Tofacitinib Regulates Endostatin via Effects on CD147 and Cathepsin S.

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin' s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

The Roles of GDF-9, BMP-15, BMP-4 and EMMPRIN in Folliculogenesis and In Vitro Fertilization.

Growth differentiation factor 9 (GDF-9) contributes to early ovarian development and oocyte survival. Higher concentrations of GDF-9 in follicular fluid (FF) are associated with oocyte nuclear maturation and optimal embryo development. In in vitro fertilization (IVF), GDF-9 affects the ability of the oocyte to fertilize and subsequent embryonic development. Bone morphogenetic protein 15 (BMP-15) is involved in the regulation of ovarian function and affects oocyte development. During IVF, BMP-15 contributes to the formation of competent blastocysts. BMP-15 may play a role in embryo implantation by affecting endometrial receptivity. Bone morphogenetic protein 4 (BMP-4) is involved in the regulation of follicle growth and development and affects granulosa cell (GC) differentiation. In relation to IVF, BMP-4 is important for embryonic development, influences cell fate and differentiation, and plays a role in facilitating embryo-endometrial interactions during the implantation process. Extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with ovulation and follicle rupture, promotes the release of mature eggs, and affects the modification of the extracellular matrix of the follicular environment. In IVF, EMMPRIN is involved in embryo implantation by modulating the adhesive properties of endometrial cells and promotes trophoblastic invasion, which is essential for pregnancy to occur. The purpose of the current article is to review the studies and recent findings of GDF-9, BMP-15, BMP-4 and EMMPRIN as fundamental factors in normal follicular development and in vitro fertilization.

Pseudolaric Acid B Targets CD147 to Selectively Kill Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive blood cancer. With low survival rates, new drug targets are needed to improve treatment regimens and patient outcomes. Pseudolaric acid B (PAB) is a plant-derived bioactive compound predicted to interact with cluster of differentiation 147 (CD147/BSG). CD147 is a transmembrane glycoprotein overexpressed in various malignancies with suggested roles in regulating cancer cell survival, proliferation, invasion, and apoptosis. However, the detailed function of PAB in AML remains unknown. In this study, AML cell lines and patient-derived cells were used to show that PAB selectively targeted AML (IC50: 1.59 ± 0.47 µM). Moreover, proliferation assays, flow cytometry, and immunoblotting confirmed that PAB targeting of CD147 resulted in AML cell apoptosis. Indeed, the genetic silencing of CD147 significantly suppressed AML cell growth and attenuated PAB activity. Overall, PAB imparts anti-AML activity through transmembrane glycoprotein CD147.

EMMPRIN promotes spheroid organization and metastatic formation: comparison between monolayers and spheroids of CT26 colon carcinoma cells.

Background: In vitro studies often use two-dimensional (2D) monolayers, but 3D cell organization, such as in spheroids, better mimics the complexity of solid tumors. To metastasize, cancer cells undergo the process of epithelial-to-mesenchymal transition (EMT) to become more invasive and pro-angiogenic, with expression of both epithelial and mesenchymal markers. Aims: We asked whether EMMPRIN/CD147 contributes to the formation of the 3D spheroid structure, and whether spheroids, which are often used to study proliferation and drug resistance, could better model the EMT process and the metastatic properties of cells, and improve our understanding of the role of EMMPRIN in them. Methods: We used the parental mouse CT26 colon carcinoma (CT26-WT) cells, and infected them with a lentivirus vector to knock down EMMPRIN expression (CT26-KD cells), or with an empty lentivirus vector (CT26-NC) that served as a negative control. In some cases, we repeated the experiments with the 4T1 or LLC cell lines. We compared the magnitude of change between CT26-KD and CT26-WT/NC cells in different metastatic properties in cells seeded as monolayers or as spheroids formed by the scaffold-free liquid overlay method. Results: We show that reduced EMMPRIN expression changed the morphology of cells and their spatial organization in both 2D and 3D models. The 3D models more clearly demonstrated how reduced EMMPRIN expression inhibited proliferation and the angiogenic potential, while it enhanced drug resistance, invasiveness, and EMT status, and moreover it enhanced cell dormancy and prevented CT26-KD cells from forming metastatic-like lesions when seeded on basement membrane extract (BME). Most interestingly, this approach enabled us to identify that EMMPRIN and miR-146a-5p form a negative feedback loop, thus identifying a key mechanism for EMMPRIN activities. These results underline EMMPRIN role as a gatekeeper that prevents dormancy, and suggest that EMMPRIN links EMT characteristics to the process of spheroid formation. Conclusions: Thus, 3D models can help identify mechanisms by which EMMPRIN facilitates tumor and metastasis progression, which might render EMMPRIN as a promising target for anti-metastatic tumor therapy.

Glucose Promotes EMMPRIN/CD147 and the Secretion of Pro-Angiogenic Factors in a Co-Culture System of Endothelial Cells and Monocytes.

Vascular complications in Type 2 diabetes mellitus (T2DM) patients increase morbidity and mortality. In T2DM, angiogenesis is impaired and can be enhanced or reduced in different tissues ("angiogenic paradox"). The present study aimed to delineate differences between macrovascular and microvascular endothelial cells that might explain this paradox. In a monoculture system of human macrovascular (EaHy926) or microvascular (HMEC-1) endothelial cell lines and a monocytic cell line (U937), high glucose concentrations (25 mmole/L) increased the secretion of the pro-angiogenic factors CD147/EMMPRIN, VEGF, and MMP-9 from both endothelial cells, but not from monocytes. Co-cultures of EaHy926/HMEC-1 with U937 enhanced EMMPRIN and MMP-9 secretion, even in low glucose concentrations (5.5 mmole/L), while in high glucose HMEC-1 co-cultures enhanced all three factors. EMMPRIN mediated these effects, as the addition of anti-EMMPRIN antibody decreased VEGF and MMP-9 secretion, and inhibited the angiogenic potential assessed through the wound assay. Thus, the minor differences between the macrovascular and microvascular endothelial cells cannot explain the angiogenic paradox. Metformin, a widely used drug for the treatment of T2DM, inhibited EMMPRIN, VEGF, and MMP-9 secretion in high glucose concentration, and the AMPK inhibitor dorsomorphin enhanced it. Thus, AMPK regulates EMMPRIN, a key factor in diabetic angiogenesis, suggesting that targeting EMMPRIN may help in the treatment of diabetic vascular complications.

Paradoxical Changes: EMMPRIN Tissue and Plasma Levels in Marfan Syndrome-Related Thoracic Aortic Aneurysms.

Background: Thoracic aortic aneurysms (TAAs) associated with Marfan syndrome (MFS) are unique in that extracellular matrix metalloproteinase inducer (EMMPRIN) levels do not behave the way they do in other cardiovascular pathologies. EMMPRIN is shed into the circulation through the secretion of extracellular vesicles. This has been demonstrated to be dependent upon the Membrane Type-1 MMP (MT1-MMP). We investigated this relationship in MFS TAA tissue and plasma to discern why unique profiles may exist. Methods: Protein targets were measured in aortic tissue and plasma from MFS patients with TAAs and were compared to healthy controls. The abundance and location of MT1-MMP was modified in aortic fibroblasts and secreted EMMPRIN was measured in conditioned culture media. Results: EMMPRIN levels were elevated in MFS TAA tissue but reduced in plasma, compared to the controls. Tissue EMMPRIN elevation did not induce MMP-3, MMP-8, or TIMP-1 expression, while MT1-MMP and TIMP-2 were elevated. MMP-2 and MMP-9 were reduced in TAA tissue but increased in plasma. In aortic fibroblasts, EMMPRIN secretion required the internalization of MT1-MMP. Conclusions: In MFS, impaired EMMPRIN secretion likely contributes to higher tissue levels, influenced by MT1-MMP cellular localization. Low EMMPRIN levels, in conjunction with other MMP analytes, distinguished MFS TAAs from controls, suggesting diagnostic potential.

Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S.

During progression of rheumatoid arthritis (RA), angiogenesis provides oxygen and nutrients for the cells' increased metabolic demands and number. To turn on angiogenesis, pro-angiogenic factors must outweigh anti-angiogenic factors. We have previously shown that CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) can induce the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) in a co-culture of the human HT1080 fibrosarcoma and U937 monocytic-like cell lines. However, whether CD147 influences anti-angiogenic factors was not known. We now show that relative to single cultures, the co-culture of these cells not only enhanced pro-angiogenic factors but also decreased the anti-angiogenic factors endostatin and thrombospondin-1 (Tsp-1), generally increasing the angiogenic potential as measured by a wound assay. Using anti-CD147 antibody, CD147 small interfering RNA (siRNA), and recombinant CD147, we demonstrate that CD147 hormetically regulates the generation of endostatin but has no effect on Tsp-1. Since endostatin is cleaved from collagen XVIII (Col18A), we applied different protease inhibitors and established that MMP-9 and proteasome 20S, but not cathepsins, are responsible for endostatin generation. MMP-9 and proteasome 20S collaborate to synergistically enhance endostatin generation, and in a non-cellular system, CD147 enhanced MMP-9 activity and hormetically regulated proteasome 20S activity. Serum samples obtained from RA patients and healthy controls mostly corroborated these findings, indicating clinical relevance. Cumulatively, these findings suggest that secreted CD147 mediates a possibly allosteric effect on MMP-9 and proteasome 20S activities and can serve as a switch that turns angiogenesis on or off, depending on its ambient concentrations in the microenvironment.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

A Single-Center Retrospective Analysis of Kaposi's Sarcoma: Is There a Relationship Between Emmprin/CD147 Expression and Biological Behavior?

OBJECTIVES: Emmprin (CD147/BSG) protein is estimated to play a key role in cell migration and chemoresistance in viral carcinogenesis. However, there are very limited studies investigating the CD147 in the oncogenesis of Kaposi's sarcoma-associated herpesvirus. This study aims to reveal the relationship between CD147 expression with histopathological parameters, disease pattern, and recurrence in Kaposi's sarcoma (KS). METHODS: The study included 67 patients diagnosed with KS between January 1982 and September 2023. Clinical and histopathological features were analyzed retrospectively. HHV-8, CD31, and CD147 expressions were evaluated by immunohistochemistry. RESULTS: Sixteen (24%) female and 51 (76%) male patients with median age of 64 (10-86) were included in the study. CD147 was positive in 57 (85%) cases and associated with nodular pattern (P = .001), presence of solid/fibrosarcomatous area (P = .005), and high mitotic activity (P = .035). The disease relapsed in 17 (27%) of the 63 patients with median 2 (0-12) years follow-up. While a 5-year relapse-free survival was 48.5% in the CD147 diffuse positive group, it was 83.4% in focal positive and 100% in negative cases (P = .029). CONCLUSION: Our study exhibited the relationship between CD147 overexpression and recurrence in KS, but the inhomogeneity of the treatment groups and the small number of patients should also be considered. These findings may provide insight into the pathogenesis of KS and the development of targeted therapies in the future.

Intervention with extracellular matrix metalloproteinase inducer in osteoclasts attenuates periodontitis-induced bone resorption.

Extracellular matrix metalloproteinase inducer (EMMPRIN) plays critical roles in the regulation of inflammation and bone metabolism. The roles of EMMPRIN signaling in osteoclasts are worthy of deep study. The present study aimed to investigate bone resorption in periodontitis through the intervention of EMMPRIN signaling. The distribution of EMMPRIN in human periodontitis was observed. RANKL-induced osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) were treated with EMMPRIN inhibitor in vitro. Rats with ligation-induced periodontitis were treated with EMMPRIN inhibitor and harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, and double immunofluorescence analysis. Positive expressions of EMMPRIN could be found in the CD68+-infiltrating cells. Downregulated EMMPRIN restrained osteoclast differentiation of BMMs in vitro, which also inhibited MMP-9 expression (*P < 0.05). In vivo, EMMPRIN inhibitor restrained ligation-induced bone resorption by decreasing tartrate-resistant acid phosphatase-positive osteoclasts. Both EMMPRIN-positive and MMP-9-positive osteoclasts were less common in the EMMPRIN inhibitor groups than in the control groups. Intervention of EMMPRIN signaling in osteoclasts could probably provide a potential therapeutic target for attenuating ligation-induced bone resorption.

Extracellular matrix metalloproteinases inducer gene polymorphism and reduced serum matrix metalloprotease-2 activity in preeclampsia patients.

Preeclampsia increases the risk of pregnancy-related complications, nevertheless a successful spiral vessel remodeling, and trophoblast invasion reduces disorders of pregnancy. Matrix metalloproteinase-2 (MMP-2) clears the path for trophoblast invasion, and activation of MMP-2 largely depends on extracellular matrix metalloproteinases inducer (EMMPRIN) protein. This study aimed to investigate EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia patients. Archival whole blood and serum samples of 74 preeclampsia and 66 normotensive pregnant women age-matched were used in this case-control study. Genomic DNA was extracted from the whole blood samples and EMMPRIN gene amplified with specific primers following fragments sequence mutation analysis. Serum MMP-2 activity was determined using enzyme-linked immunosorbent assay (ELISA) and socio-demographic data of participants retrieved from the database. Age of preeclampsia patients (32.78 ± 6.39) years and body mass index (BMI) (33.09 ± 7.27) kg/m2 compared with the normotensive counterparts (32.33 ± 5.56) years and (32.33 ± 5.56) kg/m2,respectively, were not statistically significant (P > 0.05). Serum matrix metalloprotease-2 (MMP-2) activity was significantly reduced in preeclampsia group (16.34 ± 7.07) compared with the normotensives (25.63 ± 4.56) (P < 0.001), and rs424243T/G variant (55.6%) was overrepresented among the cases compared with the normotensives (16.7%). The single-nucleotide polymorphism T/G was found to be associated with preeclampsia (odds ratio [OR] = 7.63; 95% confidence interval [CI] = 3.95-14.75; P < 0.0001). Decreased activity of MMP-2 and rs424243T/G SNP of EMMPRIN gene was reported in preeclampsia. These preliminary data warrant a further investigation into the relationship between EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia.

CD147: an integral and potential molecule to abrogate hallmarks of cancer.

CD147 also known as EMMPRIN, basigin, and HAb18G, is a single-chain type I transmembrane protein shown to be overexpressed in aggressive human cancers of CNS, head and neck, breasts, lungs, gastrointestinal, genitourinary, skin, hematological, and musculoskeletal. In these malignancies, the molecule is integral to the diverse but complimentary hallmarks of cancer: it is pivotal in cancerous proliferative signaling, growth propagation, cellular survival, replicative immortality, angiogenesis, metabolic reprogramming, immune evasion, invasion, and metastasis. CD147 also has regulatory functions in cancer-enabling characteristics such as DNA damage response (DDR) and immune evasion. These neoplastic functions of CD147 are executed through numerous and sometimes overlapping molecular pathways: it transduces signals from upstream molecules or ligands such as cyclophilin A (CyPA), CD98, and S100A9; activates a repertoire of downstream molecules and pathways including matrix metalloproteinases (MMPs)-2,3,9, hypoxia-inducible factors (HIF)-1/2α, PI3K/Akt/mTOR/HIF-1α, and ATM/ATR/p53; and also functions as an indispensable chaperone or regulator to monocarboxylate, fatty acid, and amino acid transporters. Interestingly, induced loss of functions to CD147 prevents and reverses the acquired hallmarks of cancer in neoplastic diseases. Silencing of Cd147 also alleviates known resistance to chemoradiotherapy exhibited by malignant tumors like carcinomas of the breast, lung, pancreas, liver, gastric, colon, ovary, cervix, prostate, urinary bladder, glioblastoma, and melanoma. Targeting CD147 antigen in chimeric and induced-chimeric antigen T cell or antibody therapies is also shown to be safer and more effective. Moreover, incorporating anti-CD147 monoclonal antibodies in chemoradiotherapy, oncolytic viral therapy, and oncolytic virus-based-gene therapies increases effectiveness and reduces on and off-target toxicity. This study advocates the expedition and expansion by further exploiting the evidence acquired from the experimental studies that modulate CD147 functions in hallmarks of cancer and cancer-enabling features and strive to translate them into clinical practice to alleviate the emergency and propagation of cancer, as well as the associated clinical and social consequences.

Combined Biomarker System Predicts Prognosis in Patients with Metastatic Oral Squamous Cell Carcinoma.

BACKGROUND: Metastatic oral squamous cell carcinoma (OSCC) is associated with poor patient prognosis. Metastasis is a complex process involving various proteins, tumor cell alterations, including changes attributable to the epithelial-to-mesenchymal transition (EMT) process, and interactions with the tumor microenvironment (TME). In this study, we investigate a combined protein marker system consisting of connexin 43 (Cx43), EMMPRIN (CD147), E-cadherin, and vimentin, with a focus on their roles in the invasive metastatic progression of OSCC and their potential utility in predicting prognosis. METHODS: We conducted an immunohistochemical analysis to assess the protein expression profiles of Cx43, EMMPRIN, E-cadherin, and vimentin using tissue samples obtained from 24 OSCC patients. The metastatic process was mapped through different regions of interest (ROIs), including adjacent healthy oral mucosa (OM), center of primary OSCC, invasive front (IF), and local cervical lymph node metastases (LNM). The primary clinical endpoints were disease-free survival (DFS) and overall survival (OS). RESULTS: Substantial changes in the expression profiles of the different marker proteins were observed among the different ROIs, with all p-values < 0.05, signifying statistical significance. Multivariable Cox regression analysis results showed a significant effect of increased EMMPRIN expression toward the IF on DFS (p = 0.019) and OS (p = 0.023). Furthermore, the combined predictive analysis showed a significant predictive value of the marker system for DFS (p = 0.0017) and OS (p = 0.00044). CONCLUSIONS: The combined marker system exhibited a significant ability to predict patient prognosis. An increase in EMMPRIN expression toward the IF showed the strongest effect and could be an interesting new antimetastatic therapy approach.

S-nitrosylation of EMMPRIN influences the migration of HSCs and MMP activity in liver fibrosis.

The mechanism of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of liver fibrosis has not been clarified. This study aims to investigate the role of EMMPRIN S-nitrosylation (SNO) in the regulation of hepatic stellate cell (HSC) migration and matrix metalloproteinase (MMP) activities in liver fibrosis. The results from the tissue microarrays and rat/mouse liver tissues suggest that EMMPRIN mRNA and protein levels in the fibrotic livers are lower than those in the corresponding normal control livers, but higher SNO level of EMMPRIN in fibrotic liver area was shown by immunohistochemistry, immunofluorescence staining, and biotin-switch assay conversely in vivo. Primary EMMPRIN comes from hepatocytes and liver sinus epithelial cells (LSECs) rather than quiescent HSCs. To mimic the uptake of extrinsic EMMPRIN, supernatants from mouse primary hepatocytes/293 cells transfected with EMMPRIN wild-type plasmids (WT) and EMMPRIN SNO site (cysteine 87) mutation plasmids (MUT) were collected and added to JS-1/LX2 cell medium. The MUT EMMPRIN diminishes SNO successfully, enhances the activities of MMP2 and MMP9, and subsequently increases HSC migration. In conclusion, SNO of EMMPRIN influences HSC migration and MMP activities in liver fibrosis. This finding may shed light on the possible regulatory mechanism of MMPs in ECM accumulation in liver fibrosis.

MMP-2 Regulation of Emmprin on Tumour Cells and CD73 on Fibroblasts During Tumour-Stromal Interaction.

BACKGROUND/AIM: We previously found that binding between CD73 and extracellular matrix metalloproteinase (MMP) inducer (emmprin) and suppression of CD73 in both tumour cells and fibroblasts suppressed MMP-2 production when co-cultured. However, the importance of CD73 expression in either fibroblasts or cancer cells for cancer invasion remains unknown. In this study, we used siRNA to separately down-regulate CD73 in individual cells, and then performed a 3D co-culture to investigate tumour invasion. MATERIALS AND METHODS: siRNA was used for suppression of CD73 in either fibroblasts (ST353i, HDF) or tumour cells (FU-EPS-1, A431, CRL-2095). Immunoblotting was performed for detecting MMP-2 production after CD73 suppression. 3D-co-cultures were performed for examining tumour invasion. RESULTS: CD73 suppression revealed that CD73 expression on fibroblasts and emmprin on tumour cells were important in regulating MMP-2 production, suggesting that emmprin on tumour cells does not bind CD73 at the cis-manner, but rather at the trans-manner to CD73 present on fibroblasts. CD73 suppression also reduced MMP-2 production at the transcription level and reduced tumour invasion. CONCLUSION: CD73 on fibroblasts acts as a receptor for emmprin, which forms a complex that increases MMP-2 production, possibly resulting in increased invasiveness.

Comprehensive analysis of glycoprotein VI-mediated platelet activation signaling pathway for predicting pan-cancer survival and response to anti-PD-1 immunotherapy.

Platelets play a vital role in cancer and immunity. However, few comprehensive studies have been conducted on the role of platelet-related signaling pathways in various cancers and their responses to immune checkpoint blockade (ICB) therapy. In the present study, we focused on the glycoprotein VI-mediated platelet activation (GMPA) signaling pathway and comprehensively evaluated its roles in 19 types of cancers listed in The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Cox regression and meta-analyses showed that for all 19 types of cancers, patients with high GMPA scores tended to have a good prognosis. Furthermore, the GMPA signature score could serve as an independent prognostic factor for patients with skin cutaneous melanoma (SKCM). The GMPA signature was linked to tumor immunity in all 19 types of cancers, and was correlated with SKCM tumor histology. Compared to other signature scores, the GMPA signature scores for on-treatment samples were more robust predictors of the response to anti-PD-1 blockade in metastatic melanoma. Moreover, the GMPA signature scores were significantly negatively correlated with EMMPRIN (CD147) and positively correlated with CD40LG expression at the transcriptomic level in most cancer patient samples from the TCGA cohort and on-treatment samples from anti-PD1 therapy cohorts. The results of this study provide an important theoretical basis for the use of GMPA signatures, as well as GPVI-EMMPRIN and GPVI-CD40LG pathways, to predict the responses of cancer patients to various types of ICB therapy.

Extracellular Vesicles Potentiate Medulloblastoma Metastasis in an EMMPRIN and MMP-2 Dependent Manner.

Extracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.

Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

Metastasis in colorectal cancer is responsible for most of the cancer-related deaths. For metastasis to occur, tumor cells must first undergo the epithelial-to-mesenchymal transition (EMT), which is driven by the transcription factors (EMT-TFs) Snail, Slug twist1, or Zeb1, to promote their migration. In the distant organs, tumor cells may become dormant for years, until signals from their microenvironment trigger and promote their outgrowth. Here we asked whether CD147/EMMPRIN controls entry and exit from dormancy in the aggressive and proliferative (i.e., non-dormant) CT26 mouse colon carcinoma cells, in its wild-type form (CT26-WT cells). To this end, we knocked down EMMPRIN expression in CT26 cells (CT26-KD), and compared their EMT and cellular dormancy status (e.g., proliferation, pERK/pP38 ratio, vimentin expression, expression of EMT-TFs and dormancy markers), and angiogenic dormancy (e.g., VEGF and MMP-9 secretion, healing of the wounded bEND3 mouse endothelial cells), to the parental cells (CT26-WT). We show that knocking-down EMMPRIN expression reduced the pERK/pP38 ratio, enhanced the expression of vimentin, the EMT-TFs and the dormancy markers, and reduced the proliferation and angiogenic potential, cumulatively indicating that cells were pushed towards dormancy. When macrophages were co-cultured with both types of CT26 cells, the CT26-WT cells increased their angiogenic potential, but did not change their proliferation, state of EMT, or dormancy, whereas the CT26-KD cells exhibited values mostly similar to those of the co-cultured CT26-WT cells. Addition of recombinant TGFß or EMMPRIN that simulated the presence of macrophages yielded similar results. Combinations of low concentrations of TGFß and EMMPRIN had a minimal additive effect only in the CT26-KD cells, suggesting that they work along the same signaling pathway. We conclude that EMMPRIN is important as a gatekeeper that prevents cells from entering a dormant state, and that macrophages can promote an exit from dormancy.

Elevated EMMPRIN Serum Levels Indicate Plaque Vulnerability in Patients With Asymptomatic High Grade Carotid Stenosis.

OBJECTIVE: Carotid atherosclerosis is an important cause of cerebral ischaemic stroke. Sonographic plaque characteristics are inappropriate for exact prediction of possible future ischaemic events. Additional markers are needed to predict the clinical outcome in high grade carotid stenosis. This study aimed to test extracellular matrix metalloproteinase inducer (EMMPRIN), due to its involvement in plaque formation and destabilisation, as a potential marker of high risk vulnerable plaques. METHODS: EMMPRIN was analysed in pre-operative serum samples from patients with symptomatic and asymptomatic carotid artery stenosis by a specific ELISA. Pre-operative duplex sonography classified the atherosclerotic plaque due to echogenicity. Histopathological analysis of vulnerable and non-vulnerable plaques was based on the American Heart Association (AHA) classification. RESULTS: The study included 265 patients undergoing carotid endarterectomy: 90 (m:f, 69:21) patients with symptomatic and 175 (m:f, 118:57) with asymptomatic disease. Analysis of circulating EMMPRIN revealed significantly higher levels in patients with echolucent plaques (4 480; IQR 3 745, 6 144 pg/mL) compared with echogenic plaques (4 159; IQR 3 418, 5 402 pg/mL; p = .025). Asymptomatic patients with vulnerable plaques had significantly higher levels of EMMPRIN (4 875; IQR 3 850, 7 016 pg/mL) compared with non-vulnerable plaques (4 109; IQR 3 433, 5 402 pg/mL; p < .001). In logistic regression analysis, duplex sonography combined with age, gender, and clinical risk factors predicted vulnerable plaques in asymptomatic patients with an AUC of 0.71 (95% CI 0.61 - 0.80). EMMPRIN significantly improved the AUC in asymptomatic patients (AUC 0.79; 95% CI 0.71 - 0.87; p = .014). CONCLUSION: Patients with high risk plaques according to ultrasound and histopathological characteristics demonstrated increased serum EMMPRIN levels. EMMPRIN on top of clinical risk factors, including age, gender, and duplex sonography may be used for pre-operative risk stratification in asymptomatic patients.

CD147 and MMPs as key factors in physiological and pathological processes.

Cluster of differentiation 147 (CD147) or extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that induces the synthesis of matrix metalloproteinases (MMPs). MMPs, as zinc-dependent proteases and versatile enzymes, play critical roles in the degradation of the extracellular matrix (ECM) components, cleaving of the receptors of cellular surfaces, signaling molecules, and other precursor proteins, which may lead to attenuation or activation of such targets. CD147 and MMPs play essential roles in physiological and pathological conditions and any disorder in the expression, synthesis, or function of CD147 and MMPs may be associated with various types of disease. In this review, we have focused on the roles of CD147 and MMPs in some major physiological and pathological processes.