Use of membrane protein CMTM3 as a drug target for treating inflammatory bowel disease and its associated colorectal cancer

Abstract

The present application relates to the field of biological medicine, and specifically, the present application provides a kind of membrane protein CMTM3 as the application of drug target for treating inflammatory bowel disease and its related colorectal cancer.The present application first finds and confirms that the deletion of Cmtm3 can protect mice from AOM / DSS-induced colorectal cancer (CAC).Our further research results confirm that endothelial cell-derived CMTM3 promotes the development of intestinal inflammation.Cmtm3 gene knockout and imatinib mesylate can both target the regulation of vascular permeability and have comparable efficacy.And the present application also discloses that CMTM3 promotes intestinal inflammation by stabilizing CLTC and increasing the clathrin-mediated endocytosis of VE-cadherin in endothelial cells, thereby promoting the development of inflammation-related colon cancer, indicating that CMTM3 can be used as a potential therapeutic target for intestinal inflammation and colon cancer (CAC).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine biology, in particular, the present application relates to the application of membrane protein CMTM3 as a drug target for treating inflammatory bowel disease and its related colorectal cancer. BACKGROUND

[0002] Colorectal cancer (CRC) is the third leading cause of cancer death worldwide. Chronic inflammatory conditions, such as inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), increase the risk of developing inflammation-associated colon cancer (CAC). It has been reported that CAC patients have a poorer prognosis compared with sporadic colorectal cancer patients, leading to increased mortality. In recent years, epigenetic changes, particularly changes in DNA methylation, have been observed in the process of inflammation and CAC. However, compared with sporadic CRC, less is known about the molecular mechanisms of CAC. Given the increasing incidence of IBD, inhibiting the development of intestinal inflammation can help to intervene early in CAC, which may ultimately reduce the mortality rate of CRC.

[0003] Currently, IBD treatment mainly induces and maintains basic remission, prevents disease recurrence and progression to colorectal cancer, and avoids surgery and complications. The use of biological agents targeting immune response and inflammatory factors, such as anti-TNF-α antibodies, provides a new solution for the treatment of moderate to severe patients. However, many patients do not respond to the drug or develop immunogenicity in some patients after long-term use, followed by drug resistance. In recent years, ustekinumab targeting interleukin (IL)-12 and IL-23p40 subunit, vedolizumab specifically binding to α4β7 integrin, and JAK kinase (Janus kinase, JAK) inhibitors, all of which inhibit the production of inflammatory factors or inhibit the entry of immune cells into the intestinal tissue of the inflammatory site, increase the risk of serious infections in patients due to the inhibition of immune system function, and may increase the risk of malignancy. The pathogenesis of intestinal inflammation is multifactorial, including the immune system and the intestinal epithelium and endothelium. The intestinal vascular endothelium is the second important barrier of the intestinal tract, and endothelial cells are the most important component. Studies have shown that the intestinal vascular endothelium is related to the pathogenesis of DSS (sodium dextran sulfate) induced intestinal inflammation and CAC in mice. Due to the key role of vascular endothelium in the development of intestinal inflammation, its potential regulatory mechanisms need to be further elucidated.

[0004] CMTM (CKLF-like MARVEL transmembrane domain-containing family) is a kind of chemokine-like factor superfamily containing MARVEL transmembrane domain, which is encoded by 9 genes (CKLF and CMTM1-8) of human. It is a gene family that the inventors have been studying for a long time. CKLF and CMTM1-CMTM8 genes are located on human chromosomes 3 (CMTM6, CMTM7, CMTM8), 14 (CMTM5) and 16 (CMTM1, CMTM2, CMTM3, CMTM4) and mouse chromosomes 8, 9 and 14. CMTM plays a key role in many biological processes, such as regulating the stability of epidermal growth factor receptor (EGFR), VE-cadherin, PD-L1, NEMO, CD58, beta-catenin, etc., and is involved in the occurrence of tumors. CMTM has different expression profiles in human tumors and normal tissues, and different CMTM members play different roles in the occurrence and development of tumors. CMTM3 is a member of the CMTM family and is located at the key tumor suppressor locus 16q22.1. In some cancer cell lines and primary tumors including gastric cancer, breast cancer and colon cancer, CMTM3 is silenced or down-regulated due to CpG methylation. CMTM3 inhibits the proliferation and migration of various tumor cells. However, there is no relevant report on the relationship between CMTM3 and inflammatory bowel disease and its related colorectal cancer. SUMMARY

[0005] In view of the deficiencies in the existing treatments, the present application finds that the membrane protein CMTM3 promotes vascular permeability by disrupting VE-cadherin in endothelial cells, thereby becoming a potential therapeutic target for intestinal inflammation and colon cancer (CAC). Therefore, the purpose of the present application is to provide the application of the membrane protein CMTM3 as a drug target for treating inflammatory bowel disease and its related colorectal cancer. To achieve the purpose of the present application, the following technical solutions are adopted:

[0006] In a first aspect of the present application, the application of the membrane protein CMTM3 and / or CLTC as a target in screening drugs for treating inflammatory bowel disease and / or related colorectal cancer is provided.

[0007] In an embodiment, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0008] In an embodiment, the CMTM3 promotes the occurrence of inflammatory bowel disease by disrupting VE-cadherin in endothelial cells under inflammatory stimulation.

[0009] In a second aspect of the present application, there is provided a use of a membrane protein CMTM3 inhibitor in the preparation of a medicament for treating inflammatory bowel disease and / or associated colorectal cancer, wherein the membrane protein CMTM3 inhibitor can reduce the expression of a gene encoding membrane protein CMTM3 in a patient.

[0010] In one embodiment, the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.

[0011] In a third aspect of the present application, there is provided a use of an agent for modulating the expression of a gene encoding membrane protein CMTM3 and / or CLTC in the preparation of a medicament for inhibiting the expression of a protein molecule associated with intestinal inflammation, wherein the agent can reduce the expression of a gene encoding membrane protein CMTM3 in a patient.

[0012] In a fourth aspect of the present application, there is provided a medicament for treating inflammatory bowel disease, wherein the medicament comprises a membrane protein CMTM3 inhibitor and a pharmaceutically acceptable excipient, wherein the membrane protein CMTM3 inhibitor can reduce the expression of a gene encoding membrane protein CMTM3 in a patient.

[0013] In one embodiment, the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.

[0014] In a fifth aspect of the present application, there is provided a use of a membrane protein CMTM3 and / or CLTC in the preparation of a medicament for modulating vascular permeability.

[0015] In a sixth aspect of the present application, there is provided a use of a membrane protein CMTM3 and / or CLTC as a drug target for modulating VE-cadherin in endothelial cells and vascular permeability. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Construction of Cmtm3 knockout mice, wherein: (A) Cmtm3 knockout mice were constructed by targeting the first exon of mouse Cmtm3 gene using a guide RNA. (B) PCR genotyping of mouse tails. (C) Western blot analysis of WT and Cmtm3 - / - Expression of CMTM3 protein in mouse colon tissue.

[0017] Figure 2 Cmtm3 deletion inhibits AOM / DSS-induced mouse CAC, wherein: (A) Schematic diagram of CAC model. After the first injection of AOM (10 mg / kg) for 7 days, 2% DSS was given for 5 days, followed by recovery of regular drinking water for 14 days, and this cycle was repeated three times. Analysis was performed on day 100. Seven WT mice and six Cmtm3 - / - Mice were treated. (B, C) Analysis of WT and Cmtm3- / - Colonic length of mice. (D-F) Measurement of tumor number and tumor burden. Red arrows indicate obvious tumors. (G) WT and Cmtm3 - / - Developed tumors in the colon of mice. Scale bar = 100 pm (left panel); inset scale bar = 20 pm (right panel). Data represent two independent experiments with similar results. Data are presented as mean ± s.d., statistical significance was determined by (C, E, F) two-tailed Student's t-test. *P < 0.05.

[0018] Figure 3 .CMTM3 is significantly upregulated in UC patients and DSS-induced mouse colitis, wherein: (A-C) Whole genome transcriptional profiling data from patient colonic biopsies. Data sets: GSE38713; GSE9452; GSE13367. (D, E) Expression data of DSS-induced mouse colitis model. Data sets: GSE22307; GSE34874. (F) Tissue sections of normal and UC patients (n = 5 each) were stained with CMTM3 (red) antibody and DAPI (blue) and observed by confocal microscopy. Scale bar = 50 pm. (G) Positive cells were counted in 130 high-power fields of normal or colitis. (H) Cmtm3 - / - CMTM3 (red) protein levels in mice and WT mice. Positive cells were counted in 15 high-power fields per mouse. n = 3, 4 per group. Scale bar = 50 pm. (I) Quantitative assessment is shown on the right of representative pictures. Data are presented as mean ± s.d., statistical significance was determined by (A-E, G, I) two-tailed Student's t-test. *P < 0.05, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0019] Figure 4 .Cmtm3 deletion protects mice from colitis, wherein: (A) WT and Cmtm3 - / - Body weight loss measurement of mice; n = 3, 4. (B) WT and Cmtm3 - / - DAI score of mice; n = 3, 4. (C, D) WT and Cmtm3 - / - Colonic length of mice; n = 3, 4. (E) Representative micrographs of hematoxylin and eosin staining in colon. Scale bar = 100 pm. (F) WT and Cmtm3 - / -Histological scores of mice; n = 3, 4. (G-J) Representative pictures of MPO (G) and F4 / 80 (I) staining in the colon. Scale bar = 20 μm. The number of MPO-positive (H) and F4 / 80-positive (J) cells was analyzed in 5 areas of each colon tissue section; n = 3. (K) Appearance of Evans blue in the colon of mice. (L) Evans blue dye content extracted from colon tissues normalized to colon tissue weight; n = 3, 4. (M) WT and Cmtm3 - / - Mice were intravenously injected with 70 kDa FITC-dextran (50 mg / kg); n = 3. Accumulation in intestinal crypts indicates vascular permeability. Scale bar = 50 μm. (N) Quantitative assessment, ratio of FITC signal within crypts to total FITC signal was calculated. 15 crypts were analyzed per mouse. Data represent three independent experiments with similar results. Data are expressed as mean ± s.d., statistical significance was determined by (A, D, F, H, J, L, N) one-way ANOVA (Tukey’s multiple comparison test) and (B) two-tailed Student’s t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001; ns indicates no significant difference.

[0020] Figure 5 Non-hematopoietic cell-derived CMTM3 plays an important role in DSS-induced colitis, wherein: (A) Four groups of mice (WT-WT, WT-KO, KO-WT and KO-KO; n = 3, 4 per group) generated by bone marrow transplantation received 3% DSS treatment for 4 days. (B) DAI scores of four groups of mice were monitored daily. (C) Representative pictures of hematoxylin and eosin staining of colon tissues. Scale bar = 100 μm. Data represent two independent experiments with similar results. Data are expressed as mean ± s.d., statistical significance was determined by (B) one-way ANOVA (Tukey’s multiple comparison test). **P < 0.01, ***P < 0.001; ns indicates no significant difference.

[0021] Figure 6 CMTM3 expression is upregulated in endothelial cells during colitis, wherein: (A) Immunofluorescence staining of CMTM3 (green), EpCAM (red) and DAPI (blue) in colon tissues of WT mice treated with water or DSS. Scale bar = 25 μm. (B) Number of cells expressing CMTM3 and EpCAM; n = 5. (C) Immunofluorescence staining of CD31 (green), CMTM3 (red) and DAPI (blue) in colon tissues of WT mice treated with water or DSS. Yellow arrows indicate CD31 +CMTM3 expression detected in cells. Data represent one experiment repeated three times. Scale bar = 25 μm. (D) Number of cells expressing CMTM3 and CD31; n = 3. (E) CD31 (green), CMTM3 (red), and DAPI (blue) staining in colon sections of normal subjects and UC patients. Yellow arrows indicate CD31 + CMTM3 expression detected in cells. Scale bar = 25 μm. (F) Number of cells expressing CMTM3 and CD31; n = 5. (G) scRNA-Seq-based analysis of CMTM3 expression in healthy and inflamed human colonic endothelial cells. The size of each circle reflects the percentage of cells in which CMTM3 was detected, and the color reflects the average CMTM3 expression level in each cluster. Data are presented as mean ± SD, and statistical significance was determined by a two-tailed Student's t-test (B, D, F). ***P < 0.001, ****P < 0.0001; ns indicates no significant difference.

[0022] Figure 7 .Construction of endothelial cell-specific Cmtm3 null mice, including: (A) Restriction maps of the wild-type Cmtm3 allele targeting vector and the Cmtm3 allele after Cre-mediated deletion of exons 2-4. (B) PCR analysis of the Cmtm3 floxed allele and the Cdh5-Cre transgene. Numbered from left to right, lane 1: DNA ladder; lane 2: negative control. The upper part of panel B shows the PCR product sizes of the wild-type (+ / +) (lane 3) and homozygous floxed Cmtm3 gene (fl / fl) (lanes 4 and 5). The lower part of panel B shows the PCR product sizes without (lanes 3 and 5) and with (lane 4) the Cdh5-Cre transgene. All experimental mice were analyzed by tail genomic DNA. Cmtm3 fl / fl -Cdh5-Cre and Cmtm3 fl / fl Littermate mice were treated with acute or chronic DSS-induced colitis. (C) From Cmtm3 fl / fl and Cmtm3 fl / fl -Cmtm3 mRNA expression in endothelial cells sorted from colonic tissue of Cdh5-Cre mice. Data were normalized to β-Actin and expressed as Cmtm3. fl / fl -Cdh5-Cre group was used as the baseline to calculate the fold change. (D) Cmtm3 treated with 3% DSS for 4 days fl / fl and Cmtm3 ΔECImmunofluorescence staining of CMTM3 (red) and VE-cadherin (green) in mouse colon tissues. DAPI (blue) counterstaining. Scale bar = 25 pm. n = 3. Data are represented as mean ± s.d., statistical significance was determined by (C) two-tailed Student’s t-test. ***p < 0.001.

[0023] Figure 8 Deletion of Cmtm3 in endothelial cells suppresses DSS-induced acute colitis, wherein: (A) Cmtm3 fl / fl and Cmtm3 ΔEC Body weight loss measurement of mice; n = 7, 4 per group. (B) Cmtm3 fl / fl and Cmtm3 ΔEC DAI score of mice. (C, D) Cmtm3 fl / fl and Cmtm3 ΔEC Colon length of mice. (E) Representative micrographs of hematoxylin and eosin staining in colon. Scale bar = 200 pm (top panel); scale bar = 100 pm (bottom panel). (F) Cmtm3 fl / fl and Cmtm3 ΔEC Histological score of mice. (G-J) Representative pictures of MPO (G) and F4 / 80 (I) staining in colon. Scale bar = 100 pm (top panel); scale bar = 20 pm (bottom panel). Number of MPO-positive (H) and F4 / 80-positive (J) cells was analyzed in 8 regions of each colon tissue section; n = 3. (K) Cmtm3 fl / fl and Cmtm3 ΔEC Mice were injected intravenously with 70 kDa FITC-dextran (50 mg / kg); n = 3. Accumulation in intestinal crypts indicates vascular permeability. Scale bar = 30 pm. (L) Quantitative assessment, ratio of FITC signal within crypts to total FITC signal was calculated. 8 crypts were analyzed per mouse; n = 3. Data are representative of three independent experiments with similar results. Data are represented as mean ± s.d., statistical significance was determined by (A, B, D, F, H, J) two-tailed Student’s t-test and (L) one-way ANOVA (Tukey’s multiple comparison test). *P < 0.05, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0024] Figure 9Cmtm3 deletion in endothelial cells suppresses DSS-induced chronic colitis, wherein: (A) Schematic of chronic colitis model. Mice were given 2% DSS for 7 days, then returned to regular water for 14 days, followed by another 7-day treatment of 2% DSS; n = 7, 5. (B) Cmtm3 fl / fl and Cmtm3 ΔEC Body weight loss measurement of mice. (C) Cmtm3 fl / fl and Cmtm3 ΔEC DAI score of mice. (D, E) Cmtm3 fl / fl and Cmtm3 ΔEC Colon length of mice. (F) Representative micrographs of hematoxylin and eosin staining in colon. Scale bar = 200 pm (top panel); scale bar = 100 pm (bottom panel). (G) Cmtm3 fl / fl and Cmtm3 ΔEC Histological score of mice. Data represent three independent experiments with similar results. Data are presented as mean ± s.d., statistical significance was determined by (B, C, E, G) two-tailed Student’s t test. *P < 0.05, **P < 0.01.

[0025] Figure 10 CMTM3 disrupts VE-cadherin in colon tissue endothelial cells, wherein: (A) Representative staining of CD31 (green), VE-cadherin (red), and DAPI (blue) in colon tissue of WT and Cmtm3 - / - mice treated with water or DSS. Scale bar = 50 pm (left panel); inset scale bar = 10 pm (right panel). Data represent three independent experiments with similar results. (B) Co-localization of CD31 with VE-cadherin was classified as high, medium, or negative / weak; n = 3. (C) Representative immunofluorescence images of CD31 (green), VE-cadherin-Y731 phosphorylation (red), and DAPI (blue) in colon sections of WT and Cmtm3 - / - mice treated with water or DSS. Scale bar = 50 pm (left panel); inset scale bar = 10 pm (right panel). Data represent three independent experiments with similar results. (D) Bar graph showing the number of VE-cadherin-Y731 phosphorylation positive cells per field (n = 3, 4; 12 fields per mouse). (E) Representative immunofluorescence images of CD31 (green), VE-cadherin-Y731 phosphorylation (red), and DAPI (blue) in colon sections of Cmtm3 fl / fl and Cmtm3 ΔECWestern blot analysis of p-VE-cadherin (Y731) and VE-cadherin expression in mouse colon tissues. β-Actin as a loading control. 3 mice per group. (F) Quantification of VE-cadherin expression normalized to β-Actin. (G) Quantification of p-VE-cadherin (Y731) expression normalized to VE-cadherin. Data were repeated three times. Data are presented as mean ± s.d., statistical significance was determined by (D, F, G) one-way ANOVA (Tukey’s multiple comparison test) and (B) Chi-square test (Fisher’s exact test). *P < 0.1, **P < 0.01, ****P < 0.0001; ns indicates no significant difference.

[0026] Figure 11 .CMTM3 disrupts VE-cadherin in HUVECs, wherein: (A) Delivery efficiency of fluorescently labeled siRNA (FAM-siRNA) in HUVECs was analyzed by flow cytometry. (B) HUVECs were transfected with Scr siRNA, siCMTM3-1# or siCMTM3-2#. CMTM3 expression in HUVECs stimulated with LPS (1 μg / mL) for 24 hours or unstimulated was analyzed by real-time quantitative PCR (RT-PCR). (C) HUVECs were transfected with Scr or siCMTM3-1#. Representative immunofluorescence images of VE-cadherin (green) and DAPI (blue) in HUVECs stimulated with LPS (1 μg / mL) for 24 hours or unstimulated. Yellow arrows indicate VE-cadherin detected in the plasma membrane. Scale bar = 10 μm. Data are representative of three independent experiments with similar results. (D) HUVECs were transfected with Scr or siCMTM3-1#. Immunofluorescence images of VE-cadherin-Y731 phosphorylation (green) and DAPI (blue) in HUVECs stimulated with LPS (1 μg / mL) for 24 hours or unstimulated. Scale bar = 50 μm. (E) Fluorescence intensity of VE-cadherin-Y731 phosphorylation (green) per cell was quantified normalized to the lowest expression. Data are representative of three independent experiments with similar results. Data are presented as mean ± s.d., statistical significance was determined by (E) one-way ANOVA (Tukey’s multiple comparison test). ****P < 0.0001; ns indicates no significant difference.

[0027] Figure 12 .CMTM3 promotes clathrin-mediated endocytosis (CME) in endothelial cells by upregulating CLTC expression, wherein: (A) CLTC expression in WT and Cmtm3 - / -Immunofluorescence staining of CD31 (red), CLTC (green) and DAPI (blue) in mouse colon tissues. Data represent one experiment, repeated three times. Scale bar = 50 pm. (B) Statistical plot showing the percentage of CLTC expression (high, medium or negative) in CD31 -positive cells. n = 3; 10 fields per mouse. (C) HUVECs were transfected with internal ribosome entry site (IRES)-EGFP-encoding adenovirus (Mock) and CMTM3-IRES-EGFP-encoding adenovirus (ad-CMTM3) at MOI 100. CMTM3 expression was detected by Western blot. (D) HUVECs were infected with adenovirus at MOI 0, 10, 50, 100, and EGFP expression (green) was detected after 24 or 48 hours. Scale bar = 200 pm. (E) Representative immunofluorescence images of CLTC (red) and DAPI (blue) staining in HUVECs transfected with Mock and ad-CMTM3 (MOI 10). Data represent one experiment, repeated three times. Scale bar = 50 pm. (F) Quantification of the fluorescence intensity of CLTC (red) in each cell. Quantification results of immunofluorescence images are shown. (G) Pictures of siRNA-mediated CLTC knockdown in HUVECs, CLTC (green) and DAPI (blue) staining. Scale bar = 50 pm. (H) HUVECs were transfected with Scr or siCLTC, and then infected with adenovirus at MOI 10 after 24 hours. EGFP (green), VE-cadherin (red) and DAPI (blue) were observed by confocal microscopy. Yellow arrows indicate VE-cadherin detected in the plasma membrane. Scale bar = 50 pm. Data represent three independent experiments with similar results. (I) Expression of CLTC in healthy and inflamed human colon endothelial cells based on scRNA-Seq analysis. The size of each circle reflects the percentage of endothelial cells in which CLTC was detected, and the color reflects the average expression level of CLTC. Data are presented as mean ± s.d., and statistical significance was determined by (B) Chi-square test (Fisher’s exact test) and (F) two-tailed Student’s t test. ****p < 0.0001; ns indicates no significant difference.

[0028] Figure 13CMTM3 interacts with CLTC and inhibits ubiquitination and proteasome-dependent degradation of CLTC in HEK293T cells, wherein: (A) HEK293T cells transfected with Flag or Flag-CMTM3 expression plasmid were treated with 25 μg / mL cycloheximide (CHX) for 0, 6, 12 hours. The expression of CLTC was detected by Western blot. (B) Data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (C) HEK293T cells transfected with Flag-CMTM3 expression plasmid were treated with 25 μg / mL CHX alone, or with 25 μg / mL CHX plus 10 μM MG132 or 25 μg / mL CHX plus 20 μM chloroquine (CQ) for 12 hours. Subsequently, cell lysates were subjected to Western blot detection with anti-Flag, anti-CLTC and anti-β-Actin antibodies. (D) Data were normalized to β-Actin and the relative fold change compared to the control group was calculated. (E) HEK293T cells transfected with Flag or Flag-CMTM3 and HA-ubiquitin were treated with MG132 (10 μM) for 12 hours, and then lysed with IP lysis buffer containing protease inhibitors. CLTC was subjected to immunoprecipitation, and the precipitates were detected with anti-HA, anti-Flag, anti-CLTC and anti-β-Actin antibodies. (F) HEK293T cells transfected with Flag or Flag-CMTM3 were subjected to immunoprecipitation with anti-Flag beads. Subsequently, cell lysates were subjected to Western blot detection with anti-Flag, anti-CLTC and anti-β-Actin antibodies. (G) HEK293T cells transfected with Flag or Flag-CMTM3 were subjected to immunoprecipitation with anti-CLTC antibody. Subsequently, cell lysates were subjected to Western blot detection with anti-Flag, anti-CLTC and anti-β-Actin antibodies.

[0029] (H) Representative immunofluorescence images of CLTC (green), Flag-CMTM3 (red) and DAPI (blue) in HEK293T cells transfected with Flag-CMTM3 for 48 hours. An isotype antibody was used as a control. Scale bar = 5 μm. (I, J) In HEK293T cells transfected with Flag-CMTM3, CLTC was labeled with FITC (donor) and Flag-CMTM3 was labeled with TRITC (acceptor). The fluorescence intensity (I) and lifetime (J) of FITC-labeled CLTC in the donor alone group and the donor-acceptor group were shown. (K, L) GST and GST-CMTM3 proteins expressed in E. coli BL21 (DE3) and purified with glutathione sepharose beads were incubated with in vitro translated CLTC (K) or Flag-CMTM3 (L) in the presence of ATP or ATP plus CMTM3. Subsequently, the mixtures were subjected to Western blot detection with anti-Flag or anti-CLTC antibody. HA-CLTC proteins produced in the rapid coupling transcription / translation system were incubated. Binding proteins were identified by Western blot with anti-HA and anti-GST antibodies. Data are representative of three experiments with similar results. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student's t-test (B, J) and one-way ANOVA with Tukey's multiple comparison test (D). *p < 0.1; **p < 0.01; ****p < 0.0001; ns indicates no significant difference.

[0030] Figure 14 CMTM3 interacts with CLTC and inhibits proteasome-dependent degradation of CLTC in HUVECs, wherein: (A) HUVECs transfected with Flag or CMTM3-Flag expression adenovirus were treated with 25 μg / mL cycloheximide (CHX) for 0, 6, 12 hours. The expression of CLTC, Flag and β-Actin was detected by Western blot. (B) Data were normalized to β-Actin and the relative change fold compared with the control group was calculated. (C) HUVECs transfected with CMTM3-Flag expression adenovirus were treated with 25 μg / mL CHX alone, or with 25 μg / mL CHX plus 10 μM MG132 or 25 μg / mL CHX plus 20 μM chloroquine (CQ) for 12 hours. Subsequently, cell lysates were subjected to Western blot with anti-Flag, anti-CLTC and anti-β-Actin antibodies. (D) Data were normalized to β-Actin and the relative change fold compared with the control group was calculated. (E) HUVECs transfected with Flag or CMTM3-Flag were subjected to immunoprecipitation with anti-Flag beads. Subsequently, cell lysates were subjected to Western blot with anti-Flag, anti-CLTC and anti-β-Actin antibodies. (F) HUVECs transfected with Flag or CMTM3-Flag were subjected to immunoprecipitation with anti-CLTC antibody. Subsequently, cell lysates were subjected to Western blot with anti-Flag, anti-CLTC and anti-β-Actin antibodies. Data are representative of three experiments with similar results. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student's t-test (B) and one-way ANOVA with Tukey's multiple comparison test (D). *p < 0.1; ***p < 0.001; ****p < 0.0001; ns indicates no significant difference.

[0031] Figure 15.CMTM3 is a potential therapeutic target for ulcerative colitis (UC), wherein: (A) Schematic of Imatinib treatment. Imatinib mesylate (50 mg / kg) was dissolved in phosphate-buffered saline (PBS) and intraperitoneally injected once a day from the first day of DSS treatment until the last day of DSS treatment. PBS was used as a control. (B) Percentage of body weight loss. Initial body weight was measured (day 0) and monitored daily throughout the experiment. (C) Disease activity index (DAI) scores of the four groups. (D) Representative images of hematoxylin-eosin staining of colonic tissue. Scale bar = 100 pm. n = 3-5. Data are representative of two independent experiments with similar results. Data are presented as mean ± standard deviation (SD), and statistical significance was determined by one-way analysis of variance (ANOVA) combined with Tukey’s multiple comparison test. **P <.01 and ***P <.001; ns indicates no significant difference. DETAILED DESCRIPTION

[0032] The schemes of the present application will be explained below in connection with examples. Those skilled in the art will appreciate that the examples below are for illustrative purposes only and should not be construed as limiting the scope of the present application. Unless otherwise indicated in the examples, the techniques or conditions described in the literature or according to the manufacturer’s instructions were used. Unless otherwise indicated, the reagents or instruments used were conventional products available on the market.

[0033] Example 1 Role of CMTM3 in colon cancer and intestinal inflammation

[0034] 1. Cmtm3 deletion suppresses AOM / DSS-induced colon cancer (CAC)

[0035] To clarify the exact role of CMTM3 in CAC, Cmtm3 knockout (Cmtm3 - / - ) mice ( Figure 1 ) were generated by CRISPR-Cas9 gene editing technology. Deletion of Cmtm3 did not affect the reproduction and development of mice, nor did it affect major organs. Subsequently, we established an inflammation-driven colon cancer model by injecting AOM and then performing three rounds of DSS treatment, and the experimental subjects included wild-type (WT) and Cmtm3 - / - mice ( Figure 2 A). As shown in Figure 2 B to G, Cmtm3 - / - mice exhibited less colon shortening ( Figure 2 B and C), and reduced tumor number and volume ( Figure 2 D to F). Histologically, Cmtm3 - / - mice had lower canceration than WT mice carrying tumors ( Figure 2G). These results demonstrate that CMTM3 promotes the development of CAC.

[0036] 2. CMTM3 is significantly upregulated in inflammatory intestinal tissues of humans and mice

[0037] Since inflammation is a major driver of CAC, we further determined whether CMTM3 is involved in the intestinal inflammation stage. First, we analyzed the expression of CMTM3 in colon biopsy samples using whole-genome transcriptional profiling data in the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / ). As shown in Figure 3 A to C, CMTM3 was significantly higher in biopsy samples of active UC patients compared to healthy controls, non-lesional active ulcerative colitis (UC), and UC in remission. Similarly, mouse Cmtm3 was also increased during the development of DSS-induced intestinal inflammation Figure 3 D and E).

[0038] We also detected the protein level of CMTM3 in human samples and experimental intestinal inflammation mice by immunofluorescence staining. As shown in Figure 3 F and G, CMTM3 was significantly increased in the colon tissues of UC patients compared to healthy controls. In addition, CMTM3 upregulation was also detected in the colon tissues of DSS-treated mice, but not in untreated mice Figure 3 H and I), indicating the specificity of CMTM3 staining. These results suggest that CMTM3 is significantly upregulated in the inflammatory intestinal tissues of UC patients and DSS-induced intestinal inflammation mouse model.

[0039] 3. Cmtm3 deficiency protects mice from intestinal inflammation

[0040] Expression analysis suggests that CMTM3 can be involved in the development of CAC from the intestinal inflammation stage. Therefore, we investigated its role in intestinal inflammation using the DSS-induced intestinal inflammation model. First, there were no differences in body weight, disease activity index (DAI), colon length, tissue structure, and immune cell infiltration between WT and Cmtm3 - / - mice ( Figure 4 ). Interestingly, compared to WT littermates, Cmtm3 - / - mice showed significantly reduced body weight loss ( Figure 4 A) and DAI ( Figure 4 B) after DSS treatment. Cmtm3 - / - mice also exhibited longer colon length ( Figure 4 C and D), less epithelial damage, and fewer inflammatory cell infiltrations ( Figure 4E and F). Moreover, Cmtm3 - / - Mice DSS-induced colonic tissue showed a significant reduction in myeloperoxidase (MPO) positive Figure 4 G and H) and F4 / 80 positive Figure 4 I and J) cells compared to WT mice. These results suggest that Cmtm3 deletion has a protective effect in intestinal inflammation.

[0041] To determine whether CMTM3 regulates the vascular barrier in the intestine, we treated WT and Cmtm3 - / - mice with intravenous injection of Evans Blue (EB) dye or 70 kDa fluorescein isothiocyanate (FITC)-dextran. In resting conditions, Cmtm3 deletion did not alter the integrity of the vascular barrier. As reported, DSS promoted colonic vascular permeability Figure 4 K to N) in WT mice. However, after DSS treatment, Cmtm3 - / - mice showed a significant reduction in the leakage of Evans blue dye Figure 4 K and L) or FITC-dextran Figure 5 M and N), indicating a decrease in vascular permeability. Taken together, these data suggest that CMTM3 promotes the development of intestinal inflammation and regulates vascular permeability.

[0042] 4. CMTM3 of non-hematopoietic cell origin is responsible for DSS-induced intestinal inflammation

[0043] To further clarify the cellular origin of the relief of intestinal inflammation in the absence of Cmtm3, we took advantage of irradiated WT or Cmtm3 - / - recipient mice by transplanting bone marrow cells from WT or Cmtm3 - / - donor mice to prepare chimeric mice Figure 5 A). After 8 weeks of hematopoietic recovery, mice were challenged with 3% DSS. As shown in Figure 5 B, Cmtm3 - / - recipient mice (red line) had lower DAI than WT recipient mice that received bone marrow cell transplantation from WT mice (black line). However, WT recipient mice that received bone marrow cell transplantation from WT mice (black line) and Cmtm3 - / - donor mice (blue line) showed comparable DAI. In addition, the damage of the villus structure and the infiltration of inflammatory cells were consistent with the results of DAI Figure 6 C). These data suggest that CMTM3 of non-hematopoietic cell origin plays a pro-inflammatory role in the progression of intestinal inflammation.

[0044] 5. CMTM3 deletion in endothelial cells inhibits DSS-induced acute and chronic intestinal inflammation

[0045] Both the intestinal epithelium and endothelium play an important role in intestinal inflammation. Since CMTM3 from non-hematopoietic cells plays a pro-inflammatory role, we first detected the expression of CMTM3 in epithelial cells and endothelial cells. Figure 6 As shown in A and B, CMTM3 is barely expressed in epithelial cells of normal and DSS-induced enteritis mice. Interestingly, we detected increased CMTM3 in endothelial cells of DSS-treated mice ( Figure 6 C and D). In addition, the CMTM3 protein level in endothelial cells of colon tissues from patients with enteritis was also upregulated compared with tissues from normal individuals ( Figure 6 E and F). We further verified the expression of CMTM3 in colonic endothelial cells using single-cell transcriptome mapping (http: / / cells.ucsc.edu / ?bp=colon&ds=human-colon). Figure 7 G shows that CMTM3 expression levels are increased in endothelial cells of UC patients compared with healthy individuals. These results suggest that endothelial cell-derived CMTM3 may promote the development of intestinal inflammation.

[0046] To test this hypothesis, we knocked down Cmtm3 specifically in endothelial cells.

[0047] (Cmtm3 fl / fl Cdh5-Cre, Cmtm3 ΔEC ) and Cmtm3 fl / fl Mice served as controls ( Figure 8 A to C). Cmtm3 ΔEC and Cmtm3 fl / fl The mice developed normally, and no macroscopic differences were observed in the major organs. fl / fl Compared with mice (black line), after DSS treatment, Cmtm3 ΔEC Mice (red line) showed significantly reduced weight loss ( Figure 8 A), lower disease activity index (DAI) ( Figure 8 B) and longer colon length ( Figure 8 C and D). Histological analysis of the colon showed that Cmtm3 fl / fl Compared with mice, Cmtm3 ΔEC The destruction of glandular crypts and inflammatory cell infiltration were significantly reduced in mice ( Figure 8 E and F). In addition, Cmtm3 ΔEC Myeloperoxidase (MPO)-positive cells of mice ( Figure 8 G and H) and F4 / 80-positive cells ( Figure 8 I and J) were significantly reduced. To verify the regulatory effect of CMTM3 on the intestinal vascular barrier, we gave Cmtm3ΔEC and Cmtm3 fl / fl Mice were injected intravenously with 70 kDa FITC-dextran Figure 9 K and 8L). In the resting state, the absence of Cmtm3 in endothelial cells did not change the integrity of the vascular barrier. As observed in Cmtm3 - / - Mice, after DSS treatment, Cmtm3 ΔEC FITC-dextran leakage in mice was significantly reduced, indicating decreased vascular permeability. Taken together, these data demonstrate that endothelial cell-derived CMTM3 modulates vascular permeability and promotes the development of colitis.

[0048] In addition, we also verified the role of CMTM3 in a chronic intestinal inflammation model Figure 9 A). First, mice received 2% DSS for 7 days; 2 weeks later, they received 2% DSS again for 7 days, and then were analyzed. Compared with Cmtm3 fl / fl Mice, Cmtm3 ΔEC Mice exhibited significantly reduced weight loss Figure 9 B), lower DAI Figure 9 C), and longer colon length Figure 9 D and E). Cmtm3 ΔEC Mice had a protective effect on the colon epithelium and reduced inflammatory cell infiltration in the crypt Figure 10 F and G). Taken together, our data suggest that Cmtm3 ΔEC Mice were less susceptible to severe inflammation. These results indicate that the absence of Cmtm3 in endothelial cells inhibits the development of acute and chronic intestinal inflammation, which in turn can affect the occurrence of colon cancer (CAC).

[0049] Example 2 Mechanism of action of CMTM3

[0050] 1. CMTM3 disrupts VE-cadherin in endothelial cells

[0051] VE-cadherin is an important adhesion molecule that maintains the stability of endothelial junctions. To determine whether endothelial cell-derived CMTM3 regulates the intestinal vascular barrier through VE-cadherin, we detected the distribution of VE-cadherin in endothelial cells of wild-type (WT) and Cmtm3 - / - Mice. As shown in Figure 10 A and B, VE-cadherin was more obviously co-localized with platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in DSS-treated Cmtm3 - / - Mice compared with WT mice, indicating that Cmtm3 - / -More continuous adherens junctions formed in the colonic tissue of mice, potentially leading to reduced vascular permeability. Therefore, CMTM3 may disrupt VE-cadherin in endothelial cells.

[0052] Since the destruction of VE-cadherin begins with its tyrosine phosphorylation, we further examined the phosphorylation levels of tyrosine residues. In DSS-induced intestinal inflammation tissues of WT mice, the phosphorylation of VE-cadherin Y731 was significantly increased, while it was reduced in Cmtm3 knockout mice ( Figure 10 C and D), indicating that the upregulation of VE-cadherin Y731 phosphorylation is dependent on the presence of CMTM3. We also examined the ΔEC and Cmtm3 fl / fl Expression of VE-cadherin and its phosphorylation level at Y731 in mouse colon tissue. In the absence of DSS treatment, knockout of Cmtm3 in endothelial cells had no effect on the expression of VE-cadherin and its phosphorylation level at Y731. However, knockout of Cmtm3 inhibited the downregulation of VE-cadherin and reduced the upregulation of VE-cadherin-Y731 phosphorylation level ( Figure 11 E to 10G).

[0053] Next, we used human umbilical vein endothelial cells (HUVECs) to study the effect of CMTM3 on VE-cadherin in vitro. We verified the efficient silencing of CMTM3 in HUVECs ( Figure 11 A and B). Under untreated conditions, CMTM3 depletion did not affect VE-cadherin at adherens junctions. Because lipopolysaccharide (LPS)-induced inflammatory responses are associated with the pathology of ulcerative colitis (UC), we treated HUVECs with LPS. Interestingly, knockdown of CMTM3 inhibited LPS-induced loss of VE-cadherin on the cell membrane ( Figure 11 C). Consistent with the in vivo results, the phosphorylation level of VE-cadherin Y731 was upregulated after LPS treatment, but CMTM3 inhibition significantly reduced its phosphorylation response to LPS stimulation ( Figure 12 D and E). Thus, CMTM3 disrupts VE-cadherin in endothelial cells in response to inflammatory stimuli.

[0054] 2. CMTM3 promotes clathrin-mediated endocytosis of VE-cadherin in endothelial cells by upregulating CLTC expression

[0055] Previous studies have shown that CMTM3 regulates the endocytosis and trafficking of VE-cadherin, but there is no interaction between the two, and the underlying mechanism remains unclear. Since VE-cadherin is mainly internalized through a clathrin-dependent pathway, we hypothesized that CMTM3 may promote clathrin-mediated endocytosis.Figure 12 A and B, CLTC expression of CD31 positive endothelial cells in mouse colon tissue was significantly increased after DSS treatment, while it was decreased in Cmtm3 knockout mice. We further confirmed that overexpression of CMTM3( Figure 12 C and D) enhanced the expression level of CLTC in HUVECs( Figure 12 E and F), while knockdown of CLTC( Figure 12 G) could rescue the downregulation of VE-cadherin on the cell membrane caused by CMTM3( Figure 12 H). In addition, we analyzed the single cell transcriptome atlas and found that CLTC expression was upregulated in endothelial cells of UC patients( Figure 13 I), which further confirmed the involvement of endothelial cell-derived CLTC in intestinal inflammation. These results suggest that CMTM3 promotes clathrin-mediated endocytosis of VE-cadherin in endothelial cells by upregulating CLTC expression, thereby exacerbating intestinal inflammation.

[0056] 3. CMTM3 interacts with CLTC and inhibits ubiquitination and proteasome-dependent degradation of CLTC

[0057] CMTM3 stabilizes NEMO by inhibiting its ubiquitination. To our knowledge, there is currently no report on the regulation of CLTC protein stability. To explore the mechanism of CMTM3 upregulating CLTC, we treated control and CMTM3 overexpressing cells with the protein synthesis inhibitor cycloheximide (CHX). As shown in Figure 13 A, the protein level of CLTC was significantly decreased after 12 hours of CHX treatment, and this effect was reversed by proteasome inhibitor MG132, but not by lysosome inhibitor chloroquine (CQ) Figure 13 B). These results suggest that CLTC is degraded through the proteasome pathway.

[0058] Next, we verified whether CMTM3 could inhibit the ubiquitination of CLTC. As shown in Figure 13 C, CMTM3 significantly reduced the ubiquitination level of CLTC. These data confirm that CMTM3 stabilizes CLTC by antagonizing its ubiquitination and proteasome-dependent degradation.

[0059] In addition, we further determined whether CMTM3 interacts with CLTC. As shown in Figure 13 D to F, co-immunoprecipitation (IP) Figure 13 D and E) and high-resolution imaging Figure 13 F) confirmed the interaction of CMTM3 with CLTC protein. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Figure 13G and H) and glutathione S-transferase (GST) pull-down assay ( Figure 14 I and J) further confirmed the direct interaction between CMTM3 and CLTC. Therefore, CMTM3 interacts with CLTC and inhibits the ubiquitination and proteasome-dependent degradation of CLTC. We further verified the above conclusion in HUVECs. Figure 14 As shown in A and 14B, when cells were treated with CHX, the protein level of CLTC in the control group was significantly decreased compared with the CMTM3 overexpression group. The degradation of CLTC could be reversed by MG132, but not by CQ ( Figure 14 C and 14D). Subsequently, we confirmed the interaction between CLTC and CMTM3 in HUVECs ( Figure 15 E and F), which is consistent with our observations in HEK293T cells.

[0060] Example 3 CMTM3 is a potential therapeutic target for ulcerative colitis

[0061] Imatinib is a tyrosine kinase inhibitor, and an increasing number of reports have shown that imatinib can restore VE-cadherin junctions. To determine whether CMTM3 can be used as a therapeutic target and compare its efficacy with imatinib, we performed a double-blind double-blind double-stranded ... - / - Mice were treated with PBS or imatinib daily ( Figure 15 A). During intestinal inflammation, imatinib significantly attenuated the body weight loss of WT mice compared with PBS treatment ( Figure 15 B)、DAI( Figure 15 C) and histological damage ( ​ D and E), which is consistent with previous reports. Interestingly, Cmtm3 knockout (red line) significantly alleviated intestinal inflammation compared with WT mice (black line), while weight loss, DAI, and histological damage were comparable in imatinib-treated WT mice (blue line) and Cmtm3 knockout mice (green line), indicating that imatinib is ineffective in Cmtm3 knockout mice. These results indicate that both CMTM3 and imatinib target vascular permeability and have comparable efficacy. Given that imatinib primarily acts by restoring VE-cadherin junctions, our study further confirms that CMTM3 promotes the development of intestinal inflammation by disrupting VE-cadherin junctions in endothelial cells and promoting vascular permeability.

[0062] Therefore, CMTM3 may promote vascular permeability by disrupting VE-cadherin in endothelial cells, thus becoming a potential therapeutic target for enteritis and colon cancer (CAC).

[0063] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. Application of membrane protein CMTM3 as a target in screening drugs for the treatment of ulcerative colitis.

2. The use according to claim 1, characterized in that The CMTM3 promotes the occurrence of inflammatory bowel disease by destroying VE-cadherin in endothelial cells under inflammatory stimulation.

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