Prognostic value and immunological role of MMRN1: a rising star in cancer.

BACKGROUND: Multimerin 1 (MMRN1) is a factor V binding protein, which can support platelet adhesion and thrombus formation. In recent years, the role of MMRN1 in cancer has begun to attract attention. But systematic studies in this area are lacking. Therefore, we used bioinformatics methods to analyze MMRN1 in tumors to reveal the possible role of MMRN1. METHODS: Using the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database, we obtained relevant data for analyzing MMRN1. Using Gene Expression Profiling Interactive Analysis (GEPIA), Human Protein Atlas (HPA), TCGA, GeneMANIA, and cBioPortal, we explored the potential role of MMRN1 in different types of tumors. Tumor Immune System Interactions and Drug Bank (TISIDB) and Sangerbox were used to analyze the correlation between MMRN1 and tumor immunity. Gene set cancer analysis (GSCA) and UALCAN were used to analyze the methylation of MMRN1. GSCA was also used to analyze the drug sensitivity of MMRN1. RESULTS: MMRN1 is down-regulated in most cancer types and is closely related to the prognosis of cancer patients. Interestingly, in most tumors, MMRN1 is positively correlated with immune -related genes. In addition, we observed different levels of methylation and mutations in different types of tumors. Drug sensitivity analysis found that MMRN1 was negatively correlated with several drugs, including GW-2580 and TL-1-85, suggesting that it can be used to develop potential anticancer therapies. CONCLUSION: Our analysis demonstrated a significant relationship between MMRN1 and prognosis, tumor immunity, and drug sensitivity of several tumors. As a rising star in cancer, it needs further research.

Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis.

OBJECTIVES: CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed. METHODS: By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot. RESULTS: MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation. CONCLUSION: CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.

Bioinformatics Approaches to Predict Mutation Effects in the Binding Site of the Proangiogenic Molecule CD93.

The transmembrane glycoprotein CD93 has been identified as a potential new target to inhibit tumor angiogenesis. Recently, Multimerin-2 (MMRN2), a pan-endothelial extracellular matrix protein, has been identified as a ligand for CD93, but the interaction mechanism between these two proteins is yet to be studied. In this article, we aim to investigate the structural and functional effects of induced mutations on the binding domain of CD93 to MMRN2. Starting from experimental data, we assessed how specific mutations in the C-type lectin-like domain (CTLD) affect the binding interaction profile. We described a four-step workflow in order to predict the effects of variations on the inter-residue interaction network at the PPI, based on evolutionary information, complex network metrics, and energetic affinity. We showed that the application of computational approaches, combined with experimental data, allowed us to gain more in-depth molecular insights into the CD93-MMRN2 interaction, offering a platform for developing innovative therapeutics able to target these molecules and block their interaction. This comprehensive molecular insight might prove useful in drug design in cancer therapy.

Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion.

BACKGROUND: Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo. OBJECTIVES: To examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion. METHODS: Mmrn1-deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple-helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion. RESULTS: MMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple-helical collagen peptides (P < .05). Mmrn1-/- and Mmrn1+/- mice were viable and fertile, with complete and partial platelet Mmrn1 deficiency, respectively. Relative to wild-type mice, Mmrn1-/- and Mmrn1+/- mice did not have overt bleeding, increased median bleeding times, or increased wound blood loss (P ≥ .07); however, they both showed significantly impaired platelet adhesion and thrombus formation in the ferric chloride injury model (P ≤ .0003). Mmrn1-/- platelets had impaired adhesion to GPAGPOGPX peptides and fibrillar collagen (P ≤ .03) and formed smaller aggregates than wild-type platelets when captured onto collagen, triple-helical collagen mimetic peptides, von Willebrand factor, or fibrinogen (P ≤ .008), despite preserved, low shear, and high shear aggregation responses. CONCLUSIONS: Multimerin 1 supports platelet adhesion and thrombus formation and binds to highly conserved, GPAGPOGPX motifs in fibrillar collagens that synergistically enhance platelet adhesion.

Analysis of Multimerin 1 (MMRN1) expression in ovarian cancer.

Ovarian cancer, the most lethal gynecological cancer, is the fifth most common cause of cancer-related deaths in women. A cost-effective and non-invasive early screening method for ovarian cancer is required to reduce the high mortality rate. Saliva is a clinically informative unique fluid, which is useful for novel approaches to prognosis, clinical diagnosis, and monitoring for non-invasive detection of disease. Multimerin1 (MMRN1) is a di-sulfide linked homo-polymeric glycoprotein from EMILIN family. Altered expression of MMRN1 has been reported in hepatocellular carcinoma, cervical cancer, and ovarian cancer. But, its role in epithelial ovarian cancer (EOC) is not clear and well documented. In this study, expression of Multimerin 1 was validated in saliva and tissues of EOC patients and age-matched controls by western blotting, ELISA, RT-PCR, and immunohistochemistry. Significant over expression of MMRN1 was observed by western blot and ELISA in saliva samples of EOC patients. The average concentration of MMRN1 in the saliva of healthy controls was 28.7 pg/ml (SE ± 1.76), 42.53 pg/ml (SE ± 4.06) in low grade and 52.91 pg/ml (SE ± 4.24) with p < 0.01 in high-grade EOC. Upregulated cytoplasmic expression of MMRN1 was observed in EOC tissue by immunohistochemistry. Our results suggest that MMRN1 expression is associated with EOC progression and MMRN1 may be potential biomarker candidates for early-stage EOC detection however further experiments are required in a large cohort to establish this proposition. Also, saliva can be explored as a novel medium for ovarian cancer diagnosis.

Increased Expression of the Multimerin-1 Gene in α-Synuclein Knokout Mice.

Multimerin-1 (Mmrn-1) is a soluble protein, also known as elastin microfibril interfacer 4 (EMILIN-4), found in platelets and in the endothelium of blood vessels. Its function and role in pathology are still not fully understood. Genetic modifications in alpha-synuclein gene (Snca) locus that mapped 160 Kb apart from Mmrn-1 in mouse genome, could weigh with regulatory elements of Mmrn-1 gene. We have studied the Mmrn-1 expression in brain cortex of three mouse lines with Snca knock-out: B6(Cg)-Sncatm1.2Vlb/J, B6;129-Sncatm1Sud/J, and B6;129X1-Sncatm1Rosl/J. The 35-fold increase for Mmrn-1 mRNA level have been found in B6;129X1-Sncatm1Rosl/J mice that carry in their genome foreign sequences including bacterial gene neo and a strong promoter of a mouse phosphoglycerate kinase (Pgk1) oriented towards Mmrn-1 gene. This effect on regulatory elements of Mmrn-1 gene as a result of modifications in Snca locus should be taken into consideration when using B6;129X1-Sncatm1Rosl/J line, that is widely applied for study of neurodegeneration mechanisms.

Identification of potential cervical cancer serum biomarkers in Thai patients.

Cervical cancer is one of the most common causes of cancer-associated mortality in females worldwide. Serum biomarkers are important tools for diagnosis, disease staging, monitoring treatment and detecting recurrence in different types of cancer. However, only a small number of established biomarkers have been used for clinical diagnosis of cervical cancer. Therefore, the identification of minimally invasive, sensitive and highly specific biomarkers for detection of cervical cancer may improve outcomes. In the present pilot study, changes in disease-relevant proteins in 31 patients with cervical cancer were compared with 16 healthy controls. The Human 14 Multiple Affinity Removal system was used to deplete the 14 most abundant serum proteins to decrease sample complexity and to enrich proteins that exhibited decreased levels of abundance in the serum samples. Immunoaffinity-depleted serum samples were analyzed by in-gel digestion, followed by liquid chromatography mass spectrometry analysis and data processing. Automated quantitative western blot assays and receiver operating characteristic (ROC) curves were used to evaluate the differential protein expression levels between the two groups. Capillary electrophoresis-based western blot analysis was performed to quantitatively determine serum levels of the candidate biomarkers. Significantly increased levels of α-1-antitrypsin (A1AT) and pyrroline-5-carboxylate reductase 2 (PYCR2) were detected, whereas the levels of transthyretin (TTR), apolipoprotein A-I (ApoA-I), vitamin D binding protein (VDBP) and multimerin-1 (MMRN1) were significantly decreased in patients with cervical cancer compared with the healthy controls. ROC curve analysis indicated that the sensitivity and specificity was improved through the combination of the 6 candidate biomarkers. In summary, the results demonstrated that 6 candidate biomarkers (A1AT, PYCR2, TTR, ApoA-I, VDBP and MMRN1) exhibited significantly different expression between serum samples from healthy controls and patients with cervical cancer. These proteins may represent potential biomarkers for distinguishing patients with cervical cancer from healthy controls and for differentiation of patient subgroups.

Expression patterns and the prognostic value of the EMILIN/Multimerin family members in low-grade glioma.

Managing low-grade gliomas (LGG) remains a major medical challenge due to the infiltrating nature of the tumor and failure of surgical resection to eliminate the disease. EMILIN/Multimerins contain the gC1q signature, which is involved in many tumor processes. However, the expression and prognostic value of EMILIN/Multimerins in LGG remains unclear. This study used integrated bioinformatics analysis to investigate the expression pattern, prognostic value and function of EMILIN/Multimerins in patients with LGG. We analyzed the transcription levels and prognostic value EMILIN/Multimerins in LGG using the ONCOMINE, Gene Expression Profiling Interactive Analysis (GEPIA) and UALCAN databases. The mutation and co-expression rates of neighboring genes in EMILIN/Multimerins were studied using cBioPortal. TIMER and Metascape were used to reveal the potential function of EMILIN/Multimerins in LGG. According to our analysis, most EMILIN/Multimerins were overexpressed in LGG and shared a clear association with immune cells. GEPIA analysis confirmed that high levels of EMILIN/Multimerins, not including MMRN2, were associated with a poor prognosis in disease-free survival of patients with LGG. Additionally, we discovered that EMILIN/Multimerins may regulate LGG and we found a correlation between their expression patterns and distinct pathological grades. We found that EMILIN/Multimerins serve as possible prognostic biomarkers and high-priority therapeutic targets patients with LGG.

Urinary biomarkers for the diagnosis of cervical cancer by quantitative label-free mass spectrometry analysis.

Due to the invasive procedure associated with Pap smears for diagnosing cervical cancer and the conservative culture of developing countries, identifying less invasive biomarkers is of great interest. Quantitative label-free mass spectrometry was performed to identify potential biomarkers in the urine samples of patients with cervical cancer. This technique was used to study the differential expression of urinary proteomes between normal individuals and cancer patients. The alterations in the levels of urinary proteomes in normal and cancer patients were analyzed by Progenesis label-free software and the results revealed that 60 proteins were upregulated while 73 proteins were downregulated in patients with cervical cancer. This method could enrich high molecular weight proteins from 100 kDa. The protein-protein interactions were obtained by Search Tool for the Retrieval of Interacting Genes/Proteins analysis and predicted the biological pathways involving various functions including cell-cell adhesion, blood coagulation, metabolic processes, stress response and the regulation of morphogenesis. Two notable upregulated urinary proteins were leucine-rich α-2-glycoprotein (LRG1) and isoform-1 of multimerin-1 (MMRN1), while the 3 notable downregulated proteins were S100 calcium-binding protein A8 (S100A8), serpin B3 (SERPINB3) and cluster of differentiation-44 antigen (CD44). The validation of these 5 proteins was performed by western blot analysis and the biomarker sensitivity of these proteins was analyzed individually and in combination with receiver operator characteristic curve (ROC) analysis. Quantitative mass spectrometry analysis may allow for the identification of urinary proteins of high molecular weight. The proteins MMRN1 and LRG1 were presented, for the first time, to be highly expressed urinary proteins in cervical cancer. ROC analysis revealed that LRG1 and SERPINB3 could be individually used, and these 5 proteins could also be combined, to detect the occurrence of cervical cancer.

Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).

C-type lectin family 14, member A (CLEC14A), is a single-pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid-like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid-like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or -3. Site-directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2-fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3-fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.-Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).