CD146 Promotes EMT-Mediated Migration and Invasion of NSCLC via PI3K/Akt Signaling Pathway.

BACKGROUND: Recurrence and metastasis are the main causes of non-small cell lung cancer (NSCLC)-related death. CD146 has been identified as a potential risk factor for poor prognosis, closely related to the distant metastasis and drug resistance in various cancers. However, the clinical significance of CD146 in NSCLC requires further investigation. MATERIALS AND METHODS: This study explored the correlation between CD146 expression and clinical variables using tumor tissue samples collected from our hospital. CD146 expression levels in NSCLC cell lines and tissues were assessed and compared using immunohistochemistry, real-time polymerase chain reaction (RT-qPCR), flow cytometry, and western blot analysis. The invasion and migration capabilities of tumor cells were determined using transwell and wound healing assays. The levels of proteins related to epithelial-mesenchymal transition (EMT) as well as the underlying PI3K/Akt signaling pathway was measured by western blotting. RESULTS: We discovered that CD146 expression is significantly associated with the EMT signaling pathway. High CD146 expression predicted lymph node metastasis, metastasis to distant organs, advanced Tumor, Node, Metastasis (TNM) staging, and poor survival in NSCLC patients. Wound healing and transwell assays showed that knocking down CD146 significantly suppressed cell migration along with cell invasion in NSCLC, whereas overexpressing CD146 notably enhanced these processes. Western blot analysis revealed significantly reduced levels of N-cadherin, vimentin, snail, twist, PI3K, and AKT phosphorylation in shCD146 H460 cells compared to vector control cells. Treatment with PI3K inhibitor PI3K-IN-1 increased E-cadherin expression levels but reduced N-cadherin, Twist, Vimentin, PI3K, and AKT phosphorylation levels in pcDNA3.1-CD146 A549 cells compared with the vector control cells. CONCLUSIONS: CD146 expression acts as a prognostic risk factor for adverse outcomes in NSCLC, promoting invasion and metastasis by activating the EMT through the PI3K/Akt signaling pathway. These findings underscore the potential therapeutic strategies targeting CD146, offering new treatment options for NSCLC patients, especially those at risk of metastasis.

CD146 as a prognostic marker in breast cancer: A meta-analysis.

BACKGROUND: CD146, a cell adhesion molecule, was first discovered in melanoma. Since then, it has been established as a promoter of tumor progression and metastasis. Many recent clinical studies have associated CD146 overexpression with poor prognosis in various cancers. However, clinical relevance of CD146 in prognosis of breast cancer has been poorly studied. METHODS: We performed meta-analysis of data of all clinical studies associated with the prognostic value of CD146 expression in breast cancer. Relevant studies were retrieved from PubMed database as per the inclusion and exclusion criteria, data were extracted independently and carefully by two reviewers with the help of standardized form, and meta-analysis was performed to correlate CD146 expression with molecular subtypes, lymph node metastasis, and overall survival in breast cancer. RESULTS: Our findings suggest that CD146 expression is predominantly found in triple-negative breast cancer subtype (pooled odds ratio = 2.98, 95% confidence interval [CI] =2.19-4.05, P < .00001) and breast tumors overexpressing CD146 have a higher risk of lymph node metastasis (pooled relative risk = 1.64, 95% CI = 1.44-1.87, P < .00001). Furthermore, high expression of CD146 was associated with poor prognosis in breast cancer (pooled hazard ratio = 1.51, 95% CI = 1.21-1.87, P = .0002). CONCLUSION: Overall results suggested that CD146 may be a potential prognostic marker to predict metastatic potential and disease outcomes in breast cancer and can be used as a therapeutic target.

CD146/MCAM links doxorubicin-induced epigenetic dysregulation to the impaired fatty acid transportation in H9c2 cardiomyoblasts.

CD146/MCAM has garnered significant attention for its potential contribution to cardiovascular disease; however, the transcriptional regulation and functions remain unclear. To explore these processes regarding cardiomyopathy, we employed doxorubicin, a widely used stressor for cardiomyocytes. Our in vitro study on H9c2 cardiomyoblasts highlights that, besides impairing the fatty acid uptake in the cells, doxorubicin suppressed the expression of fatty acid binding protein 4 (Fabp4) along with the histone deacetylase 9 (Hdac9), bromodomain and extra-terminal domain proteins (BETs: Brd2 and Brd4), while augmented the production of CD146/MCAM. Silencing and chemical inhibition of Hdac9 further augmented CD146/MCAM and deteriorated fatty acid uptake. In contrast, chemical inhibition of BETs as well as silencing of MCAM/CD146 ameliorated fatty acid uptake. Moreover, protein kinase C (PKC) inhibition abrogated CD146/MCAM, particularly in the nucleus. Taken together, our results suggest that epigenetic dysregulation of Hdac9, Brd2, and Brd4 alters CD146/MCAM expression, deteriorating fatty acid uptake by downregulating Fabp4. This process depends on the PKC-mediated nuclear translocation of CD146. Thus, this study highlights a pivotal role of CD146/MCAM in doxorubicin-induced cardiomyopathy.

CD146 deficiency aggravates chronic obstructive pulmonary disease via the increased production of S100A9 and MMP-9 in macrophages.

Chronic obstructive pulmonary disease (COPD) is a leading cause of global death. As a molecule beyond adhesion, CD146 is involved in COPD pathogenesis. However, the mechanisms of CD146 in COPD remain largely elusive. We hypothesized that CD146 regulates the production of matrix metalloproteinase-9 (MMP-9) in macrophages and thereby contributes to COPD. Here, we constructed a murine model of COPD using lipopolysaccharide (LPS) and porcine pancreatic elastase (PPE). In COPD-like mice, LPS and PPE decreased the pulmonary expression of CD146. MMP-9 expression and bioactivity were increased in CD146 knockout COPD-like mice. In vitro, LPS decreased CD146 expression in macrophages. With or without LPS challenge, CD146-defective macrophages produced more MMP-9. Transcriptome analysis based on next-generation sequencing (NGS) revealed that S100A9 regulated MMP-9 production in CD146-defective macrophages. Targeting S100A9 with paquinimod decreased lung inflammation and alleviated alveolar destruction in COPD-like mice. Collectively, our study suggests that CD146 negatively regulates MMP-9 production in macrophages via the S100A9 pathway in COPD.

Investigating changes in the post-transcriptional pattern of VEGF and CD146 genes carrying miR-573 in breast cancer cells treated with tamoxifen.

BACKGROUND: Recent developments regarding the contribution of microRNAs (miRNAs) to tumor angiogenesis and the oncogenic effects of miRNAs point to their potential role in breast cancer angiogenesis. Tumor-derived exosomes are considered a rich source of miRNAs that can regulate the function of other cells in the tumor microenvironment, including vascular endothelial cells. This study analyzes the effect of tamoxifen chemotherapy on the expression of a key miRNA, miR-573, involved in the angiogenesis of the tumor exosomes and introduces a regulatory link between this miRNA and the CD146 gene associated with the vascular endothelial growth factor (VEGF) messaging pathway. MATERIALS AND METHODS: MCF-7 breast cancer cells were purchased and cultured in a complete culture medium. These cells were treated with tamoxifen and then their exosomes were extracted from the culture medium. The RNAs of the exosomes were isolated and the expression of miR-573, VEGF, and CD146 genes in the exosomes was investigated using the real-time polymerase chain reaction (PCR) method. RESULTS: The results of this study showed that tamoxifen treatment increased the expression of miR-573 in exosomes derived from MCF-7 cancer cells. The expression of CD146 and VEGF genes in drug-treated cell exosomes had a downward pattern. CONCLUSION: The results of this experiment demonstrated that the treatment of breast cancer cells with tamoxifen reduces the expression of VEGF and CD146 by increasing miR-573. Thus, angiogenesis is reduced and, therefore, its anti-tumor effects are applied.

CD146 promotes malignant progression of breast phyllodes tumor through suppressing DCBLD2 degradation and activating the AKT pathway.

BACKGROUND: As a rapid-progressing tumor, breast malignant phyllodes tumors (PTs) are challenged by the lack of effective therapeutic strategies and suitable prognostic markers. This study aimed to clarify the role and mechanism of CD146 on promoting PTs malignant progression, and to identify a novel prognosis marker and treatment target of breast malignant PTs. METHODS: The expression and prognostic significance of CD146 in PTs was detected through single-cell RNA-sequencing (scRNA-seq), immunostaining, real-time PCR and other methodologies. Functional experiments including proliferation assay, colony formation assay, transwell assay, and collagen contraction assay were conducted to validate the role of CD146 in malignant progression of PTs. The efficacy of anti-CD146 monoclonal antibody AA98 against malignant PTs was corroborated by a malignant PT organoid model and a PT patient-derived xenograft (PDX) model. Transcriptome sequencing, proteomic analysis, co-immunoprecipitation, and pull-down assay was employed to identify the modulating pathway and additional molecular mechanism. RESULTS: In this study, the scRNA-seq analysis of PTs disclosed a CD146-positive characteristic in the α-SMA+ fibroblast subset. Furthermore, a progressive elevation in the level of CD146 was observed with the malignant progression of PTs. More importantly, CD146 was found to serve as an independent predictor for recurrence in PT patients. Furthermore, CD146 was found to augment the viability and invasion of PTs. Mechanistically, CD146 acted as a protective "shield" to prevent the degradation of Discoidin, CUB, and LCCL domain-containing protein 2 (DCBLD2), thereby activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and enhancing malignant behaviors of PT cells. In the malignant PT organoid and PDX model, a significant suppression of malignant PT growth was observed after the application of AA98. CONCLUSIONS: These findings suggested that CD146 served as an efficacious marker for predicting PT malignant progression and showed promise as a prognosis marker and treatment target of breast malignant PTs. The study further unveiled the essential role of the CD146-DCBLD2/PI3K/AKT axis in the malignant progression of PTs.

CD146+ Endometrial-Derived Mesenchymal Stem/Stromal Cell Subpopulation Possesses Exosomal Secretomes with Strong Immunomodulatory miRNA Attributes.

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1ß, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.

METCAM/MUC18 Plays a Tumor Suppressor Role in the Development of Nasopharyngeal Carcinoma Type I.

From previous studies of negatively correlating the expression of human METCAM/MUC18 with the pathology of nasopharyngeal carcinoma (NPC), we have suggested that human METCAM/MUC18 (huMETCAM/MUC18) might play a tumor suppressor role in the development of nasopharyngeal carcinoma. To scrutinize this hypothesis, we investigated the effects of huMETCAM/MUC18's over-expression on in vitro cellular behavior and on the in vivo tumorigenesis of one NPC cell line (NPC-TW01). HuMETCAM/MUC18 cDNA was first transfected into the NPC-TW01 cell line, which was established from NPC type I, and many G418-resistant clones were obtained. Then, two NPC-TW01 clones, which expressed high and medium levels of huMETCAM/MUC18, respectively, and one empty vector (control) clone were used to test the effects of huMETCAM/MUC18's over-expression on in vitro behaviors and on in vivo tumorigenesis (via subcutaneous injection) in athymic nude mice (Balb/cAnN.Cg-Foxnlnu/Cr1Nar1). The time course of tumor proliferation and the final tumor weights were determined. Tumor sections were used for the histology and immunohistochemistry (IHC) studies. Tumor lysates were used for determining the expression levels of huMETCAM/MUC18 and various downstream key effectors. HuMETCAM/MUC18's over-expression reduced in vitro motility and invasiveness and altered growth behaviors in 3D basement membrane culture assays, and it decreased the in vivo tumorigenicity of the NPC-TW01 cells. The tumor cells from a high-expressing clone were clustered and confined in small areas, whereas those from a vector control clone were more spread out, suggesting that the tumor cells from the high-expressing clone appeared to stay dormant in micro-clusters. Expression levels of the proliferation index, an index of the metabolic switch to aerobic glycolysis, angiogenesis indexes, and survival pathway indexes were reduced, whereas the pro-apoptosis index increased in the corresponding tumors. The over-expression of huMETCAM/MUC18 in the NPC-TW01 cells decreased the epithelial-to-mesenchymal transition and the in vitro and in vitro tumorigenesis, suggesting that it plays a tumor suppressor role in the development of type I NPC, perhaps by increasing apoptosis and decreasing angiogenesis, proliferation, and the metabolic switch to aerobic glycolysis.

Full spectrum flow cytometry reveals mesenchymal heterogeneity in first trimester placentae and phenotypic convergence in culture, providing insight into the origins of placental mesenchymal stromal cells.

Single-cell technologies (RNA-sequencing, flow cytometry) are critical tools to reveal how cell heterogeneity impacts developmental pathways. The placenta is a fetal exchange organ, containing a heterogeneous mix of mesenchymal cells (fibroblasts, myofibroblasts, perivascular, and progenitor cells). Placental mesenchymal stromal cells (pMSC) are also routinely isolated, for therapeutic and research purposes. However, our understanding of the diverse phenotypes of placental mesenchymal lineages, and their relationships remain unclear. We designed a 23-colour flow cytometry panel to assess mesenchymal heterogeneity in first-trimester human placentae. Four distinct mesenchymal subsets were identified; CD73+CD90+ mesenchymal cells, CD146+CD271+ perivascular cells, podoplanin+CD36+ stromal cells, and CD26+CD90+ myofibroblasts. CD73+CD90+ and podoplanin + CD36+ cells expressed markers consistent with cultured pMSCs, and were explored further. Despite their distinct ex-vivo phenotype, in culture CD73+CD90+ cells and podoplanin+CD36+ cells underwent phenotypic convergence, losing CD271 or CD36 expression respectively, and homogenously exhibiting a basic MSC phenotype (CD73+CD90+CD31-CD144-CD45-). However, some markers (CD26, CD146) were not impacted, or differentially impacted by culture in different populations. Comparisons of cultured phenotypes to pMSCs further suggested cultured pMSCs originate from podoplanin+CD36+ cells. This highlights the importance of detailed cell phenotyping to optimise therapeutic capacity, and ensure use of relevant cells in functional assays.

Knockdown of CD146 promotes endothelial-to-mesenchymal transition via Wnt/ß-catenin pathway.

PURPOSE: Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins and leads to the maladaptive changes in myocardium. Endothelial cells (ECs) undergoing mesenchymal transition contributes to the occurrence and development of cardiac fibrosis. CD146 is an adhesion molecule highly expressed in ECs. The present study was performed to explore the role of CD146 in modulating endothelial to mesenchymal transition (EndMT). METHODS: C57BL/6 mice were subjected to subcutaneous implantation of osmotic minipump infused with angiotensin II (Ang Ⅱ). Adenovirus carrying CD146 short hairpin RNA (shRNA) or CD146 encoding sequence were infected into cultured human umbilical vein endothelial cells (HUVECs) followed by stimulation with Ang II or transforming growth factor-ß1 (TGF-ß1). Differentially expressed genes were revealed by RNA-sequencing (RNA-Seq) analysis. Gene expression was measured by quantitative real-time PCR, and protein expression and distribution were determined by Western blot and immunofluorescence staining, respectively. RESULTS: CD146 was predominantly expressed by ECs in normal mouse hearts. CD146 was upregulated in ECs but not fibroblasts and myocytes in hearts of Ang II-infused mice and in HUVECs stimulated with Ang Ⅱ. RNA-Seq analysis revealed the differentially expressed genes related to EndMT and Wnt/ß-catenin signaling pathway. CD146 knockdown and overexpression facilitated and attenuated, respectively, EndMT induced by Ang II or TGF-ß1. CD146 knockdown upregulated Wnt pathway-related genes including Wnt4, LEF1, HNF4A, FOXA1, SOX6, and CCND3, and increased the protein level and nuclear translocation of ß-catenin. CONCLUSIONS: Knockdown of CD146 exerts promotional effects on EndMT via activating Wnt/ß-catenin pathway and the upregulation of CD146 might play a protective role against EndMT and cardiac fibrosis.

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling.

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

Comparison of CD146 +/- mesenchymal stem cells in improving premature ovarian failure.

BACKGROUND: Mesenchymal stem cells (MSCs) are a heterogeneous group of subpopulations with differentially expressed surface markers. CD146 + MSCs correlate with high therapeutic and secretory potency. However, their therapeutic efficacy and mechanisms in premature ovarian failure (POF) have not been explored. METHODS: The umbilical cord (UC)-derived CD146 +/- MSCs were sorted using magnetic beads. The proliferation of MSCs was assayed by dye670 staining and flow cytometry. A mouse POF model was established by injection of cyclophosphamide and busulfan, followed by treatment with CD146 +/- MSCs. The therapeutic effect of CD146 +/- MSCs was evaluated based on body weight, hormone levels, follicle count and reproductive ability. Differential gene expression was identified by mRNA sequencing and validated by RT-PCR. The lymphocyte percentage was detected by flow cytometry. RESULTS: CD146 +/- MSCs had similar morphology and surface marker expression. However, CD146 + MSCs exhibited a significantly stronger proliferation ability. Gene profiles revealed that CD146 + MSCs had a lower levels of immunoregulatory factor expression. CD146 + MSCs exhibited a stronger ability to inhibit T cell proliferation. CD146 +/- MSCs treatment markedly restored FSH and E2 hormone secretion level, reduced follicular atresia, and increased sinus follicle numbers in a mouse POF model. The recovery function of CD146 + MSCs in a reproductive assay was slightly improved than that of CD146 - MSCs. Ovary mRNA sequencing data indicated that UC-MSCs therapy improved ovarian endocrine locally, which was through PPAR and cholesterol metabolism pathways. The percentages of CD3, CD4, and CD8 lymphocytes were significantly reduced in the POF group compared to the control group. CD146 + MSCs treatment significantly reversed the changes in lymphocyte percentages. Meanwhile, CD146 - MSCs could not improve the decrease in CD4/8 ratio induced by chemotherapy. CONCLUSION: UC-MSCs therapy improved premature ovarian failure significantly. CD146 +/- MSCs both had similar therapeutic effects in repairing reproductive ability. CD146 + MSCs had advantages in modulating immunology and cell proliferation characteristics.

CD146 at the Interface between Oxidative Stress and the Wnt Signaling Pathway in Systemic Sclerosis.

CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

Overexpression of MCAM induced by SMYD2-H3K36me2 in breast cancer stem cell properties.

BACKGROUND: Melanoma cell adhesion molecule (MCAM) is highly expressed in various malignancies. However, studies on the effects of MCAM on stemness of cancer stem cells are limited. Here, we aimed to explore the relationship between MCAM and stem cell phenotype in breast cancer (BC). METHODS: We analyzed the genes differentially expressed in BC from the oncomine database, followed by TCGA-BRCA database validation. We then used gene set enrichment analysis to analyze the signaling pathways enriched to the relevant genes, followed by loss-of-function experiments to analyze the role of MCAM in the growth of BC cells and the maintenance of stem cell properties. We analyzed the cause for the MCAM overexpression using ChIP-seq and clarified the upstream mechanism by constructing SE-Deleted cells. Finally, the role of SMYD2 in the growth of BC cells and the maintenance of stem cell properties were verified by rescue experiments. RESULTS: MCAM was significantly overexpressed in BC, which predicted somber prognosis in patients. Knockdown of MCAM drastically hindered the growth and metastasis of BC cells in vitro and in vivo. Subsequently, the MCAM promoter was observed to have significant H3K36me2 modification and that SMYD2 could significantly promote the expression of MCAM. In addition, further overexpression of SMYD2 in cells with MCAM knockdown increased MCAM expression and promoted the growth as well as stemness of BC cells. CONCLUSION: SMYD2 can elevate the expression of MCAM by promoting its H3K36me2 modification, which in turn expedites the growth and stem cell properties of BC cells.

Comparison of the Angiogenic Ability between SHED and DPSC in a Mice Model with Critical Limb Ischemic.

BACKGROUND: Regenerative medicine by using stem cells from dental pulp is promising for treating patients with critical limb ischemic (CLI). Here, we investigated the difference in the angiogenetic ability of stem cells from human exfoliated deciduous teeth (SHED) and human dental pulp stem cells (DPSC). METHODS: SHED and DPSC were harvested from dental pulp and analyzed in flow- cytometry for detecting the expression of surface markers. Levels of angiogenetic marker were examined by RT-PCR and Western-blot. Eighteen immunodeficient mice of critical limb ischemic model were divided into three groups: SHED, DPSC and saline, which was administered with SHED, DPSC or saline intramuscularly. Histological examination was performed to detect the regenerative results. RESULTS: A highly expression of CD146 was detected in SHED. Moreover, cells with negative expression of both CD146 and CD31 in SHED were more in comparison with those in DPSC. Expression of angiogenesis factors including CXCL12, CXCR4, Hif-1a, CD31, VEGF and bFGF were significant higher in SHED than DPSC by the RT-PCR and Western-Blot results. SHED induced more CD31 expression and less fibrous tissue formation in the critical limb ischemic model as compare with DPSC and saline. CONCLUSION: Both SHED and DPSC possessed the ability of repairing CLI. With expressing more proangiogenesis factors, SHED may have the advantage of repairing CLI.

The CD146-HIF-1α axis regulates epithelial cell migration and alveolar maturation in a mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common challenge in preterm neonates. Retardation of alveolar development characterizes the pulmonary pathology in BPD. In the present study, we explored the roles of the CD146-HIF-1α axis in BPD. We demonstrated that the levels of reactive oxygen species (ROS) and soluble CD146 (sCD1146) were increased in the peripheral blood of preterm neonates with BPD. In alveolar epithelial cells, hyperoxia promoted the expression of HIF-1α and CD146, which reinforced each other. In a mouse model of BPD, by exposing pups to 65% hyperoxia, HIF-1α and CD146 were increased in the pulmonary tissues. Mechanistically, CD146 hindered the migration of alveolar epithelial cells; in contrast, movement was significantly enhanced in CD146-knockout alveolar epithelial cells. As expected, CD146-knockout ameliorated alveolarization and improved BPD disease severity. Taken together, our findings imply that the CD146-HIF-1α axis contributes to alveolarization and that CD146 may be a novel candidate in BPD therapy.

Reversing the Epithelial-Mesenchymal Transition in Metastatic Cancer Cells Using CD146-Targeted Black Phosphorus Nanosheets and a Mild Photothermal Treatment.

Cancer metastasis leads to most deaths in cancer patients, and the epithelial-mesenchymal transition (EMT) is the key mechanism that endows the cancer cells with strong migratory and invasive abilities. Here, we present a nanomaterial-based approach to reverse the EMT in cancer cells by targeting an EMT inducer, CD146, using engineered black phosphorus nanosheets (BPNSs) and a mild photothermal treatment. We demonstrate this approach can convert highly metastatic, mesenchymal-type breast cancer cells to an epithelial phenotype (i.e., reversing EMT), leading to a complete stoppage of cancer cell migration. By using advanced nanomechanical and super-resolution imaging, complemented by immunoblotting, we validate the phenotypic switch in the cancer cells, as evidenced by the altered actin organization and cell morphology, downregulation of mesenchymal protein markers, and upregulation of epithelial protein markers. We also elucidate the molecular mechanism behind the reversal of EMT. Our results reveal that CD146-targeted BPNSs and a mild photothermal treatment synergistically contribute to EMT reversal by downregulating membrane CD146 and perturbing its downstream EMT-related signaling pathways. Considering CD146 overexpression has been confirmed on the surface of a variety of metastatic, mesenchymal-like cancer cells, this approach could be applicable for treating various cancer metastasis via modulating the phenotype switch in cancer cells.

Involvement of Multiple Variants of Soluble CD146 in Systemic Sclerosis: Identification of a Novel Profibrotic Factor.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. METHODS: We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. RESULTS: Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/ß-catenin/WISP-1 pathway. CONCLUSION: Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

Soluble CD146 as a Potential Target for Preventing Triple Negative Breast Cancer MDA-MB-231 Cell Growth and Dissemination.

Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.