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Objective: CKLF-like MARVEL transmembrane domain containing 3 (CMTM3), as a tumor suppressor gene, plays an important role in the suppression of cell growth and apoptosis. The goal of our study is to investigate the association between CMTM3 promoter methylation and laryngeal squamous cell carcinoma (LSCC)., Design and Methods: Using the bisulfite pyrosequencing technology, DNA methylation levels of seven CpG sites in CMTM3 promoter are measured in tumor tissues and their adjacent tissues of 76 male LSCC patients., Results: Our results reveal a significantly elevated promoter methylation of CMTM3 in tumor tissues compared with their adjacent tissues (P<0.001). A breakdown analysis by age shows that significant association of CMTM3 promoter methylation with cancer risk is specific to the LSCC patients older than 55years (P<0.001) but not in the younger patients (P=0.305). Moreover, the association is only observed in the LSCC patients with smoking behavior (P=0.001). Breakdown analysis also shows that CMTM3 promoter methylation is associated with cancer risk among patients with stage I LSCC (P<0.001)., Conclusion: In conclusion, our study indicates that elevated CMTM3 methylation is a risk factor in male LSCC patients, especially in the patients with age over 55years and with smoking behavior., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate whether chemokine like factor (CKLF)-like myelin and lymphocyte and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing protein 2 (CMTM2) is involved in spermatogenesis in varicocele induced subfertility rats and to discuss the possible mechanisms., Methods: Forty male SD rats (body weight: 220-330 g, age: 6-7 weeks) were randomly divided into 4 groups: varicocele for 4 weeks, varicocele for 12 weeks, sham operation for 4 weeks and sham operation for 12 weeks, with 10 rats in each group. These rats were introduced by partially ligating left kidney veins for the experimental groups, and the sham surgery groups as controls were executed with exactly the same surgery as in the experimental groups except for the ligation. The rats in control and experimental groups for 4 and 12 weeks were killed after laparotomy at the end of 4 and 12 weeks, respectively, the left testes and epididymis were taken out for counting the sperm, observing the seminiferous tubule change and immunochemistry for CMTM2. The changes included sperm density and motility, the outer diameter and inner diameter change and the changes of epithelium and the CMTM2 expression in immunochemistry., Results: Compared with the control groups, the sperm density [(63.9±7.1)×10 6 /mL vs.(74.3±5.0)×10 6 /mL] and motility [(58.7%±7.9%) vs.(66.1%±4.3%)] were reduced slightly in group of varicoele for 4 weeks, respectively (t=1.432, 1.563; P=0.076, 0.059, respectively). Varicocele significantly caused a decrease in sperm concentration [(40.5±7.2) ×10 6 /mL vs.(71.1±4.5)×10 6 /mL] and motility [(35.2%±8.5%)vs. (63.4%±4.1%)] at 12 weeks, compared with the related sham groups (t=3.754, 3.933; P=0.004, 0.002, respectively). Additionally, testis CMTM2 exhibited the same disparity, that is, the CMTM2 protein expression in varicocele group was significantly reduced, with the ratio of sham group to varicocele group at the end of 12 weeks 2.3±0.4 (t=1.978; P=0.039). In the evaluation of seminiferous tubules diameter, the external [(198.2±10.2) μm vs. (255.8±12.7) μm, t=2.125, P=0.003] and epithelium diameter [(54.1±1.5) μm vs. (75.5±4.1) μm, t=2.246, P=0.021] were decreased compared with the sham-related groups and previous varicocele groups. In all the varicocele groups, all types of sperm motility decreased compared with the related sham-operated group (P<0.05)., Conclusion: This study suggests varicocele has a detrimental effect on CMTM2 levels and decreases spermatogonia cell number, seminiferous tubules diameter, and sperm indices. CMTM2 is associated with sperm changes in rats with varicocele, and further studies are needed to study the mechanism.

Objective: To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets., Methods: The research includes two groups: sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line; shN is the control group in which CMTM3 is negatively knocked down. The expression of CMTM3 was detected by Western blot. The migration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay. The invasion ability of PC3 was detected by Matrigel assay. RESULTS were obtained from at least three individual experiments., Results: The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0±0.000 4 vs. 0.490 0±0.055 7, P<0.001) detected by Western blot. It also had statistical significance in Matrigel assays (248.6±4.5 vs. 113.0± 3.3), Transwell (203.6±1.9 vs. 103.0±1.2) and Wound healing assays (95.0±2.9 vs. 33.0±1.5) that knockdown of CMTM3 promoted migration, and invasion of PC3 cells in vitro (P<0.001)., Conclusion: Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells, and knocking down CMTM3 affects migration, and invasion ability in PC3 cells.

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS's) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse. In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease., Competing Interests: PDD: None; POR: None; HGC: None; RTG: None; DBL: None; PGJ: Previous Founder and Shareholder of Almac Diagnostics; CV6 Therapeutics: Expert Advisor and Shareholder; Chugai Pharmaceuticals: Consultant; MST: None; ML: None; DMA: None.

CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted.

Previous research revealed that CMTM8 acts as a tumor suppressor gene in variety cancers. However, the role of CMTM8 in bladder cancer has never been reported. In this study, the expression profile of CMTM8 was examined in bladder cancer tissues and bladder cancer cell lines. The effects of CMTM8 on bladder cancer cell proliferation, apoptosis, migration, and invasion were examined. Bladder tumor tissues from 84 cases were examined for CMTM8 expression by immunohistochemistry. Disease-specific survival was investigated using a Kaplan-Meier analysis, and Cox proportional hazards analysis was assessed. Our results showed that upregulation of CMTM8 in the T24 cell line could suppress T24 cells proliferation, migration and invasion and enhance the sensitivity to Epirubicin. Kaplan-Meier analysis revealed that the expression of CMTM8 was correlated with the survival time of bladder cancer patients. Altogether, our data suggested that CMTM8 is an important tumor suppressor gene in human bladder cancer and qualified as a useful prognostic indicator for patients with bladder cancer.

Chemokine-like factor 1 (CKLF1) is a newly cloned chemotactic cytokine with CCR4 being its functional receptor. Recent evidence demonstrates a role of CKLF1 in arthritis. The aim of this study was to quantify the expression of CKLF1 as well as assess the correlation between CKLF1 and plasma acute-phase markers. Synovium was obtained from 16 osteoarthritis (OA), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients undergoing total joint arthroplasty, with other 11 patients treated for meniscal tears during sport accidents serving as normal controls. Levels of CKLF1 and CCR4 mRNA were detected by qRT-PCR, and the expression of CKLF1 was investigated by immunohistochemistry staining, subsequently analyzed with semiquantitative scores. Plasma acute-phase markers of inflammation were determined by ELISA. CKLF1 was found with a particularly up-regulated expression in synovim from AS and RA patients, and CCR4 mRNA levels increased in RA patients, not in OA or AS patients. Elevated levels of plasma markers of inflammation including CRP, ESR and D-dimer were observed in RA. Further, significantly positive correlations between relative expression levels of CKLF1 and CRP/ESR in RA patients and a positive correlation between CKLF1 and ESR in AS patients were found. There was no detectable correlation between CKLF1 and plasma D-dimer. This study confirms an increased but different level of CKLF1 in RA, OA and AS patients, all significantly higher than that in controls. Additionally, the significant positive correlations between CKLF1 levels and CRP/ESR in RA and between CKLF1 and ESR suggest that CKLF1 might contribute to the inflammation state and clinical symptoms in these rheumatic diseases. Further studies are required to investigate the utility of targeting specific CKLF1 for symptom control or disease modification in RA and AS.

Objective: To elucidate the correlation between the single nucleotide polymorphism of CKLF-like MARVEL transmembrane member 5 (CMTM5) gene rs723840 and the occurrence of high on aspirin platelet reactivity (HAPR)., Methods: The present study is a case-control study. A total of 210 hospitalized patients in Peking University First Hospital were enrolled. Aspirin response was assessed by 0.5 g/L arachidonic acid (AA)-induced platelet aggregation ratio (PR), and ≥ 3/4 quartile of PR of the population was defined as HAPR. Accordingly all the enrolled 210 coronary artery diseases (CAD) patients were divided into HAPR group and No-HAPR group. The genotypes were determined by polymerase chain reaction (PCR) and sequencing analysis for rs723840 of CMTM5 gene., Results: The genotype frequencies in rs723840 C>T of CMTM5 gene conformed well to the Hardy-Weinberg equilibrium in both HAPR group and No-HAPR group. Between the two groups, the genotypes frequencies in HAPR and No-HAPR groups were 48.4%, 51.6%, 0.0% and 73.7%, 22.9%, 0.034%, respectively (P=0.004). The C, T allele frequencies were significantly different in the two groups (P=0.031,OR=0.501, 95% CI: 0.264-0.947)., Conclusion: Our study finds a significant correlation between CMTM5 gene rs723840 polymorphism and high on aspirin platelet reactivity.

Bladder cancer is the most common tumor of the urinary tract. The incidence of bladder cancer has increased in the last few decades, thus novel molecular markers for early diagnosis and more efficacious treatment are urgently needed. Chemokine‑like factor (CKLF)‑like MARVEL transmembrane domain containing 8 (CMTM8) is downregulated in several types of cancers and is associated with tumor progression. However, CMTM8 expression has been unexplored in bladder cancer to date. Our results revealed that the expression of CMTM8 was negative in 46 of 74 (62.2%) bladder cancer samples via immunohistochemistry assay. CMTM8 downregulation was associated with advancing tumor stage and tumor grade. CMTM8 was successfully overexpressed by lentivirus in EJ and T24 cells, and the CCK‑8 and Transwell assays showed that CMTM8 overexpression decreased cell proliferation, migration and invasion in vitro. In tumor xenografts upregulation of CMTM8 inhibited tumor growth and lymph node metastasis in vivo. In conclusion, overexpression of CMTM8 in bladder cancer results in reduced malignant cell growth, migration and invasion, which could make it a potential therapeutic target in the treatment of bladder cancer.

The dysregulation of epidermal growth factor receptor (EGFR) signaling has been well documented to contribute to the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer death in the world. EGF-stimulated EGFR activation induces receptor internalization and degradation, which plays an important role in EGFR signaling. This process is frequently deregulated in cancer cells, leading to enhanced EGFR levels and signaling. Our previous study on CMTM7 is only limited to a brief description of the relationship of overexpressed CMTM7 with EGFR-AKT signaling. The biological functions of endogenous CMTM7 and its molecular mechanism remained unclear. In this study, we show that the stable knockdown of CMTM7 augments the malignant potential of NSCLC cells and enhances EGFR-AKT signaling by decreasing EGFR internalization and degradation. Mechanistically, CMTM7 knockdown reduces the activation of Rab5, a protein known to be required for early endosome fusion. In NSCLC, the loss of CMTM7 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, our findings highlight the role of CMTM7 in the regulation of EGFR signaling in tumor cells, revealing CMTM7 as a novel molecule related to Rab5 activation.

Background: Chemokine-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., Methods: An 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., Results: By 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., Conclusions: CMTM4 is downregulated and exhibits tumour-suppressor activities in ccRCC, and could be exploited as a target for ccRCC treatment.

Downregulation of CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) has been reported in a number of human tumors. However, the role of CMTM3 in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we showed that the expression of CMTM3 was significantly reduced in OSCC cell lines and primary tumor specimens (P < 0.001). Methylation-specific PCR showed hypermethylation in CMTM3 promoter in a significant proportion of tumor tissues (61 %). The expression of CMTM3 was associated with T stage, lymph node metastasis, tumor node metastasis (TNM) stage, and recurrence of OSCC patients (P < 0.05, n = 201). More importantly, CMTM3 expression was associated with the prognosis of OSCC patients (P < 0.001) and was an independent prognostic factor (hazard ratio = 0.593, 95 % confidence interval, 0.272-1.292; P = 0.039). Overexpression of CMTM3 inhibited the growth and migration of OSCC cells. In vivo experiments also showed that the growth of OSCC xenografts in nude mice was significantly inhibited by CMTM3 overexpression. These findings indicate that downregulation of CMTM3 due to promoter hypermethylation contributed to the proliferation and migration of OSCC cells and suggest that CMTM3 is an independent prognostic factor for the evaluation of the survival of OSCC patients.

A leucine-rich protein, ARR19 (androgen receptor corepressor-19 kDa), is highly expressed in male reproductive organs and moderately in others. Previously, we have reported that ARR19 is differentially expressed in adult Leydig cells during the testis development and inhibits steroidogenesis by reducing the expression of steroidogenic enzymes. Whereas in prostate, ARR19 represses the transcriptional activity of AR (androgen receptor), it is important for male sexual differentiation and maturation in prostate and epididymis, through the recruitment of HDAC4. In this study we show that long term adenovirus mediated overexpression of ARR19 in mice testis has the potential of inhibiting the differentiation of testicular and prostatic cells by reducing the size of testis and prostate but has no effect on the growth of seminal vesicles. Further, it reduces the level of progesterone and testosterone by reducing the steroidogenic enzymes such as 3HSD, P450c17 and StAR. This is the first study reporting a time-course analysis of the implications of long term overexpression of ARR19 in mice testis and its effect on other organs such as prostate and seminal vesicles. Taken together, these results suggest that ARR19 may play an important role in the differentiation of male reproductive organs such as testis and prostate.

Proteolipid protein 2 (PLP2) has been shown to be upregulated in several cancers, including breast cancer, hepatocellular carcinoma, osteosarcoma, and melanoma. PLP2 specifically binds to phosphatidylinositol 3 kinase to activate the protein kinase B pathway to enhance cell proliferation, adhesion, and invasion in melanoma cells. Therefore, we speculated that PLP2 exhibits oncogenic potential. However, the regulatory mechanisms of PLP2 in cancer cells remain unclear.Herein, we found that microRNA (miR)-664 expression was significantly downregulated in cutaneous malignant melanoma (CMM) cells and tissues compared with normal human melanocytes and benign melanocytic naevi. MiR-664 expression level was significantly correlated with patient survival. Ectopic expression of miR-664 reduced CMM cell proliferation and anchorage-independent growth, whereas the inhibition of miR-664 induced these effects. Furthermore, inhibition of miR-664 in CMM cells resulted in modulation of their entry into the G1/S transitional phase, which was caused by downregulation of the cyclin-dependent kinase inhibitor P21 and upregulation of the cell-cycle regulator cyclin D1. Moreover, we demonstrated that miR-664 downregulated PLP2 expression by directly targeting the PLP2 untranslated region.Taken together, our results suggest that miR-664 may play an important role in suppressing proliferation of CMM cells and present a novel mechanism of miR-mediated direct suppression of PLP2 expression in cancer cells.

Purpose: A novel tumor suppressor gene CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) is reduced or undetectable in many kinds of cancers and relates tumor malignant features. We detected its role in prostate cancer for possibility of target therapy as accumulating evidence has shown that CMTM3 is a promising tumor suppressor gene (TSG) for gene therapy., Methods: The expression of CMTM3 detected in prostate tissue microarray, specimens and cell lines were evaluated by immunohistochemistry and semi-quantitative PCR and Western blot, respectively. After being transfected with CMTM3 adenovirus or vector (mock), the proliferation and migration and invasion of LNCaP cells were detected by transwell assay and matrigel assay, respectively. Furthermore, the effects of CMTM3 on tumor growth were performed in nude mice xenograft in vivo., Results: We found CMTM3 was reduced in PCa tissues and cells compared with BPH tissues, and its expression in PCa tissues was related to the Gleason score. Moreover, after being transfected with adenovirus, ectopic expression of CMTM3 in LNCaP cells led to significant inhibition of cell proliferation and migration and invasion compared with the control (P < 0.05), which may be attributed to decreased Erk1/2 activity as p-Erk1/2 was remarkably reduced when CMTM3 was overexpressed. Finally, restoration of CMTM3 significantly suppressed xenograft tumor growth in vivo (P < 0.01).

The novel CKLF-like Marvel Transmembrane Domain-containing gene family (CMTM) consists of 8 members (CMTM1-8). As little is known about the oncogenic impact of these genes, we aimed to systematically investigate the relevance of CMTMs to glioblastoma pathogenesis. We performed mRNA expression analyses and survival correlations in glioblastoma patients. Moreover, we analyzed the impact of RNAi-based silencing and overexpression of CMTM family genes on tumor cell proliferation and invasion in vitro. CMTMs appeared to be widely regulated in the group of glioblastomas relative to non-neoplastic brain (NB) tissue (significant upregulation for CMTM2, 3, and 6 and significant downregulation for CMTM 4 and 8). For CMTM1, 5 and 7, we found aberrant expression levels in individual tumors. Functionally, CMTM1, 3, and 7 promoted tumor cell invasion, while CMTM1 additionally enhanced cell proliferation. In a large clinically annotated dataset, higher CMTM1 and 3 expression was significantly correlated with shorter overall survival. Our data thus suggest CMTM1 and 3 as priority targets in glioblastomas. Using a human phosphokinase protein expression profiling assay, we can provide first insights into signalling of these two genes that might be conveyed by growth factor receptor, Src family kinase and WNT activation., (© 2015 Wiley Periodicals, Inc.)

In Drosophila, myoblast fusion is a conserved process in which founder cells (FCs) and fusion competent myoblasts (FCMs) fuse to form a syncytial muscle fiber. Mutants for the myogenic regulator Myocyte enhancer factor-2 (MEF2) show a failure of myoblast fusion, indicating that MEF2 regulates the fusion process. Indeed, chromatin immunoprecipitation studies show that several genes involved in myoblast fusion are bound by MEF2 during embryogenesis. Of these, the MARVEL domain gene singles bar (sing), is down-regulated in MEF2 knockdown pupae, and has five consensus MEF2 binding sites within a 9000-bp region. To determine if MEF2 is an essential and direct regulator of sing during pupal muscle development, we identified a 315-bp myoblast enhancer of sing. This enhancer was active during myoblast fusion, and mutation of two MEF2 sites significantly decreased enhancer activity. We show that lack of sing expression resulted in adult lethality and muscle loss, due to a failure of fusion during the pupal stage. Additionally, we sought to determine if sing was required in either FCs or FCMs to support fusion. Interestingly, knockdown of sing in either population did not significantly affect fusion, however, knockdown in both FCs and FCMs resulted in muscles with significantly reduced nuclei numbers, provisionally indicating that sing function is required in either cell type, but not both. Finally, we found that MEF2 regulated sing expression at the embryonic stage through the same 315-bp enhancer, indicating that sing is a MEF2 target at both critical stages of myoblast fusion. Our studies define for the first time how MEF2 directly controls fusion at multiple stages of the life cycle, and provide further evidence that the mechanisms of fusion characterized in Drosophila embryos is also used in the formation of the more complex adult muscles., (Copyright © 2015 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. However, the role of microRNAs in leukaemia, particularly T-cell acute lymphoblastic leukaemia (T-ALL), has remained elusive. Here, we identified miR-664 and its predicted target gene PLP2 were differentially expressed in T-ALL using bioinformatics methods. In T-ALL cell lines, CCK-8 proliferation assay indicated that the cell proliferation was promoted by miR-664, while miR-664 inhibitor could significantly inhibited the proliferation. Moreover, migration and invasion assay showed that overexpression of miR-664 could significantly promoted the migration and invasion of T-ALL cells, whereas miR-664 inhibitor could reduce cell migration and invasion. luciferase assays confirmed that miR-664 directly bound to the 3'untranslated region of PLP2, and western blotting showed that miR-664 suppressed the expression of PLP2 at the protein levels. This study indicated that miR-664 negatively regulates PLP2 and promotes proliferation and invasion of T-ALL cell lines. Thus, miR-664 may represent a potential therapeutic target for T-ALL intervention., (Copyright © 2015. Published by Elsevier Inc.)

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 1 (CMTM1) consists of at least 23 alternatively spliced isoforms designated CMTM1&#95;v1-v23. In the present study, we detected CMTM1&#95;v17 expression in multiple human normal and tumor tissues and found that CMTM1&#95;v17 was highly expressed in testis and many tumor tissues including breast tumor. The overexpression of CMTM1&#95;v17 in the breast cancer cell line MDA-MB-231 promoted cell proliferation and resistance to tumor necrosis factor-α (TNF-α)-induced apoptosis. Moreover, siRNA-mediated silencing of CMTM1&#95;v17 sensitized MDA-MB-231 cells to TNF-α-induced apoptosis. We propose that CMTM1&#95;v17 may be a novel potential target for therapy in breast cancer patients. The present study provides insight into a novel mechanism by which CMTM1&#95;v17 enhances cellular proliferation and abrogates TNF-α-induced apoptosis. These findings also have implications for clinical practice as they highlight the potential for therapeutic targeting of CMTM1&#95;v17 for the treatment of breast and other cancers in which CMTM1&#95;v17 impacts cellular proliferation and survival.

Tumor protein D52 (TPD52) is overexpressed in different cancers, but its molecular functions are poorly defined. A large, low-stringency yeast two-hybrid screen using full-length TPD52 bait identified known partners (TPD52, TPD52L1, TPD52L2, MAL2) and four other preys that reproducibly bound TPD52 and TPD52L1 baits (PLP2, RAB5C, GOLGA5, YIF1A). PLP2 and RAB5 interactions with TPD52 were confirmed in pull down assays, with interaction domain mapping experiments indicating that both proteins interact with a novel binding region of TPD52. This study provides insights into TPD52 functions, and ways to maximise the efficiency of low-stringency yeast two-hybrid screens.

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.

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 was down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human CMTM3 has been proposed as a putative tumor suppressor gene. The loss of CMTM3 has been found in several carcinomas. However, the regulation of CMTM3 expression and its function in tumor progression remain largely unknown. Here, we investigated the regulation of CMTM3 expression, function and molecular mechanism in human testicular cancer cells. CMTM3 was frequently downregulated or silenced in testicular cancer cell lines and tumor tissues but highly expressed in normal testis tissues. The re-expression of CMTM3 significantly suppressed the colony formation, proliferation, and migration capacity of testicular cancer cells by inducing a G2 cell cycle arrest and apoptosis. Moreover, the re-expression of CMTM3 activated the transcription of p53, induced p53 accumulation, up-regulated the expression of p21, and increased the cleavage of caspase 9, 8, 3, and PARP. The downregulation of CMTM3 in clinical tumor tissues was associated with the methylation of a single CpG site located within the Sp1/Sp3-responsive region of the core promoter. These results indicate that CMTM3 can function as tumor suppressor through the induction of a G2 cell cycle arrest and apoptosis. CMTM3 is thus involved in testicular cancer pathogenesis, and it is frequently at least partially silenced by the methylation of a single, specific CpG site in tumor tissues.

The CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) gene is a novel tumor suppressor with frequent epigenetic inactivation. In this study, we showed the role played by CMTM3 in gastric cancer cells as a tumor suppressor gene, and examined the correlation between CMTM3 expression and clinicopathological parameters using immunohistochemistry in gastric cancer patients with different pathological stages (n = 350). We found that CMTM3 expression was reduced or silenced by epigenetic regulation in gastric cell lines, and dramatically downregulated in primary gastric cancer tissues. Restoration of CMTM3 significantly affected migration and invasion of AGS and SGC-7901 cells (P < 0.001). In vivo experiments showed that peritoneal disseminated metastases were significantly suppressed by CMTM3 (P < 0.001). We further showed that the expression of MMP2 and the phosphorylation of Erk1/2 were decreased when CMTM3 was restored. In addition, by immunohistochemical staining, we found that the expression of CMTM3 was remarkably weaker in gastric cancer tissues than in normal mucosae (P = 0.008), and was significantly correlated with gender (P = 0.033), tumor depth (P = 0.049), stage (P = 0.021), and histological grade (P = 0.022). More importantly, CMTM3 expression was associated with prognosis in gastric cancer patients (P = 0.041), and was a significant independent prognostic indicator (hazard ratio = 0.704, 95% confidence interval, 0.498-0.994; P = 0.046). Our findings indicate that CMTM3 regulates migration and invasion of gastric cancer cells. Moreover, CMTM3 is a candidate marker for prognosis of gastric cancer in the clinic., (© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Unlabelled: Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis., Importance: The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene, NCE102, caused a strong defect in formation of invasive hyphal growth in vitro and decreased virulence in mice. The nce102Δ mutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

Objective: To investigate the expression and clinical pathological significance of CMTM8 and E-cadherin in primary and metastatic clear-cell renal cell carcinoma., Methods: The immunohistochemistry was used to detect the expressions of CMTM8 and E-cadherin in 17 cases of primary clear-cell renal cell carcinoma, 8 cases of normal renal tissue, 9 cases of metastatic renal cell carcinoma in lungs and 10 cases of metastatic renal cell carcinoma in bones., Results: The membranous staining intensities of CMTM8 and E-cadherin in normal renal tissue were strong, but reduced in low-grade clear-cell renal cell carcinoma. There were positive cytoplasmic stainings of CMTM8 and E-cadherin in high-grade clear-cell renal cell carcinoma. Increased cytoplasmic expression of CMTM8 was frequent in metastatic renal cell carcinoma, accompanied with reduced cell surface staining. The expression of E-cadherin could be negative or weakly positive at membrane and cytoplasma. CMTM8 and E-cadherin expressions were negatively correlated with development and metastasis in clear-cell renal cell carcinoma (r=-0.841 and r=-0.732, P<0.001). Moreover, CMTM8 was also correlated with the expression of E-cadherin (r=0.694, P<0.001)., Conclusion: CMTM8 and E-cadherin are negatively correlated with tumorgenesis and development in clear-cell renal cell carcinoma. The location and intensity of their expressions have significant association with the prognosis.

Background: CKLF-like MARVEL transmembrane domain-containing 7 (CMTM7) located at 3p22.3, is a frequent deletion site and a tumor suppressor gene (TSG) locus in many cancer, which suggests CMTM7 may be a potential TSG. The aim of this study was to investigate the correlations of CMTM7 expression and survival rate in patients with non-smallcell lung cancer (NSCLC)., Methods: Surgical specimens of 180 cases with pathologically confirmed NSCLC were grouped into 18 tissue microarray slides. CMTM7 expression in these specimens were detected by immunohistochemistry staining and representative cases were confirmed by Western blotting. Univariate and multivariate analyses were performed to identify the association of CMTM7 expression with pathological features and survival of patients with NSCLC., Results: A total of 78.9% of the 180 patients had variations of CMTM7 protein expression, either up-regulated or down-regulated. Univariate analysis showed that the patients' survival rate after surgery was highly correlated with CMTM7 expression (P = 0.0091). In addition, prognostic factors were examined by multivariate Cox regression analysis, and results suggested that CMTM7 expression was a unique prognostic factor in NSCLC survival., Conclusions: The CMTM7 expression may be related to survival of patients with NSCLC and a unique prognostic factor. CMTM7 may play an important role in NSCLC development.

A chemokine-like factor 1 (CKLF1) is a recently discovered chemokine with broad-spectrum biological functions in inflammation and autoimmune diseases. C19 as a CKLF1's C-terminal peptide has been reported to exert inhibitory effects in a variety of diseases. However, the roles of CKLF1 and C19 on vascular smooth muscle cell (VSMC) migration and neointima formation still remain elusive. The effects of CKLF1 and C19 on VSMC migration and neointimal formation were investigated in cultured VSMCs and balloon-injured rat carotid arteries based on techniques including adenovirus-induced CKLF1 overexpression, gel based perivascular administration of C19, Boyden chamber, scratch-wound assay, real-time PCR, western blot and immunohistochemical analysis. CKLF1 was noticed to accumulate preferentially in neointima after the injury and colocalize with VSMCs. Luminal delivery of CKLF1 adenovirus to arteries exacerbated intimal thickening while perivascular administration of C19 to injured arteries attenuated this problem. In cultured primary VSMCs, CKLF1 overexpression up-regulated VSMC migration, which was down-regulated by C19. These data suggest that CKLF1 has a pivotal role in intimal hyperplasia by mediating VSMC migration. C19 was demonstrated to inhibit CKLF1-mediatated chemotaxis and restenosis. Thus further studies on C19 may provide a new treatment perspective for atherosclerosis and post-angioplasty restenosis.

Objective: To observe the effects of CMTM2 on cyclophosphamide (CP)-induced reproductive toxicity and the expression of steroidogenic acute regulatory (StAR) protein in the transgenic mouse model., Methods: Twenty CMTM2 transgenic mice were equally divided into a CMTM2 + CP and a CMTM2 + NS group, the former intraperitoneally injected with CP at 50 mg per kg per d, while the latter with the equivalent dose of normal saline, both for 7 days. Another 20 wild C57BL/6J mice were randomly assigned to a WT + CP and a WT + NS group, treated the same way above. After 30 days, all the mice were sacrificed and their epididymides and testes removed for measurement of the serum testosterone level by radioimmunoassay, determination of sperm concentration and motility by light microscopy and detection of the expression of StAR by Western blot., Results: The levels of serum testosterone, sperm concentration and sperm motility were significantly decreased in the CMTM2 + CP group as compared with the CMTM2 + NS group ([42.98 +/- 3.25] nmol/L vs [46.74 +/- 3.38] nmol/L, [16.89 +/- 1.17 ] x 10(6)/ml vs [24.68 +/- 0.95 ] x 10(6)/ml, [72.75 +/- 1.25]% vs [85.14 +/- 1.12]%, P < 0.05), but remarkably less than in the WT + CP group ([37.97 +/- 4.17] nmol/L, [12.75 +/- 1.02] x 10(6)/ml, [50.52 +/- 1.37] %) (P < 0.05). However, the expression of StAR was significantly higher in the CMTM2 + CP than in the WT + CP group (1.16 +/- 0.07 vs 0.69 +/- 0.08, P < 0.05)., Conclusion: CMTM2 antagonizes cyclophosphamide-induced reproductive toxicity via regulating the expression of StAR, and hence plays a protective role in the reproductive system.

Emerging evidence indicates that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in human normal and pathological cells. We have already shown that exposure of MDA-MB231 breast cancer cells to 5 μM CdCl(2) for 96 h, apart from significantly affecting mitochondrial metabolism, induces modifications of the expression level of genes coding for members of stress response-, mitochondrial respiration-, MAP kinase-, NF-κB-, and apoptosis-related pathways. In the present study, we have expanded the knowledge on the biological effects of Cd-breast cancer cell interactions, indicating PLP2 (proteolipid protein-2) as a novel member of the list of Cd-upregulated genes by MDA-MB231 cancer cells and, through the application of transfection techniques with specific antisense oligonucleotides, we have demonstrated that such over-expression may be an upstream event to some of the changes of gene expression levels already observed in Cd-treated cells, thus unveiling new possible molecular relationship between PLP2 and genes linked to the stress and apoptotic responses.

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

CKLF-like MARVEL transmembrane domain containing member(CMTM)is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM5 belongs to this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily(TM4SF). CMTM5 is broadly expressed in normal adult and fetal human tissues, but is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM5 may inhibit the proliferation, migration, and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear, CMTM5 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM5 may be a new target in the gene therapies for tumors, while further studies on CMTM5 and its anti-tumor mechanisms are warranted.

Objective: To explore the effects of chemokine-like factor 1 (CKLF1) on the growth and apoptosis of human vascular smooth muscle cell and understand its mechanism for the induction of cell apoptosis., Methods: After the transfection of human vascular smooth muscle cell with CKLF1, CCK8 assay was used to observe the growth of cell. The cell cycle and apoptosis was assessed by flow cytometry (FCM). And the mRNA and protein expressions of Bcl-2, Bax and Caspase-3 were detected by real-time polymerase chain reaction (PCR) and Western blot respectively., Results: At 24, 48 and 72 h, the growth proliferation (15.8% ± 3.4%, 25.5% ± 9.8% and 37.2% ± 3.3%, P < 0.05) and inhibited apoptosis (5.5% ± 1.3%, 7.6% ± 0.7% and 11.4% ± 5.5%, P < 0.05) of vascular smooth muscle cell were detected. CKLF1 also down-regulated the level of mRNA expression of Bax (0.96 ± 0.14, 0.61 ± 0.15 and 0.37 ± 0.05), protein expression of Caspase-3 (1.94 ± 0.30, 1.09 ± 0.17 and 0.73 ± 0.09) and elevated the mRNA expression of Bcl-2 (1.59 ± 0.18, 2.05 ± 0.51 and 2.96 ± 0.27)., Conclusion: CKLF1 can induce proliferation and inhibit apoptosis of human vascular smooth muscle cell through the down-regulation of Bax and Caspase-3 and the up-regulation of Bcl-2 expression.

Objective: To establish a transgenic mouse model systemically expressing the CMTM2 gene and study the effect of the CMTM2 expression on the reproductive system of mice in vivo., Methods: Transgenic mice were generated by microinjection of pRevTRE-CMTM2 and the genotype was detected by PCR. The expression of CMTM2 was determined by RT-PCR, Western blot and immunohistochemistry, and the serum testosterone level was measured by radioimmunoassay., Results: The CMTM2 gene was highly expressed in the testis of the transgenic mouse models and in their offspring as well. The level of serum testosterone was significantly increased in the transgenic models as compared with the wild-type mice ([46.04 +/- 3.72] vs [42.43 +/- 3.80] nmol/L, P < 0.05)., Conclusion: The transgenic mouse model was established successfully, which could highly express the CMTM2 gene. It is indicated that CMTM2 may influence steroidogenesis and testosterone secretion in transgenic mice.

Objective: To investigate the inhibitory effect of CKLF-like MARVEL transmembrane domain containing 5 (CMTM5) on xenografted human prostatic cancer in nude mice and its action mechanism., Methods: We established a model of xenografted prostatic cancer by inoculating PC-3 cells subcutaneously into nude mice, and 3 weeks later injected CMTM5 adenovirus locally into the tumor followed by daily observation of the tumor volume and body weight of the experimental animals. All the rats were killed 2 weeks after CMTM5 injection and the tumor tissue harvested for detection of the inhibitory effect of CMTM5 on the expressions of VEGF and NF-kappaB proteins by immunohistochemistry., Results: The tumor volume was significantly smaller and body weight of the CMTM5-treated mice were (573.39 +/- 175.24) mm3 and (0.55 +/- 0.11) g, respectively, significantly decreased as compared with those of the controls ([1482.50 +/- 327.86] mm3 and [1.31 +/- 0.29] g) (P = 0.03 and P = 0.027). Immunohistochemistry showed that the expressions of VEGF and NF-kappaB were obviously down-regulated in the CMTM5 group in comparison with the control group., Conclusion: CMTM5 suppresses the growth of prostate cancer by down-regulating the expressions of VEGF and NF-kappaB.

Background: Colorectal cancer is one of the leading causes of mortality worldwide. Genome wide analysis studies have identified sequence mutations causing loss-of-function that are associated with disease occurrence and severity. Epigenetic modifications, such DNA methylation, have also been implicated in many cancers but have yet to be examined in the East Asian population of colorectal cancer patients., Methods: Biopsies of tumors and matched non-cancerous tissue types were obtained and genomic DNA was isolated and subjected to the bisulphite conversion method for comparative DNA methylation analysis on the Illumina Infinium HumanMethylation27 BeadChip., Results: Totals of 258 and 74 genes were found to be hyper- and hypo-methylated as compared to the individual's matched control tissue. Interestingly, three genes that exhibited hypermethylation in their promoter regions, CMTM2, ECRG4, and SH3GL3, were shown to be significantly associated with colorectal cancer in previous studies. Using heatmap cluster analysis, eight hypermethylated and 10 hypomethylated genes were identified as significantly differentially methylated genes in the tumour tissues., Conclusions: Genome-wide methylation profiling facilitates rapid and simultaneous analysis of cancerous cells which may help to identify methylation markers with high sensitivity and specificity for diagnosis and prognosis. Our results show the promise of the microarray technology in identification of potential methylation biomarkers for colorectal cancers.

Objective: To study the effects of rat chemokine-like factor 1 (rCklf1) on chemotaxis and proliferation of rat aortic smooth muscle cells(ASMC)., Methods: The recombinant eukaryotic expression vector pcDNA3.1/rCklf1 was transiently transfected into 293T cells. The supernatants were harvested for chemotactic and proliferation assays. pcDNA3.1/rCklf1 was also transfected into ASMC and the proliferation of transfected cells was detected by MTT assays., Results: The 10 fold diluted supernatants of pcDNA3.1/rCklf1 transfected 293T cells had chemotactic effects on rat ASMC, which could be inhibited by pertussis toxin at the concentration of 10 microg/L. Furthermore, the 10 fold diluted supernatants of rCklf1 transfected 293T cells promoted the proliferation of ASMC (1.3 fold compared with the control) and transient overexpression of rCklf1 in ASMC had promoting activities on the proliferation of rat ASMC (1.5 fold compared with the control at 72 h)., Conclusion: rCklf1 has mitogenic and GiPCR-dependent chemotactic effects on rat ASMC,indicating that rCklf1 may be involved in the pathological process of atherosclerosis.