| BACKGROUND AND OBJECTIVEThe revolution in cancer research can be summed up in a single sentence:cancer is,in essence,a genetic disease.Carcinogenesis is a multistep process attributable to both gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes.Tumor suppressor genes are genes that inhibit cell growth and have the potential to suppress cancer in normal cells.Tumor suppressor genes play an important negative regulatory role in the control of cell growth,proliferation and differentiation,which are mutually restricted with oncogenes and maintain the relative stability of positive and negative regulatory signals.In recent years,the research on the mechanism of tumor in particular has focused on the research of oncogene,and many research results have been put into practical clinical application.Targeted drugs such as gleevec have achieved certain clinical therapeutic effects.While only a small number of studies are in the exploratory stage about the application of tumor suppressor genes in cancer treatment.More attention and efforts need to be addressed to the research on tumor suppressor genes if we want to make further progress in the field of tumor research.Transmembrane and immunoglobulin domain containing(TMIGD)family proteins represent a new class of immunoglobulin(Ig)domain containing cell adhesion molecules(Ig-CAMs)newly identified in our lab.TMIGD protein is a cell surface glycoprotein composed of an extracellular segment containing one or more immunoglobulin-like domains,a single transmembrane domain,and a c-terminal domain inside the cell.The first member of the TMIGD family was identified in our laboratory as immunoglobulin and proline rich receptor-1(IGPR-1,which is also called TMIGD2).Expression of IGPR-1 in endothelial cells regulates cell-cell adhesion,barrier function and angiogenesis.IGPR-1 expression in human colon cancer is increased and through promotion of multicellular aggregation it promotes tumor growth.Recently the third member of the family-transmembrane and immunoglobulin(Ig)domain containing 3(TMIGD3)has also been found.Swathi and his team found that TMIGD3 acts as a tumor suppressor in osteosarcoma.By inhibiting PKA-Akt-NF-kB pathway,TMIGD3 inhibits proliferation,migration,invasion and risistance to the outside undesirable stimulus of osteosarcoma cells.We have identified TMIGD1 as a second member of TMIGD family proteins,which is located on chromosome 17 with seven putative exons that encode for a protein with 262 amino acids.IGPR-1 is expressed in human and some other species,while abscent in mouse.The amino acid sequence of TMIGD1 is highly conserved in humans and mice.TMIGD family proteins are composed of three major domains:extracellular,transmembrane and intracellular.One of the main differences between TMIGD1 and IGPR-1 is that TMIGD1 has a shorter cytoplasmic domain with no significant proline-rich sequences.In addition,the extracellular domain of TMIGD1 contains two putative Ig domains,whereas IGPR-1 has one.TMIGD1 is highly expressed in the kidney tissue of human and mouse.Renal tubular epithelial cells were highly positive for TMIGD1,whereas podocytes of the glomerulus were negative.Stomach,intestine and colon epithelial tissues were also positive for TMIGD1,while the expression is much lower in other tissues.In our previous study,we found that extracellular domain mediates the adhesive function of TMIGD family proteins via homophilic transdimerization.HEK-293 cells that expressed TMIGD1 formed large aggregates of cells compared with HEK-293 cells that expressed an empty vector.In addition,mixing of HEK-293 cells that expressed TMIGD1 with HEK-293 cells that expressed the empty vector reduced formation of large cell aggregates.Overexpression of TMIGD1 in HEK-293 cells increased TEER,whereas it reduced HEK-293 cell permeability.In HEK-293 cells that expressed TMIGD1,actin stress fibrils were distinctively assembled at the periphery of cells,whereas in HEK-293 cells that expressed empty vector,actin fibrils were distributed central and to the periphery of cells.Expression of TMIGD1 in HEK-293 cells reduced the growth rate of HEK-293 cells.In addition,overexpression of TMIGD1 in HEK-293 cells inhibited cell migration.But TMIGD1 could protects human kidney epithelial cells from hydrogen peroxide-induced cell injury and nutrient deprivation.TMIGD1 expression was transiently upregulated in a hypertensive mouse model,and an acute renal IR model,suggesting its protective function in renal injury.To sum up,TMIGD1 as a CAM,could stabilize cell membrane structure by forming a connection structure with different ligands and extracellular segment thus enhancing the barrier function of cell membrane or epithelium formed by cells by restricting cell proliferation and motion,and finally increase cell resistance to adverse environmental factors.As TMIGD1 is down-regulated or undetectable in multiple tumor cells and itsfunction in inhibiting cell proliferation and migaration,we hyperthesized that TMIGD1 might play a tumor suppressor role in cancer cells.TMIGD1 expression in the kidney tissue is significantly higher than others and TMIGD1 expressed in renal malignant tumor cell lines were reduced,plus the protective role of TMIGD1 in renal epithelial cells have been identified in previous studies.Thus,in our study,we firstinvestigat the expression of TMIGD1 and its tumor suppressing function in renal cancer.We illustrated the regulating mechanism of TMIGDI by using LASAGNA-Search 2.0,an integrated web tool for transcription factor binding site search and visualization.And out of the reason TMIGD1 also has a high expression in the stomach,small intestine,colon and other digestive tract epithelial tissues,we searched for TMIGD1 binding ligands by using GST-pulldown and mass spectrometric analysis in colon cancer.We proved TMIGD1 as a tumor suppressor in renal and colon cancer cells,providing information in defining potential biomarkers and target for future targeted therapy.Part 1 TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancerMETHODS1.To examine expression of TMIGD1 in human and mouse tissues and organs,we analyzed the mRNA of TMIGD1 by quantitative PCR(qPCR)using mRNA derived from panels of human or mouse organs/tissues.Protein extract was tested by western blot for TMIGD1 expression.Renal tissue slices were immunohistochemically stained for TMIGD1.We analysed human RCC microarray data,TCGA data via online cBioPortal for Cancer Genomics.Examined TMIGD1 expression in human RCC cell lines and a cohort of human renal cancer biopsy samples.2.We reintroduced TMIGD1 into a renal tumor cell line,786-0 cells via a retroviral system and performed rhodamine-phalloidin staining for actin to observe the morphological changes.MTT and BrdU assays were used to assesse the function of TMIGD1 in cell proliferation.We examined the effect of TMIGD1 expression in the tumor formation of 786-0 cells in an athymic nude mouse.The possible role of TMIGD1 in the invasive characteristics of 786-0 cells was investigated by in vitro 3D branching morphogenesis and transwell assays.3.To figure out how dose TMIGD1 stimulate anti-proliferative responses in RCC cells,786-0 cells expressing TMIGD1 were analysed for activation of major cancer pathways via a recently developed immuno-paired-antibody detection system(ActiveS ignal Assay)analysis platform.The result was proved by western blot.4.5'-flanking non-coding region of TMIGD1 encompassing 1,241 base pairs(bp)was analysed via LASAGNA-Search 2.0,an integrated web tool for transcription factor binding site search and visualization.The predicted potential transcript factor was proved by electrophoretic mobility shift assay(EMSA)assay ofr physical binding.The function of transcript factor was proved via the method of luciferase reporter gene.5.The expression of potential transcript factor was tested by western blot and imunohistochemestry in cells and tissue.The result in slices were mathed with TMIGD1 expression.The factor was over-expressed and the expression of TMIGD1 was measured by qPCR and western blot.3D branching morphogenesis assay was performed to test the impact on malignant biological behavior of cancer cells.RESULTS1.The TMIGD1 mRNA was found to be highest in the kidney followed by the brain tissues.However,the TMIGD1 mRNA in the brain was significantly lower level than the kidney and its mRNA levels in ovary,heart,vein,lung,liver,pancreas,bone marrow and skin was either very low or undetectable.Similar to human,TMIGD1 was predominately present in the mouse kidney.Mouse intestine,stomach and salivary glands tissues were also positive for TMIGD1,though at lower levels.The result of western blot is in consist with qPCR.Furthermore,immunohistochemistry(IHC)analysis of human kidney tissue demonstrated that renal tubular epithelial cells were highly positive for TMIGD1,whereas podocytesof the glomerulus were negative.Analysis of human RCC microarray data,TCGA data via online cBioPortal for Cancer Genomics,which consists of 293 cases of papillary RCC,66 cases of chromophobe RCC and 499 cases of clear cell RCC.The TMIGD1 mRNA was downregulated in the all three major RCC types.2.Rhodamine-phalloidin staining for actin showed that 786-0 cells expressing TMIGD1 seeded on the collagencoated plate showed that in TMIGD1 expressing 786-0 cells,the actin fibrils were distinctively enriched in an asymmetrical fashion.The results of MTT and BruU showed that expression of TMIGD1 in 786-0 cells significantly inhibited cell proliferation compared to 786-0 cells expressing an empty vector.The tumor formation assay of 786-0 cells in athymic nude mouse showed that 786-0 cells expressing TMIGD1 formed significantly smaller tumors compared to 786-0 cells expressing empty vector.In vitro 3D branching morphogenesis assay and transwell assay proved that re-expression of TMIGD1 in 786-0 cells could inhibit cell migaration and invasion.3.786-0 cells expressing TMIGD1 were analysed for activation of twenty major cancer pathways consisting of 70 individual proteins via a recently developed immuno-paired-antibody detection system(ActiveSignal Assay)analysis platform.Among the major pathways that were affected by TMIGD1 in 786-0 cells were the proteins that are known to inhibit cell cycle and cell proliferation.Specifically,TMIGD1 upregulated expressions of p21CIP1 and p27KIP1.Additionally,while TMIGD1 reduced phosphorylation of CyclinDl and Cyclin-dependent kinase 1(CDK1/Cdc2),it increased phosphorylation of retinoblastoma protein(Rb),and p38MAPK(MAPK14).As treatment of cells with p38 inhibitor,SB203580 inhibited phosphorylation of Rb and induction of p21CIPI and p27KIP1,4.5'-flanking non-coding region of TMIGD1 encompassing 1,241 base pairs(bp),located upstream of the start site of transcription in the TMIGD1 gene,was cloned into a GFP(Zgreen)reporter vector and analyzed its promoter activity by monitoring the expression of GFP.HEK-293 cells transfected with full-length TMIGD1 promoter showed a moderate transcription activity(9%)compared to EF1?(49.8%).The 1241bp sequence was analysed via LASAGNA-Search 2.0,an integrated web tool for transcription factor binding site search and visualization.We considered CCAAT/enhancerbinding protein(C/EBP)as a potential transcription factor involved in the regulation of TMIGD1 as multiple C/EBP binding sites(TTGCnnAA)were predicated on the TMIGD1 promoter.Electrophoretic mobility shift assay(EMSA)assay revealed that C/EBP? strongly interacts with the TMIGD1 promoter.Co-expression of C/EBPp(LAP,transcriptionally active form of C/EBP?)with the full-length TMIGD1 promoter in HEK-293T cells could increased the promoter activity of TMIGD1 nearly by 90%.While over-expression of a naturally occurring transcriptionally inactive C/EBP?(LIP),inhibited the TMIGD1 promoter activity.5.Expression of C/EBP?(LAP)in 786-0,DLD1 and TK10 was very low or undetectable.However,C/EBP?(LAP)was readily detected in normal human renal epithelial cells.Expression of C/EBP? in renal cancer is significantly reduced and its reduced expression closely correlated with the expression of TMIGD1(5 out 5 cases).Expression of both TMIGD1 and C/EBP? in normal adjacent renal tissue were high,where expressions of both were significantly low in the tumor region.Expression of C/EBP?(LAP)in 786-0 cells,increased expression of TMIGD1 mRNA and protein.3D branching morphogenesis assay showed that 786-0 cells expressing C/EBP?(LAP)-FLAG displayed a significantly reduced branching morphogenesis.CONCLUSIONS1.TMIGD1 expression is highest in renal tissue both in human and mouse,intestine and brain are lower than that but still higher than most other tissues.The expression of TMIGD1 mainly located in renal tubular epithelial cells,whereas podocytes of the glomerulus were negative.In both renal cancer cell lines and tissue the expression of TMIGD1 is down-regulated.2.TMIGD1 could alter the arrangement of actin fibers in renal cancer cells,inhibit cell proliferation,tumor formation,migaration and invasion ability of renal cancer cells.3.Via promoting phosphoralation of p38,and many other signal transductions,TMIGD1 subsequently inhibits malignant biological behavior of renal cancer cells.4.C/EBP? regulates TMIGD1 expression by physically binding to the sequence upstream of TMIGD1 coding area.LAP,transcriptionally active form of C/EBP?,could increased expression of TMIGD1.While the naturally occurring transcriptionally inactive C/EBP?(LIP),inhibited the TMIGD1 promoter activity.5.C/EBP? inhibits malignant biological behavior of renal cancer cells by promting TMIGD1 expression.Part 2 TMIGD1 acts as a tumor suppressor through inhibiting CYR61 in colon cancerMETHODS1.Human colon cancer cell microarray data,TCGA data via online cBioPortal for Cancer Genomics,were analysed for TMIGD1.A cohort of human colon cancer biopsy samples were collected and slices were immunohistochemically stained for TMIGD1.qPCR and western blot were used to check the expression in conlon cancer cell lines.2.TMIGD1 was reintroduced into colon cancer cell lines,RKO and HCT116 cells via a retroviral system.Rhodamine-phalloidin staining for actin was performed to observe the morphological changes.MTT and BrdU assays were used to assesse the function of TMIGD1 in cell proliferation.We examined the effect of TMIGD1 expression in Tumor formation ability of TMIGD1-expressed cells were examinged by an athymic nude mouse model.Function of TMIGD1 on the invasive characteristics of cells was investigated by in vitro 3D branching morphogenesis and transwell assays.3.The expression or phosphorylation of p38,p-p38,p-rb,p21CIP1 and p27KIP1 were detected by western blot in colon cancer cell lines expressing TMIGD1.P38MAPK inhibitor SB203580 was used to treat the cells and changes of proteins was measured by western blot.4.C/EBP(LAP)was expressed in colon cancer cells,and the expression of LAP and TMIGD1 was detected by western blot.MTT was used to detect the change of proliferation of cells in each group.5.Extracellular domain of TMIGD1 was cloned in to pGEX plasmid,and the plasmid was transformed into engineered bacterium to produce TMIGD1-GST fusion protein.The purified glutathione S-transferase(GST)-fusion TMIGD1 protein subsequently was used for GST pull-down assay.The pull-down sample was subjected to SDS electrophosis and subsequently mass spectrometry analysis,looking for potential TMIGD1 ligand.The predicted ligand and TMIGD1 were co-transfected in RKO cells.Whole cell lysate was extracted for co-immunoprecipitation and GST-pulldown to prove physical binding between TMIGD1 and its ligand.6.TMIGD1 and the predicted ligand were manipulated in colon cancer cells and the cells were tested with MTT and BrdU assays for the proliferation ability of the cell lines.Transwell assays were performed to estimate the effect on cell migaration and invasion.Colony forming assay was used to test the tumor formation of the cell lines.Different concentration of foreign ligands was given to test the effects of TMIGD1 on the stimulation response of the cells.RESULTS1.Analysis of human conlon cancer microarray data,TCGA data via online cBioPortal for Cancer Genomics,which consists of 195 cases of colon cancer,proved that TMIGD1 expression was down-regulated in colon cancer.Immunohistochemistry(IHC)analysis of human colon cancer tissue demonstrated that TMIGD1 was down-regulated in tumor area,whereas the expression in normal adjacent renal tissue was higher.Both TMIGD1 mRNA and protein levels were downregulated in all the colon cancer cell lines tested.2.After TMIGD1 was expressed in human colon cancer cell line RKO,HCT116 cells,the results of rhodamine-phalloidin staining showed that HCT116 and RKO cells expressing TMIGD1 were more regulated in cell morphology,and when the cell density is similar,cells that expressed TMIGD1 formed fewer intercellular connections than those in the control group,the connection formed via pseudopod significantly decreased.The results of MTT and BruU showed that expression of TMIGD1 in RKO and HCT116 cells significantly inhibited cell proliferation.The tumor formation assay of RKO and HCT116 cells in athymic nude mouse showed that colon cancer cells expressing TMIGD1 formed significantly smaller tumors compared to cells expressing empty vector.Colony forming assay and transwell assay proved that re-expression of TMIGD1 in RKO and HCT116 cells could inhibit cell migaration and invasion.3.In the colon cancer cell line RKO that expressed TMIGD1,western blot results showed that p-p38,p-rb,p21CIP1,p27KIP1 protein increased,and p38 expression was not significantly changed.With p38MAPK inhibitor SB203580,p-rb,p21CIP1,p27KIP1 protein expression levels were reversed,and p-p38 and p38 expression levels did not change significantly.The results of MTT proliferation experiment showed that SB203580 had no significant effect on the proliferation of colon cancer cell RKO without transfection TMIGD1.After transfection of TMIGD1,SB203580 treatment of RKO cells could significantly reverse the decline of cell proliferation induced by transfection of TMIGD1.4.After expressing C/EBP(LAP)in colon cancer cell RKO,western blot test results showed that the expression of LAP and TMIGD1 was up-regulated.MTT proliferation assay showed that the proliferation of the colon cancer cells transfected with LAP was significantly inhibited.5.LC-MS/MS mass spectrometry analysis of GST-pulldown samples predicted CYR61(cysteine rich angiogenic inducer 61)to be the potential extracellular ligand for TMIGD1.The prediction was proved by co-immunoprecipitation and GST-pulldown experiments.GST fusion protein of domains of CYR61 was generated and GST-pulldown experiment proved domain VWC(Willebrand Factor-like module)to be the TMIGD1-bingding domain.6.After interfering the expression of CYR61,all kinds of malignant biological behaviors of HCT116 and RKO cells were reduced to a certain extent.After expressing TMIGD1,as in the previous experiment,the proliferation,migration,invasion and tumor formation ability of colon cancer cells were inhibited.At this time,there's no further inhibition on the malignant biological behavior of colon cancer cells by interfering expression of CYR61.In the RKO cell lines that expressed TMIGD1 and interfered expression of CYR61,the exogenous CYR61 stimulation of different concentrations was given,and the proliferation of the two groups of cells showed a delayed increase in TMIGD1 over-expressed group.CONCLUSIONS1.In both colon cancer cell lines and tissue the expression of TMIGD1 is down-regulated.2.TMIGD1 could inhibit the maliganent biological behaviors of colon cancer cells including cell proliferation,tumor formation,migaration and invasion.3.Via physically binding to the VWC domain of CYR61,TMIGD1 could abolish the increase caused by CYR61 in maliganent biological behaviors in colon cancer cell.SIGNIFICANCE1.Firstly proved the inhibitory function of TMIGD1 on maliganent biological behaviors,including cell proliferation,tumor formation,migaration and invasion,of renal and colon cancer cells,identifying TMIGD1 as a tumor suppressor.2.Illustrated the mechanism that via promoting phosphoralation of p3 8,TMIGD1 decreases the phosphoralation of CyclinDl,promotes phosphoralation of Rb,and up-regualtes p21CIP1 and p27KIP1,subsequently inhibiting malignant biological behavior of renal cancer cells.Identified C/EBP ? as transcript factor regulating TMIGD1 expression by directly binding to the sequence upstream to TMIGD1 coding area.Indentified CYR61 as extracellular ligand to TMIGD1,and demonstrated that TMIGD1 functions as tumor suppressor in colon cancer cells by directly binding to CYR61 and abolishing its tumor promoting effects.3.Firstly proved TMIGD1 as a tumor suppressor in renal and colon cancer cells and illustrated the mechanisms beneath,providing TMIGD1 has potential value as a tumor suppressor for early diagnosis of clinical tumor,and its tumor suppressive action can also provide reference and thought for the targeted therapy of clinical malignant tumor. |
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