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• W3096133051 abstract "Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target. Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target. Colorectal cancer (CRC) is the second most common malignant tumor in western countries.1Lynch H.T. de la Chapelle A. Hereditary colorectal cancer.N Engl J Med. 2003; 348: 919-932Crossref PubMed Scopus (1561) Google Scholar CRC's high mortality is associated with tumor metastasis and poor response of patients to current standard-of-care drugs.2Parkin D.M. Bray F. Ferlay J. Pisani P. Estimating the world cancer burden: Globocan 2000.Int J Cancer. 2001; 94: 153-156Crossref PubMed Scopus (3228) Google Scholar, 3Bogaert J. Prenen H. Molecular genetics of colorectal cancer.Ann Gastroenterol. 2014; 27: 9-14PubMed Google Scholar, 4Fearon E.R. Molecular genetics of colorectal cancer.Annu Rev Pathol. 2011; 6: 479-507Crossref PubMed Scopus (1105) Google Scholar The development of CRC is complex and involves multiple molecular pathways characterized by numerous genetic and epigenetic lesions.5Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5219) Google Scholar CRC can arise from hereditary and nonhereditary sporadic mutations, but >85% of CRCs are nonfamilial. The inactivation of the adenomatous polyposis coli (APC) or its downstream signaling components are common in hereditary and nonhereditary CRCs and are among the best understood pathways involved in the initiation of CRC.4Fearon E.R. Molecular genetics of colorectal cancer.Annu Rev Pathol. 2011; 6: 479-507Crossref PubMed Scopus (1105) Google Scholar However, other genetic and epigenetic alterations are required for the full development of CRC.5Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5219) Google Scholar Loss of APC function results in the accumulation of β-catenin, resulting in the transcription of a large number of cancer-causing target genes,6Dikovskaya D. Zumbrunn J. Penman G.A. Nathke I.S. The adenomatous polyposis coli protein: in the limelight out at the edge.Trends Cell Biol. 2001; 11: 378-384Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,7Hanson C.A. Miller J.R. Non-traditional roles for the Adenomatous Polyposis Coli (APC) tumor suppressor protein.Gene. 2005; 361: 1-12Crossref PubMed Scopus (88) Google Scholar alteration of cell-cell adhesion, and cell migration.8Zhang L. Shay J.W. Multiple roles of APC and its therapeutic implications in colorectal cancer.J Natl Cancer Inst. 2017; 109: djw332Crossref Scopus (127) Google Scholar Despite these noticeable functions of APC, its functional loss alone in mice or humans is not sufficient to account for the full-blown development of CRC,9Luongo C. Moser A.R. Gledhill S. Dove W.F. Loss of Apc+ in intestinal adenomas from Min mice.Cancer Res. 1994; 54: 5947-5952PubMed Google Scholar suggesting a significant involvement of other pathways in the tumorigenesis of CRC. Transmembrane and immunoglobulin domain containing (TMIGD) family proteins are a newly identified class of immunoglobulin domain containing cell adhesion molecules, which include TMIGD1, immunoglobulin and proline rich receptor-1,10Woolf N. Pearson B.E. Bondzie P.A. Meyer R.D. Lavaei M. Belkina A.C. Chitalia V. Rahimi N. Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.Oncogenesis. 2017; 6: e378Crossref PubMed Google Scholar, 11Wang Y.H.W. Meyer R.D. Bondzie P.A. Jiang Y. Rahimi I. Rezazadeh K. Mehta M. Laver N.M.V. Costello C.E. Rahimi N. IGPR-1 is required for endothelial cell-cell adhesion and barrier function.J Mol Biol. 2016; 428: 5019-5033Crossref PubMed Scopus (11) Google Scholar, 12Rahimi N. Rezazadeh K. Mahoney J.E. Hartsough E. Meyer R.D. Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis.Mol Biol Cell. 2012; 23: 1646-1656Crossref PubMed Scopus (37) Google Scholar and TMIGD3,13Iyer S.V. Ranjan A. Elias H.K. Parrales A. Sasaki H. Roy B.C. Umar S. Tawfik O.W. Iwakuma T. Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-kappaB and osteosarcoma progression.Nat Commun. 2016; 7: 13561Crossref PubMed Scopus (17) Google Scholar which also acts as a tumor suppressor.14Ranjan A. Iyer S.V. Iwakuma T. Suppressive roles of A3AR and TMIGD3 i1 in osteosarcoma malignancy.Cell Cycle. 2017; 16: 903-904Crossref PubMed Scopus (3) Google Scholar Although originally described as a protector of renal epithelial cells from oxidative cell injury,15Arafa E. Bondzie P.A. Rezazadeh K. Meyer R.D. Hartsough E. Henderson J.M. Schwartz J.H. Chitalia V. Rahimi N. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.Am J Pathol. 2015; 185: 2757-2767Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar TMIGD1 is down-regulated in human renal cancer, and its reexpression in renal tumor cells inhibits cell proliferation, migration, and tumor growth in mouse tumor xenograft.16Meyer R.D. Zou X. Ali M. Ersoy E. Bondzie P.A. Lavaei M. Alexandrov I. Henderson J. Rahimi N. TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.Oncotarget. 2018; 9: 9672-9684Crossref PubMed Scopus (9) Google Scholar More importantly, a recent whole genome sequencing of preinvasive colorectal tumor revealed that expression of TMIGD1 is progressively lost in colon cancer.17Cattaneo E. Laczko E. Buffoli F. Zorzi F. Bianco M.A. Menigatti M. Bartosova Z. Haider R. Helmchen B. Sabates-Bellver J. Tiwari A. Jiricny J. Marra G. Preinvasive colorectal lesion transcriptomes correlate with endoscopic morphology (polypoid vs. nonpolypoid).EMBO Mol Med. 2011; 3: 334-347Crossref PubMed Scopus (27) Google Scholar Although the change of TMIGD1 expression in colon cancer has been documented, the functional importance of loss of TMIGD1 in human colon cancer remains to be elucidated. This study demonstrates that the loss of TMIGD1 in mice results in the development of intestinal adenomas. The study provides evidence that TMIGD1 acts as a tumor suppressor by arresting cell cycle at G2/M. These findings provide novel insights into the pathogenesis of colorectal cancer and a possible new therapeutic target. Anti-TMIGD1 antibody was previously described.15Arafa E. Bondzie P.A. Rezazadeh K. Meyer R.D. Hartsough E. Henderson J.M. Schwartz J.H. Chitalia V. Rahimi N. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.Am J Pathol. 2015; 185: 2757-2767Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar The following antibodies was also used in this study. Proliferating cell nuclear antigen (PCNA) antibody (catalog number ab2426) was purchased from Abcam (Cambridge, MA). Zona occludens 1 (ZO1) antibody (catalog number 339,100) was purchased from Life Technologies (Carlsbad, CA). Actin (catalog number 4968S), β-catenin (catalog number 8480P), CDX2 (catalog number 3977S), and E-cadherin (catalog number 14472S) antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Villin antibody (catalog number sc-58897) was purchased from Santa Cruz Biotechnologies (Dallas, TX). TMIGD1 was cloned into the retroviral vector pMSCV-puro as previously described.15Arafa E. Bondzie P.A. Rezazadeh K. Meyer R.D. Hartsough E. Henderson J.M. Schwartz J.H. Chitalia V. Rahimi N. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.Am J Pathol. 2015; 185: 2757-2767Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar HCT116, DLD1, HT29, and RKO cells were grown in RPMI 1640 medium plus 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were purchased from ATCC (Manassas, VA). Normal human intestinal cell line (NCM460) was kindly provided by Arthur Stucchi (Department of Surgery, Boston University, Boston, MA) were grown in RPMI 1640 medium supplemented with 10% FBS penicillin/streptomycin. TMIGD1 heterozygous mice were generated at the Nanjing BioMedical Research Institute of Nanjing University (Nanjing, China), on C57BL/6J background. Homozygous TMIGD1 mice were generated with subsequent breeding. All mice used in this study were bred and maintained at Boston University Medical Center (Boston, MA) after approval from the Institutional Animal Care and Use Committee. The following primers were used for genotyping of TMIGD1 mice: forward primer: 5′-CCCTATATCCTCAGGCTCTG-3′ and reverse primer: 5′-CGTTCAGCACTACTGTAACGGAC-3′. Mice were euthanized, and the intestinal tissues were harvested. A gavage needle was used to flush the colon and small intestine with ice-cold phosphate-buffered saline (PBS). Organs were then stretched across filter paper and opened longitudinally to fix in 10% formalin overnight at 4°C. Tissues were Swiss rolled with the distal end of the intestine closest to the center of the coil and the proximal end at the outside. The Swiss-rolled intestines were paraffin embedded and sectioned at 4 μm for histologic examination. Three sections from different segments of the block were stained with hematoxylin and eosin and graded by a veterinary pathologist and a surgical pathologist (Q.Z.) in a blind manner for detection of atypical hyperplasia, adenoma, or adenocarcinoma. Additional sections were used for immunohistochemistry or immunofluorescence staining with antibodies as described in Figure 1. HCT116 and RKO cells expressing empty vector (EV) and TMIGD1 were plated onto 10-cm tissue culture plates at 70% to 80% confluence. Cells from each cell line were starved for 0 and 72 hours. For harvesting, cells were collected by trypsinization and washed with PBS. Cells were then fixed with 70% ethanol and stored at 4°C for at least 30 minutes. After fixation, cells were washed twice with PBS, and 1 × 106 cells per group were resuspended in PBS supplemented with 50 μL of RNase (100 μg/mL of stock). Ten minutes before flow cytometry analysis is performed, propidium iodide (5 μL per group, 1 mg/mL of stock) is added to the samples and briefly vortexed. Flow cytometry was performed by BD LSRII (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Beckton, Dickson and Co., Franklin Lakes, NJ). The ActivSignal assay examines phosphorylation or expression of 70 different human protein targets, which covers 20 major signaling pathways.16Meyer R.D. Zou X. Ali M. Ersoy E. Bondzie P.A. Lavaei M. Alexandrov I. Henderson J. Rahimi N. TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.Oncotarget. 2018; 9: 9672-9684Crossref PubMed Scopus (9) Google Scholar RKO cells expressing EV or TMIGD1 were plated in 96-well plates in triplicates and subjected to ActiveSignal Assay analysis as described in Figure 2. In vitro cell migration assay was performed using the Boyden chamber assay (Corning Transwell, purchased from Thermo Fisher Scientific, Waltham, MA). Briefly, cells (2 × 104 cells per well, triplicate per group) were plated on the Matrigel-coated transwells (Corning Transwell, purchased from Thermo Fisher Scientific) in which the upper chamber contained 1% FBS and the lower chamber contained 10% FBS medium. After 6 hours, the nonmigrated cells from the upper side of the membrane were removed by Q-tip, cells were fixed and stained with crystalline blue, and the number of migrated cells were counted under a light microscope. To evaluate the capacity of the CRC cell line HCT116 cell–expressing green fluorescent protein (GFP) alone or expressing TMIGD1/GFP to extravasate and grow, equal numbers of cells (5 × 105 cells per/mouse) were injected via the tail vein and metastasis to lung was evaluated after 2 weeks. Specifically, mice were sacrificed, lungs were removed, and slides of frozen tissues were prepared and viewed under a fluorescence microscope. Mice intestinal tissues were sectioned to 4 μm and baked for 1 hour. Tissue slides were then deparaffinized using decreasing alcohol gradients. Slides were then submerged into sodium citrate buffer for antigen unmasking. Tissue sections were then permeabilized using 0.3% Triton X-100. Slides were blocked using 5% bovine serum albumin in tris-buffered saline and Tween 20. Primary antibody was then added to tissue sections, and sections were incubated. Fluorescent antibody was added to the tissue sections and was incubated in the dark. Slides were then mounted using VectaShield Antifade Mounting Medium (Vector Laboraties, Burlingame, CA) with DAPI. Two human colorectal cancer tissue microarray slides (catalog numbers BC05012a and BC05118a; US Biomax, Derwood, MD) consisting of 172 tissue samples (72 on BC05012a and 100 on BC05118a) were stained with anti-TMIGD1 antibody. The patient age, tumor grade, tumor stage, and TNM status were provided for each sample on both microarrays. Six samples were excluded from the final analysis because of poor tissue quality secondary to artifact or complete absence of CRC tissue in the specimen. Immunohistochemistry was performed on both microarray slides using validate TMIGD1 antibody15Arafa E. Bondzie P.A. Rezazadeh K. Meyer R.D. Hartsough E. Henderson J.M. Schwartz J.H. Chitalia V. Rahimi N. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.Am J Pathol. 2015; 185: 2757-2767Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,16Meyer R.D. Zou X. Ali M. Ersoy E. Bondzie P.A. Lavaei M. Alexandrov I. Henderson J. Rahimi N. TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.Oncotarget. 2018; 9: 9672-9684Crossref PubMed Scopus (9) Google Scholar and polymer–horseradish peroxidase secondary antibody (Abcam). The staining intensity of each specimen was rated independently by two pathologists (Q.Z.). Each specimen was rated from 0 to 3, with 0 representing no TMIGD1 staining and 3 representing the highest intensity of TMIGD1 staining. The staining pattern was described for each specimen as granular, perinuclear, or diffuse. The mean TMIGD1 staining intensity was stratified based on patient age, tumor grade, tumor stage, and T rating in the TNM staging system. The mean intensity values of each subgroup were compared for statistical significance via analysis of variance with the Tukey post-hoc test. The α value for significance was set at P < 0.05. First, the expression of TMIGD1 in normal human and mouse intestinal tissues was examined via immunofluorescence staining using a previously validated anti-TMIGD1 antibody.15Arafa E. Bondzie P.A. Rezazadeh K. Meyer R.D. Hartsough E. Henderson J.M. Schwartz J.H. Chitalia V. Rahimi N. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.Am J Pathol. 2015; 185: 2757-2767Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,16Meyer R.D. Zou X. Ali M. Ersoy E. Bondzie P.A. Lavaei M. Alexandrov I. Henderson J. Rahimi N. TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.Oncotarget. 2018; 9: 9672-9684Crossref PubMed Scopus (9) Google Scholar TMIGD1 was detected at the membranous regions of intestinal epithelial cells in human (Supplemental Figure S1, B and C). Consistent with its previously described characteristic as a cell adhesion molecule, TMIGD1 co-localized with E-cadherin in human intestinal epithelial cells, a well-characterized marker of epithelial cell adherens junctions (Supplemental Figure S1, D–F), but not with the tight junction protein ZO-1 (Supplemental Figure S2, A–C). Similar to its expression in human intestinal tissue, TMIGD1 is also expressed in mouse intestinal epithelial cells (Supplemental Figure S3B). Next, overall TMIGD1 expression in human intestinal tissue was examined via the Genevestigator data set (HS_mRNASeq_Human_GL-0; Genevestigator, Nebion AG, Zurich Switzerland).18Hruz T. Laule O. Szabo G. Wessendorp F. Bleuler S. Oertle L. Widmayer P. Gruissem W. Zimmermann P. Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes.Adv Bioinformatics. 2008; 2008: 420747Crossref PubMed Google Scholar This analysis revealed that TMIGD1 mRNA is highly expressed in human colonic tissues, including cecum, small intestine, large intestine, and jejunum (Supplemental Figure S4A). In addition, analysis of the RNA sequence of 19 human fetus tissues (NIH Roadmap Epigenomics Mapping Consortium via EBI, https://www.ebi.ac.uk/gxa/home, last accessed November 20, 2019) similarly demonstrated that TMIGD1 mRNA (http://www.ncbi.nlm.nih.gov; GenBank accession number NM_206,832.3) is highest in the intestinal tissues followed by the kidney (Supplemental Figure S4B). The TMIGD1 mRNA in other organs, including heart, stomach, muscle, adrenal gland, and placenta, were not expressed or expressed at very low levels (Supplemental Figure S4B). To investigate the importance of TMIGD1 in the intestinal function in vivo, homozygous TMIGD1 knockout (KO) mice were generated via CRISPR/Cas9 system (Figure 1A). CRISPR/Cas9-mediated loss of TMIGD1 was confirmed by real-time quantitative PCR (Figure 1B). Although TMIGD1−/− mice are viable and fertile and pups display no apparent abnormality, as they get older (≥4 months), they develop intestinal hyperplasia (Figure 1C). Analysis of intestines of TMIGD1−/− mice revealed that 80% (8 of 10) of TMIGD1−/− mice develop polyps in small intestine, large intestine, and rectum (Figure 1C). The mean number of polyps observed was 7 to 12 per mouse. Furthermore, hematoxylin and eosin staining demonstrated the development of microtubular adenoma and tubular adenoma in TMIGD1−/− mice (Figure 1D). It is highly likely that development of adenoma in TMIGD1−/− mice is associated with aberrant cell proliferation. Therefore, intestinal tissues of wild-type and TMIGD1 KO mice were examined with PCNA, a marker for cell proliferation. Immunostaining of the intestinal tissues of TMIGD1+/+ and TMIGD1−/− mice for PCNA showed that loss of TMIGD1 in mice results in the hyperproliferation of the intestinal epithelial cells (Figure 1E). Although there was weak staining for PCNA in the intestinal epithelium (in the crypt region) of TMIGD1+/+ mice, there was strong staining for PCNA in the intestinal epithelium of TMIGD1−/− mice (Figure 1E). Furthermore, Ki-67 staining, another marker for in vivo cell proliferation, showed similar result (data not shown). TMIGD1−/− mice are currently being monitored for potential adenocarcinoma development and other potential pathologies as they get older. Taken together, loss of TMIGD1 in mice is shown to cause intestinal adenoma, with a significant potential role in human colorectal cancer. Examination of intestinal tissues of TMIGD1−/− mice further demonstrated that, in addition to development of adenoma, overall organization of the intestinal epithelium in TMIGD1−/− mice was abnormal. Specifically, hematoxylin and eosin staining revealed that brush border, an actin-based membrane protrusion known as microvilli, in TMIGD1−/− mice was distinctively reduced. Although brush border in the wild-type mouse intestine was clearly evident and well-organized, the brush border in the intestinal tissues of TMIGD1−/− mice was mostly absent (Figure 3A). To further investigate the apparent loss of brush border in TMIGD1−/− mice, the intestinal tissues of wild-type and TMIGD1−/− mice were stained with actin and villin, which are common markers for the brush border. Immunofluorescence staining demonstrated that both actin and villin are uniformly present at the brush border of wild-type mice (Figure 3, B and C). However, in TMIGD1−/− mice actin localization at the brush border was significantly reduced and was aberrantly localized at the crypt and villus regions (Figure 3B). Interestingly, villin localization to brush border was significantly reduced or mislocalized in TMIGD1−/− mice (Figure 3C). To obtain additional insight into the brush border impairment in the intestinal epithelial cells in TMIGD1 KO mice, the intestinal tissues of wild-type and the TMIGD1 KO mice were stained with atypical protein kinase C, a marker for apicobasal polarity. The data demonstrated that atypical protein kinase C is uniformly present at the brush border in the wild-type mice but not in the TMIGD1 KO mice (Figure 3D). Staining with the E-cadherin antibody demonstrated that in the wild-type mice, E-cadherin was strongly positive at the crypt and microvilli of epithelium (Figure 4A), which indicates a normal cellular junctions of epithelium. Similarly, β-catenin staining displayed a similar pattern (Figure 4B). However, although the crypt epithelial cells were positive for E-cadherin in TMIGD1−/− mice, it was diffusely present at the cell junctions, prominently detected at the apical membrane (Figure 4A), and mostly absent in the brush border (Figure 4A). Moreover, β-catenin was diffusely present at the intestinal cell junctions in TMIGD1−/− mice (Figure 4B). E-cadherin is known to localize to the lateral membrane of differentiated epithelial cells, providing the structural foundation for adherens junctions, which also promotes epithelial apical-basal polarization.19Gumbiner B.M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis.Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2848) Google Scholar Though E-cadherin is required for maturation of intestinal Paneth cells,20Schneider M.R. Dahlhoff M. Horst D. Hirschi B. Trulzsch K. Muller-Hocker J. Vogelmann R. Allgauer M. Gerhard M. Steininger S. Wolf E. Kolligs F.T. A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation.PLoS One. 2010; 5: e14325Crossref PubMed Scopus (121) Google Scholar β-catenin is thought to play a central role in maturation and morphogenesis of crypt and villus.21Andreu P. Peignon G. Slomianny C. Taketo M.M. Colnot S. Robine S. Lamarque D. Laurent-Puig P. Perret C. Romagnolo B. A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation.Dev Biol. 2008; 324: 288-296Crossref PubMed Scopus (81) Google Scholar,22Fevr T. Robine S. Louvard D. Huelsken J. Wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells.Mol Cell Biol. 2007; 27: 7551-7559Crossref PubMed Scopus (429) Google Scholar Additional staining with the ZO1 protein, a marker for tight junction that is also required for tight junction formation of epithelial cells,23Shin K. Fogg V.C. Margolis B. Tight junctions and cell polarity.Annu Rev Cell Dev Biol. 2006; 22: 207-235Crossref PubMed Scopus (547) Google Scholar demonstrated that in TMIGD1−/− mice, ZO1 is largely absent at the junctions of intestinal epithelial cells (Figure 4C). To gain further insights into a possible mechanism of aberrant junctional development of intestinal epithelium in TMIGD1 KO mice, whether loss of TMIGD1 in mice alters the intestinal epithelial cell maturation was examined. To this end, the TMIGD1−/− mouse intestinal tissue was stained with CDX2, a marker for maturation of intestinal epithelial cells, including goblet and Paneth cells.24Crissey M.A. Guo R.J. Funakoshi S. Kong J. Liu J. Lynch J.P. Cdx2 levels modulate intestinal epithelium maturity and Paneth cell development.Gastroenterology. 2011; 140: 517-528.e8Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25Gao N. White P. Kaestner K.H. Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.Dev Cell. 2009; 16: 588-599Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 26Suh E. Traber P.G. An intestine-specific homeobox gene regulates proliferation and differentiation.Mol Cell Biol. 1996; 16: 619-625Crossref PubMed Scopus (442) Google Scholar Although the CDX2-positive epithelial cells were uniformly present at the crypt and villus of the wild-type mice, in TMIGD1−/− mice, CDX2-positive cells were detected only at the lower part of the crypts, albeit weakly and inconsistently (Figure 4D). Altogether, these data demonstrate that the loss of TMIGD1 in mice impairs intestinal epithelial cell maturation and intercellular junctions. Considering that loss of TMIGD1 in mice induced intestinal adenomas and increased cell proliferation (Figure 1), whether the effect of ectopic expression of TMIGD1 in colon cancer cells can inhibit cell proliferation was investigated. TMIGD1 or EV was initially expressed through a retroviral expression system in RKO cells. Expression of TMIGD1 in RKO cells was confirmed by Western blot analysis using anti-TMIGD1 antibody (Figure 5A). Overexpression of TMIGD1 in RKO cells significantly inhibited cell proliferation (Figure 5B). Although proliferation of RKO cells expressing EV (EV/RKO) increased in a time dependent manner, proliferation of RKO cells expressing TMIGD1 (TMIGD1/RKO) was reduced by 53% at day 4 (P = 0.0033, number of cell for EV/RKO cells was 66.5 × 104 versus 30.8 × 104 for TMIGD1/RKO cells) (Figure 5B). It was postulated that TMIGD1 could inhibit cell proliferation by a mechanism that modulates the cell cycle. Therefore, the cell cycle distribution of EV/RKO and TMIGD1/RKO cells was examined by FACS (LSRII) analysis via propidium iodide staining. TMIGD1-expressing RKO cells showed a significant cell cycle arrest at the G2/M phase (25.4% versus 9.3%) in the presence of 10% FBS (Figure 5, C and D) and serum-starved (72 hours) 14.1% versus 7.2% (Figure 5, E and F) G2/M phase arrest in the cell cycle of TMIGD1/RKO cells followed by a markedly decreased G0/G1 phase of the cell cycle (Figure 5 C–F). The effect of ectopic expression of TMIGD1 on cell cycle was not limited to RKO cells because its ectopic expression in HCT116 cells (Supplemental Figure S5A) similarly resulted in the cell cycle arrest at the G2/M phase (Supplemental Figure S5B). In a complementary approach, TMIGD1 was knocked down by shRNA in normal colon epithelial cells, NMC460 (Supplemental Figure S6A), and the cell cycle profile was examined. The result showed that the knockdown of TMIGD1 in NMC460 cells reduced the serum starvation–induced cell cycle arrest at the G2/M phase (17.5% versus 10.2%) (Supplemental Figure S6B). Taken together, these data demonstrate that reintroduction of TMIGD1 in CRC cells (RKO and HCT116) promotes cell cycle arrest at the G2/M phase, and knockdown of TMIGD1 in normal intestinal epithelial cells, NMC460, decreases the serum starvation–induced cell cycle arrest at the G2/M phase. To investigate the potential mechanism by which TMIGD1 affects cell cycle, RKO cells expressing TMIGD1 for activation of 20 major cancer pathways consisting of 70 individual proteins were analyzed via an immunopaired antibody detection system (ActiveSignal Assay) analysis platform.16Meyer R.D. Zou X. Ali M. Ersoy E. Bondzie P.A. Lavaei M. Alexandrov I. Henderson J. Rahimi N. TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.Oncotarget. 2018; 9: 9672-9684Crossref PubMed Scopus (9) Google Scholar Among the major pathways that were affected by TMIGD1 in RKO cells were the proteins known to inhibit cell cycle and cell proliferation. Specifically, TMIGD1 up-regulated expressions of p21CIP1 (cyclin-dependent kinase inhibitor 1) and p2" @default.
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• W3096133051 title "Transmembrane and Immunoglobulin Domain Containing 1, a Putative Tumor Suppressor, Induces G2/M Cell Cycle Checkpoint Arrest in Colon Cancer Cells" @default.
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