2axis is involved in invasion and metastasis in colorectal cancer.Literatures report that FMNL2facilitates the lamellipodia tip actin polymerization and the extension of microfilament to promote cell movement as the effector of Cdc42proteins. FMNL2promotes cancer cell round and amoeba or mesenchymal invasive migration as the effector of RhoC protein. Visibly, FMNL2regulates actin associated with cell invasion and movement. However, what is its specific mechanism? Whether does the interaction proteins involve in the process? By what signaling pathways regulate the function? The problems above are not yet clear and studies of FMNL2interacting protein have not yet been reported.The purpose of this study is to find out and to verify the interaction protein of FMNL2, thus to select one candidate protein which is involved in movement and invasion and to further elaborate the molecular mechanism of colorectal cancer invasion and metastasis from the perspective of invadopodia. We want to reveale the new FMNL2mechanisms involved in colorectal cancer metastasis and to provide with new potential targets for clinical intervention application.Methods1. Screening and identification of protein interacted with FMNL21) For yeast two hybrid test, preliminarily screen the FMNL2interaction proteins, then choose a gene which is involved in the invasion and movement of cancer cells as the research targets combining with the literatures.2)For immunoprecipitation respectively by antiobody FMNL2, SHANK2, cortactin in SW620and HT29cells, to test the two other types of protein.3) For immune precipitation respectively by FMNL2, SHANK2, cortactin,then ot test combination of proteins by Western Blot.4) For imunnofluorescent confocal, to observe the colocalization of FMNL2and SHANK2, FMNL2and cortactin as well as FMNL2, SHANK2and cortactin it-all.5) For the Western Blot and real time Q-PCR, to verify the expression of FMNL2,SHANK2, cortactin in human colorectal cancer cell lines; For immunohistochemistry, to test the expression of FMNL2,SHANK2, cortactin in human colorectal cancer tissue, to carry on correlation analysis.2. The role of FMNL2in invadopodia formation1)For double immunofluorescences to mark cortactin and F-actin, then to observe the invadopodia morphology and number in4human colorectal cancer cell lines.2) For double immunofluorescences to mark FMNL2and F-actin,then to observe the FMNL2colocalization with invadopodia.3) To construct plasmid pEGFP-C1-actin. For the method of cationic liposome, to transiently transfect into SW620cells. For live cell imaging, to observe the formation and morphology of invadopodia;4)For scanning electron microscopy,to observe invadopodia morphology in the ventral side of cells and its degradation onto the matrigel.5)For transmission electron microscopy,to observe invadopodia morphology inside of cells.6) For the method of cationic liposome, to transiently transfect FMNL2-SiRNA into SW620and Lovo cells which highly expressed FMNL2as well as negative control SiRNA, and to transiently transfect pCDNA3-Flag-FMNL2-CT into HT29and SW480cells which lowly expressed FMNL2, as well as control vector pCDNA3.0-Flag. For Western blot and real-time Q-PCR, to verify the transfection efficiency after culturing for48h.7) For double immunofluorescences to mark cortactin and F-actin in SW620and Lovo/SiFMNL2, HT29and SW480/FMNL2, and to campare the invadopodia morphology and number between before and after transfection.8) First, to pave SW620and Lovo/SiRNA, HT29and SW480/FMNL2to the small confocal dishes delt with gelatin from pig Oregon green488.Then for in situ Zymography, to campare the difference of the gelatin degradation between before and after transfection.3. The role of FMNL2/SHANK2in invadopodia formation1) For Western Blot, to test SHANK2, cortactin, p-(tyr466/tyr421) cortactin after transfecting for48h in Lovo/SiFMNL2,SW620/SiFMNL2, SW480/FMNL2and HT29/FMNL2, and for real time Q-PCR,to test SHANK2and cortactin-mRNA.2) For the method of cationic liposome, to transiently transfect SHANK2-SiRNA or cortactin-SiRNA respectively into SW480/FMNL2and HT29/FMNL2. For Western Blot and real time Q-PCR, to verify the transfection efficiency after culturing for48h. 3) For transwell, to test invasion and migration capability in SW480/FMNL2/SiSHANK2, HT29/FMNL2/SiSHANK2and SW480/FMNL2/Sicortactin, HT29/FMNL2/Sicortactin.4) For double immunofluorescences colocalization, to observe invadopodia number between before and after transfection in SW480/FMNL2/SiSHANK2, HT29/FMNL2/SiSHANK2or SW480/FMNL2/Sicortactin or HT29/FMNL2/Sicortactin. For in situ Zymography, to compare the difference of the gelatin degradation between before and after transfection.4. Preliminarily analysis the domain of FMNL2combining to SHANK2and cortactin1) For immunoprecipitation respectively by antiobody SHANK2or cortactin in SW480/FMNL2-CT-FLAG, SW480/FMNL2-NT-FLAG and SW480/ctrl cells, to test FLAG.2) To construct cortactin and truncated mutants prokaryotic expression vector using pGEX-6p-1for the frame and to construct FMNL2(525-616a) and FMNL2(617-1092a) eukaryotic expression vector using pCDNA3.0-FLAG for the skeleton.3) To respectively transform pGEX-6p-l-cortactin and truncated mutants into BL21(DE3) bacteria, then for IPTG induction, to express cortactin-GST and truncated-mutant-GST. For the method of cationic liposome, to transiently transfect pCDNA3.0-FLAG-FMNL2(525-616a) or FMNL2(617-1092a) into SW480cell respectively. Then for GST-pull down, to test FLAG.5. hEGF/Cdc42regulates the FMNL2induced invadopodia1) For Cdc42-GTP-pull down, to test Cdc42-GTP in SW620/SiFMNL2cell stimulated with hEGF. For Western Blot to test FMNL2,SHANK2, cortactin, p-(tyr466/tyr421) cortactin and Cdc42. 2) For Cdc42-GTP-pull down, to test Cdc42-GTP in HT29/FMNL2cell stimulated with receptor tyrosine kinases inhibitors AG1478. For Western Blot to test FMNL2, SHANK2, cortactin, p-(tyr466/tyr421) cortactin and Cdc42.3) For the method of cationic liposome, to transiently transfect Cdc42-SiRNA-1/-2/-3into SW620cell, then for Western Blot and real time Q-PCR, to verify the transfection efficiency after culturing for48h.4)For the method of cationic liposome, to transiently transfect SiCdc42-1and pRK5-Cdc42Q61L into SW620cell, then for Western Blot,to test Cdc42, FMNL2, SHANK2and cortactin.5) For Western Blot, to test Cdc42, FMNL2, SHANK2and cortactin in SW620/SiCdc42cell stimulated with.hEGF.6) For double immunofluorescences colocalization, to observe invadopodia number and morphology in SW620/SiFMNL2and Lovo/SiFMNL2stimulated with hEGF. For in situ Zymography, to compare the difference of the gelatin degradation in SW620/SiCdc42and Lovo/SiCdc42cells stimulated with hEGF.7) For double immunofluorescences colocalization, to observe invadopodia number and morphology between before and after transfection in SW620/SiFMNL2/Cdc42Q61L and Lovo/SiFMNL2/Cdc42Q61L cell; For in situ Zymography, to campare the difference of the gelatin degradation between before and after transfection SW620/SiFMNL2/Cdc42Q61L cell and Lovo/SiFMNL2/Cdc42Q61L cell.8) For transwell, to test invasion and migration capability in SW620/SiFMNL2and Lovo/SiFMNL2cell with the stumilation of hEGF.9) For transwell,to test invasion and migration capability in SW620/SiFMNL2/Cdc42Q61L and Lovo/SiFMNL2/Cdc42Q61L cell.10) For double immunofluorescences colocalization, to observe invadopodia number and morphology in HT29/FMNL2and SW480/FMNL2delt with AG1478; For in situ Zymography, to campare the difference of the gelatin degradation in HT29/FMNL2and SW480/FMNL2cells delt with AG1478.RESULTS1. screening and identification of protein interacted with FMNL21) Yeast two hybrid test preliminariry screened COMMD10, EGFL6, ZNF38, ENO3, SHANK2, DNAJA1, RBM16et al, seven candidate genes which interacted with FMNL2. SHANK2called CTTNBP1(cortactin SH3domain-binding protein; cortactin-binding protein1), cortactin is the key protein of forming invadopodia, so SHANK2is chosen as the target of further research.2) SHANK2and cortactin were detected in FMNL2IP; FMNL2and cortactin were detected in SHANK2IP, SHANK2and FMNL2were detected in cortactin IP; so FMNL2interacts with SHANK2and cortactin exactly.3) FMNL2colocated with SHANK2in cell membrane and cytoplasm, FMNL2colocated with cortactin in the cell membrane and cytoplasm, FMNL2colocated trichromaticly with SHANK2and cortactin in cell membrane and cytoplasm.4) SHANK2and cortactin express highly in high metastasis potential Lovo and SW620cells and lowly express in low metastasis potential SW480and HT29cells, in accordance with the trend of FMNL2expression. FMNL2and SHANK2are significantly positive correlation (r=0.898, P=0.898), so are FMNL2and cortactin(r=0.872, P=0.872). FMNL2,SHANK2and cortactin express highly in human colorectal cancer tissues with the same trend. SHANK2and FMNL2are significant positive correlation (r=0.604, P=0.00), cortactin and FMNL2are significant positive correlation (r=0.545, P=0.002), SHANK2and cortactin are significant positive correlation (r=0.602, P=0.000) too.2. The role of FMNL2in invadopodia formation 1) Obtained the transient transfection cells SW620/SiFMNL2-1/2/3, the interfered efficiency are respectively0.65,0.57,0.61. Obtained the transient transfection cells Lovo/SiFMNL2-1/2/3, the interfered efficiency are respectively0.60,0.58,0.63, and the protein levels significantly decreased by Western Blot. Obtained the transient transfection HT29and SW480/FMNL2, FMNL2is overexpressed (111.268, P=0.024;126.630, P=0.005); and the protein levels significantly increased by Western Blot.2)4human colorectal cancer cells have invadopodia, red F-actin and green cortactin colocalized around the nucleus.18.2%of Lovo have invadopodia which is annular and circular rough particles and a lit of scattered dot-like particles.79.35%of SW620cells have invadopodia which is rosettes or scattered dot-like particles.17.26%of SW480have invadopodia which is rosettes or scattered dot-like particles, smaller than invadopodia in SW620cells significantly.11.87%of HT29have invasive pseudopodia which is characterized with scattered small dot-like particles. Compared to SW620, invadopodia content of Lovo, SW480and HT29is less with statistical difference (P=0.000; P=0.000; P=0.000), compared to SW480, invadopodia content of HT29and Lovo has no statistical difference (P=0.784).The invadopodia content has no statistical difference between SW480and HT29(P=0.123).3) Red F-actin and green FMNL2localized respectively in cell membrane as well as around the nucleus. The mass and yellow fluorescence around nuclei means the colocalization of FMNL2with invadopodia.4) Obtained the transient transfection cell SW620/EGFP-actin; the live imaging using SW620/EGFP-actin shows that granular and crumb red fluorescence, namely invadopodia, with fission and fusion behavior, the stability of the invadopodia appears halo cloud shape, which gradually formed protrusions to substrate surface. 5) SEM shows the invadopodia is in the ventral side of cells. In HT29/ctrl group, the invadopodia is thin,short and few, the cells stay on the smooth matrix surface gently, so as in Lovo/SiFMNL2. In HT29/FMNL2group, the invadopodia is coarse, long and many, the cells are firmly rooted in matrix which appeared obvious damage because of the adhesion and degradation of invadopodia, so as in Lovo/NC.6) TEM shows that a large bulky mitochondria gathered on one side of nuclei in SW620, meanwhile there are some round and irregular lumps with homogeneous high electron density, along the parallel arrangement of F-actin, invadopodia namely; In SW620/SiFMNL2cells, a little of mitochondria scattered in the cell with small chunk of high-density homogeneous invadopodia,and the structure of F-actin disappeared, some invadopodia formed a blur. In SW480cells,we did not see obviously gathered mitochondria, there is few small round and irregular lumps with homogeneous high electron density, In SW480/FMNL2cells, mitochondria gathered visibly in one side of nucleus, more bulky irregular invadopodia with homogeneous high electron density appeared too. the layered parallel arrangement of the golgi apparatus orientated toward invadopodia.7) Compared to SW620/NC invadopodia (86.52±2.61%), the invadopodia of SW620/SiFMNL2is smaller, the content reduced significantly (29.64±3.01%, P=0.000). Compared to Lovo/NC invadopodia (20.09±2.64%), the invadopodia of Lovo/SiFMNL2is smaller, the content reduced significantly (9.35±1.46%, P=0.000). Compared to HT29/ctrl(11.90±2.13%), the invadopodia of HT29/FMNL2is greater, content increased significantly (30.67±3.50%, P=0.000). Compared to SW480/ctrl (19.64±1.62%), the invadopodia of SW480/FMNL2is greater, content increased significantly (77.95±3.75%, P=0.000).8) Compared to SW620/NC gelatin degradation(89.12±4.70%), in situ Zymography, gelatin degradation of SW620/SiFMNL2cells decreased (31.90±3.23%, P=0.000). Compared to Lovo/NC gelatin degradation(18.00±3.55%), in situ Zymography, gelatin degradation of Lovo/SiFMNL2cells decreased(6.77±1.22%, P=0.007). Compared to SW480/ctrl gelatin degradation (9.96±1.23%), in situ Zymography gelatin degradation of HT29/FMNL2increased (31.20±3.63%, P=0.001), Compared to SW480/ctrl gelatin degradation (15.47±1.93%), in situ Zymography gelatin degradation of SW480/FMNL2increased (76.69±4.17%, P=0.000).3. The role of FMNL2/SHANK2in invadopodia formation1) Compared to Lovo/NC and SW620/NC respectvely, SHANK2, cortactin, and p-(tyr466/421) cortactin protein.levels in Lovo/SiFMNL2and SW620/SiFMNL2significantly decreased; SHANK2and cortactin mRNA levels in Lovo/SiFMNL2were lower(0.32, P=0.000,0.45, P=0.001). SHANK2and cortactin mRNA levels in SW620/SiFMNL2were lower (0.24, P=0.000,0.32, P=0.000). Compared to SW480and HT29/ctrl, SHANK2, cortactin, p-(tyr466/421) cortactin protein level in SW480/FMNL2and HT29/FMNL2increased significantly, SHANK2and cortactin mRNA levels in SW480/FMNL2increased (4.17, P=0.003,9.60, P=0.000), SHANK2and cortactin-mRNA levels in HT29/FMNL2increased (4.50, P=0.000,7.32, P=0.000).2) Obtained the transient transfection cells SW480/FMNL2/SiSHANK2-1/-2/-3and SW480/FMNL2/Sicortactin-1/-2/-3respectively, SHANK2-mRNA interference efficiency are respectively0.88,0.72,0.86, cortactin-mRNA interference efficiency are respectively0.36,0.56,0.58. Obtained the transient transfection cells HT29/FMNL2/SiSHANK2-1/-2/-3and HT29/FMNL2/Sicortactin-1/-2/-3respectively, SHANK2-mRNA interference efficiency are respectively0.81,0.66,0.87, cortactin-mRNA interference efficiency are respectively0.47,0.75,0.78. After SHANK2interference, SHANK2protein level significantly decreased, but cortactin didn’t change. After cortactin interference, cortactin protein level significantly decreased, but SHANK2didn’t change.3) Migration and invasion experiments results show that compared to SW480/FMNL2/NC (319.8±28.4), migration ability dropped in SW480/FMNL2/Sicortactin (78.4±12.5, P=0.000) and SW480/FMNL2/SiSHANK2(108.8±11.8,P=0.000) respectively. Compared to SW480/FMNL2/NC (284.4±28.3), invasion ability dropped in SW480/FMNL2/Sicortactin (60.8±9.1, P=0.000) and SW480/FMNL2/SiSHANK2(91.0±13.4,P=0.000) respectively. Compared to HT29/FMNL2/NC (18.8±3.8), migration ability dropped in HT29/FMNL2/Sicortactin (1.6±0.9, P=0.000) and HT29/FMNL2/SiSHANK2(3.0±0.7, P=0.000) respectively. Compared to HT29/FMNL2/NC (14.4±3.8), invasion ability dropped in HT29/FMNL2/Sicortactin (1.8±0.8, P=0.004) and HT29/FMNL2/SiSHANK2(4.0±1.0, P=0.007) respectively.4) Compared to HT29/FMNL2/NC invadopodia (30.31±3.81%), the invadopodia of HT29/FMNL2/SiSHANK2is smaller, the content (26.00±2.25%, P=0.026) reduced significantly, and the invadopodia of HT29/FMNL2/Sicortactin is smaller, the content reduced significantly (8.13±1.40%, P=0.000). Compared to SW480/FMNL2/NC invadopodia (78.52±4.06%), the invadopodia of SW480/FMNL2/SiSHANK2and SW480/FMNL2/Sicortactin is smaller, the content reduced significantly (60.72±3.03%,P=0.000;18.42±2.17%, P=0.000);5) Compared to HT29/FMNL2/NC gelatin degradation(30.07±3.02%), in situ Zymography, gelatin degradation of HT29/FMNL2/SiSHANK2cells decreased significantly (24.43±2.33%, P=0.026), gelatin degradation of HT29/FMNL2/Sicortactin cells decreased significantly (7.97±1.37%, P=0.000); Compared to SW480/FMNL2/NC gelatin degradation(81.17±2.37%), in situ Zymography, gelatin degradation of SW480/FMNL2/SiSHANK2and SW480/FMNL2/Sicortactin cells decreased significantly (61.60±3.85%, P=0.008;19.36±2.06%,P=0.000);4. Preliminarily analysis the domain of FMNL2combining to SHANK2and cortactin1) For SHANK2co-immunoprecipitation, FLAG was detected in SW480/FMNL2-CT and wasn’t detected in SW480/ctrl or SW480/FMNL2-NT. So SHANK2interacts with FMNL2-CT, not with FMNL2-NT.2) For cortactin co-immunoprecipitation, FLAG was detected in SW480/FMNL2-CT and wasn’t detected in SW480/ctrl or SW480/FMNL2-NT. So cortactin interacts with FMNL2-CT, not with FMNL2-NT.3) Constructed cortactin and truncated mutants prokaryotic expression vector pGEX-6p-l-cortactin(1-550a), pGEX-6p-l-cortactin(1-325a), pGEX-6p-l-cortactin (83-495a), pGEX-6p-1-cortactin (326-550a), pGEX-6p-l-cortactin (496-550a) and eukaryotic expression vector pCDNA3.0-FLAG-FMNL2(525-616a) and pCDNA3.0-FLAG-FMNL2(617-1092a).4) Successfully expressed GST fusion protein, GST-cortactin (1-550a),GST-cortactin (1-325a), GST-cortactin (83-495a), GST-cortactin (326-550a), GST-cortactin (496-550a) and FLAG fusion protein, FLAG-FMNL2(525-616a), FLAG-FMNL2(617-1092a).5) GST-pull down, GST-Cortactin (1-550a) combined to FMNL2, GST-cortactin (326-550a), GST-cortactin (496-550a) and GST-Cortactin (1-550a) respectively combined to FMNL2-CT, but GST-cortactin (1-325a) or GST-cortactin (83-495a) didn’t. GST-cortactin (496-550a) combined to FMNL2(525-616a) not to FMNL2(617-1092a).5. hEGF/Cdc42regulates the FMNL2involved in formation mechanism of invadopodia1) Western Blot shows that FMNL2, SHANK2, cortactin, p-(tyr466/421) cortactin, Cdc42and Cdc42-GTP protein level increased significantly after hEGF stimulated to SW620/NC and SW620/SiFMNL2.2) Western Blot shows that FMNL2, SHANK2, cortactin, p-(tyr466/421) cortactin, Cdc42and Cdc42-GTP protein level decreased significantly after administration receptor tyrosine kinase inhibitors AG1478to HT29/FMNL2and HT29/ctrl.3) Compared to SW620NC, Cdc42-mRNA interference efficiency of SW620/SiCdc42-l/-2/-3are respectively0.83,0.74,0.87by Q-PCR, and the protein level significantly decreased by Western Blot.4) Western Blot shows that FMNL2, SHANK2, cortactin, Cdc42protein level decreased significantly in SW620/SiCdc42, and FMNL2, SHANK2, cortactin, Cdc42protein level increased significantly in SW620/SiCdc42/Cdc42Q61L and in SW620/SiCdc42stimulated with hEGF.5) Compared to SW620/SiFMNL2invadopodia (36.06±5.69%), the invadopodia of SW620/SiFMNL2stimulated with hEGF is bigger, the content increased significantly (82.95±5.53%, P=0.000), the invadopodia of SW620/SiFMNL2/Cdc42Q61L is bigger, the content increased significantly (75.48±5.06%, P=0.000). Compared to Lovo/SiFMNL2invadopodia (9.37±1.67%), the invadopodia of Lovo/SiFMNL2stimulated with hEGF is bigger, the content increased significantly (19.42±2.83%, P=0.001), the invadopodia of Lovo/SiFMNL2/Cdc42Q61L is bigger, the content increased significantly (19.17±1.90%,P=0.000).6) Compared to SW620/SiFMNL2gelatin degradation(36.93±4.40%), In situ Zymography, gelatin degradation of SW620/SiFMNL2stimulated with hEGF is more obvious and increased significantly (92.24±2.43%, P=0.000), the gelatin degradation of SW620/SiFMNL2/Cdc42Q61L increased significantly (88.36±2.72%, P=0.000) as well. Compared to Lovo/SiFMNL2gelatin degradation(9.14±1.94%), in situ Zymography, gelatin degradation of Lovo/SiFMNL2stimulated with hEGF is more obvious and increased significantly (20.25±2.86%, P=0.001), the gelatin degradation of Lovo/SiFMNL2/Cdc42Q61L increased significantly (19.81±1.68%, P=0.001)as well.7) Compared to HT29/FMNL2invadopodia (30.00±3.44%), the invadopodia of HT29/FMNL2delt with AG1478decreased significantly (11.89±1.89%, P=0.000). Compared to SW480/FMNL2invadopodia (80.76±3.44%), the invadopodia of SW480/FMNL2delt with AG1478decreased significantly (18.89±2.57%, P=0.000).8) migration and invasion experiment results show that compared to SW620/SiFMNL2(193.0±15.6), migration ability increased significantly in SW620/SiFMNL2with stumilation of hEGF (304.8±14.3, P=0.000) and in SW620/SiFMNL2/Cdc42Q61L(243.0±13.9, P=0.000) respectively. Compared to SW620/SiFMNL2(109.0±11.47), invasion ability increased significantly in SW620/SiFMNL2with stumilation of hEGF (243.0±13.9, P=0.000) and in SW620/SiFMNL2/Cdc42Q61L(207.0±18.23, P=0.000) respectively.9) Migration and invasion experiments results show that compared to Lovo/SiFMNL2(156.2±14.9), migration ability increased significantly in Lovo/SiFMNL2with stumilation of hEGF (295.8±17.0, P=0.000) and in Lovo/SiFMNL2/Cdc42Q61L(277±17.2, P=0.000) respectively. compared to Lovo/SiFMNL2(77.6±8.3), invasion ability increased significantly in Lovo/SiFMNL2with stumilation of hEGF (217.8±18.1, P=0.000) and in Lovo/SiFMNL2/Cdc42Q61L(197.4±15.0, P=0.000) respectively.10) Compared to HT29/FMNL2gelatin degradation(35.19±6.00%), In situ Zymography, gelatin degradation of HT29/FMNL2delt with AG1478is unconspicuous and decreased significantly(10.11±2.06%, P=0.002). Compared to SW480/FMNL2gelatin degradation(81.69±4.12%), In situ Zymography, gelatin degradation of SW480/FMNL2delt with AG1478is unconspicuous and decreased significantly(16.69±2.86%,P=0.000).Conclusion1. FMNL2can directly interact with cortactin or form a liner complex with it via scaffold protein SHANK2;.2. FMNL2-CT directly combines to SHANK2and FMNL2directly combines to cortactin-SH3.3. FMNL2promotes invadopodia formation in colorectal cancer cell.4. SHANK2and cortactin is essential for invadopodia formation induced by FMNL2.5. FMNL2promotes invadopodia formation in colorectal cancer cell under the control of upstream hEGF/Cdc42signaling pathways.Innovation points of this paper1. The new mechanism FMNL2involved in invasion and metastasis of colorectal cancer:Namely, FMNL2directly or indirectly interracts with invadopodia key protein cortactin (through the bridge of a scaffolding protein SHANK2), FMNL2promote invadopodia formation of colorectal cancer cells by SHANK2or cortactin, which is adjusted by upstream hEGF/Cdc42signaling pathway.2. Providing with new potential applications targeting for colorectal cancer metastasis Clinical interventions.