Xiaoning Mo

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

CMTM3 can affect the transcription activity of androgen receptor and inhibit the expression level of PSA in LNCaP cells.

CMTM is a novel family of proteins linking chemokines and TM4SF. Several members of this family are highly expressed in testes and regulate androgen receptor (AR) transcription activity. One member of this family, CMTM3, has the highest expression level in testes and contains one leucine zipper and two LXXLL motifs. As assessed with the dual-luciferase reporter assay, overexpression of CMTM3 represses AR transactivation, while knocking down it can increase AR transactivation. Moreover, CMTM3 inhibits prostate-specific antigen (PSA) expression in LNCaP cells at both mRNA and protein levels with no obvious influence on AR expression. Taken together, CMTM3 may play some roles in the maturation and maintenance of the male reproduction.

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.

Identification and characterization of CMTM4, a novel gene with inhibitory effects on HeLa cell growth through Inducing G2/M phase accumulation

Human CMTM is a novel gene family consisting of CKLF and CMTM1-8. CMTM4 is the most conserved gene and has three RNA splicing forms designated as CMTM4-v1, -v2 and -v3, but in many types of tissue and cell lines, only CMTM4-v1 and -v2 could be detected. CMTM4-v2 is the full length cDNA product, which has been highly conserved during evolution. CMTM4-v1 and -v2 are broadly expressed in normal types of tissue. They are distributed on the cell membrane and across the cytoplasm in a speckled pattern. Overexpression of CMTM4-v1 and -v2 can inhibit HeLa cell growth via G2/M phase accumulation without inducing apoptosis. Therefore, CMTM4 might be an important gene involved in cell growth and cell cycle regulation.

Expressional and functional studies of CKLF1 during dendritic cell maturation.

Chemokine-like factor 1 (CKLF1) was the first member of the CKLF-like MARVEL transmembrane domain containing member (CMTM) family to be discovered. Its expression level is increased clearly in peripheral blood lymphocytes upon phytohemagglutinin stimulation, but little is known about the expression and function of CKLF1 in dendritic cells (DCs), which are the most potent antigen-presenting cells. In the present study, we showed that CKLF1 was highly expressed in monocytes. During differentiation from monocytes to immature DCs, CKLF1 was increased dramatically on day 2 and then decreased from day 3 to 5. Upon maturation with different stimuli, CKLF1 was down-regulated. Two peptides of CKLF1, C19 and C27, stimulated the effect of immature DCs (imDCs) on T-cell proliferation and IFN-gamma production. Further study on DC maturation showed that C19 and C27 up-regulated HLA-DR expression and IL-12 secretion, with no obvious effects on CD80, CD83 or CD86. Thus, CKLF1-C19 and -C27 can stimulate antigen-presenting capability of imDCs.

PC3-Secreted Microprotein Is a Novel Chemoattractant Protein and Functions as a High-Affinity Ligand for CC Chemokine Receptor 2

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

CMTM4 is frequently downregulated and functions as a tumour suppressor in clear cell renal cell carcinoma

BackgroundChemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family involved in multiple malignancies. CMTM4 is a member of this family and is located at chromosome 16q22.1, a locus that harbours a number of tumour suppressor genes. It has been defined as a regulator of cell cycle and division in HeLa cells; however, its roles in tumourigenesis remain poorly studied.MethodsAn integrated bioinformatics analysis based on the array data from the GEO database was conducted to view the differential expression of CMTM4 across multiple cancers and their corresponding control tissues. Primary clear cell renal cell carcinoma (ccRCC) and the paired adjacent non-tumour tissues were then collected to examine the expression of CMTM4 by western blotting, immunohistochemistry, and quantitative RT-PCR. The ccRCC cell lines A498 and 786-O and the normal renal tubular epithelial cell line HK-2 were also tested for CMTM4 expression by western blotting. Cell Counting Kit-8 (CCK-8) and viable cell counting assays were used to delineate the growth curves of 786-O cells after CMTM4 overexpression or knockdown. Wound healing and transwell assays were performed to assess the cells’ ability to migrate. The effects of CMTM4 on cellular apoptosis and cell cycle progression were analysed by flow cytometry, and cell cycle hallmarks were detected by western blotting and RT-PCR. The xenograft model in nude mice was used to elucidate the function of CMTM4 in tumourigenesis ex vivo.ResultsBy omic data analysis, we found a substantial downregulation of CMTM4 in ccRCC. Western blotting then confirmed that CMTM4 was dramatically reduced in 86.9 % (53/61) of ccRCC tissues compared with the paired adjacent non-tumour tissues, as well as in the 786-O and A498 ccRCC cell lines. Restoration of CMTM4 significantly suppressed 786-O cell growth by inducing G2/M cell cycle arrest and p21 upregulation, and cell migration was also inhibited. However, knockdown of CMTM4 led to a completely opposite effect on these cell behaviours. Overexpression of CMTM4 also markedly inhibited the tumour xenograft growth in nude mice.ConclusionsCMTM4 is downregulated and exhibits tumour-suppressor activities in ccRCC, and could be exploited as a target for ccRCC treatment.

Identification of FAM3D as a new endogenous chemotaxis agonist for the formyl peptide receptors.

ABSTRACT The family with sequence similarity 3 (FAM3) gene family is a cytokine-like gene family with four members FAM3A, FAM3B, FAM3C and FAM3D. In this study, we found that FAM3D strongly chemoattracted human peripheral blood neutrophils and monocytes. To identify the FAM3D receptor, we used chemotaxis, receptor internalization, Ca2+ flux and radioligand-binding assays in FAM3D-stimulated HEK293 cells that transiently expressed formyl peptide receptor (FPR)1 or FPR2 to show that FAM3D was a high affinity ligand of these receptors, both of which were highly expressed on the surface of neutrophils, and monocytes and macrophages. After being injected into the mouse peritoneal cavity, FAM3D chemoattracted CD11b+ Ly6G+ neutrophils in a short time. In response to FAM3D stimulation, phosphorylated ERK1/2 and phosphorylated p38 MAPK family proteins were upregulated in the mouse neutrophils, and this increase was inhibited upon treatment with an inhibitor of FPR1 or FPR2. FAM3D has been reported to be constitutively expressed in the gastrointestinal tract. We found that FAM3D expression increased significantly during colitis induced by dextran sulfate sodium. Taken together, we propose that FAM3D plays a role in gastrointestinal homeostasis and inflammation through its receptors FPR1 and FPR2. Highlighted Article: We find FAM3D is a new endogenous agonist of FPR1 and FPR2, and chemoattracts neutrophils and monocytes.

V‑set and transmembrane domain‑containing 1 is silenced in human hematopoietic malignancy cell lines with promoter methylation and has inhibitory effects on cell growth

Numerous leukocyte differentiation antigens act as important markers for research, diagnosis, triage and eventually treatment targets for hematopoietic malignancies. V‑set and transmembrane domain‑containing 1 (VSTM1) was identified by immunogenomic analysis as a potential leukocyte differentiation antigen gene. VSTM1 is located at 19q13.4 on human chromosomes, an important genomic region prone to genetic and epigenetic modifications in numerous hematopoietic malignancies. VSTM1‑v1, a primary splicing form encoded by VSTM1, is a type I transmembrane molecule with an extracellular immunoglobulin V‑like domain and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. In the present study, VSTM1 expression was examined in normal human peripheral leukocytes and hematopoietic tumor cell lines; in addition, the aberrant methylation of the VSTM1 gene was evaluated using methylation‑specific polymerase chain reaction (MSP). The results of the present study demonstrated that VSTM1 was widely expressed in normal human peripheral blood leukocytes, including granulocytes and monocytes, in concurrence with previous studies, as well as lymphocytes; in addition, the molecular size and expression levels of VSTM1 varied considerably between leukocytes. However, VSTM1 was undetectable in numerous hematopoietic tumor cell lines following promoter hypermethylation. The effects of pharmacologically‑induced demethylation of the VSTM1 gene and promoter region were analyzed using MSP and biosulfite genomic sequencing, and the results revealed that VSTM1 expression was restored in methylation‑silenced Jurkat cells. In addition, CKK‑8 assays revealed that VSTM1‑v1 overexpression in Jurkat cells resulted in growth suppression. Furthermore, the inhibitory effect on cell growth was enhanced following antibody‑induced cross‑linking of VSTM1‑v1. In conclusion, the results of the present study indicated that promoter methylation silenced VSTM1 and negatively regulated cell growth in human hematopoietic malignancy cell lines.

Late‐onset severe pneumonia after allogeneic hematopoietic stem cell transplantation: prognostic factors and treatments

In this study, we aimed to evaluate the prognostic factors associated with and treatments for late‐onset severe pneumonia (LOSP) in patients who underwent allogeneic hematopoietic stem cell transplantation (allo‐HSCT).