MCAM/MUC18/CD146 as a Multifaceted Warning Marker of Melanoma Progression in Liquid Biopsy.

Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide. Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease. MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146. We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression. Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression. Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series. For some patients, we also performed the time courses of molecular monitoring. Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment. We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular "warning" marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.

CD146 Regulates Growth Factor-Induced mTORC2 Activity Independent of the PI3K and mTORC1 Pathways.

The mechanistic target of rapamycin complex 2 (mTORC2) coordinates cell proliferation, survival, and metabolism with environmental inputs, yet how extracellular stimuli such as growth factors (GFs) activate mTORC2 remains enigmatic. Here we demonstrate that in human endothelial cells, activation of mTORC2 signaling by GFs is mediated by transmembrane cell adhesion protein CD146. Upon GF stimulation, the cytoplasmic tail of CD146 is phosphorylated, which permits its positively charged, juxtamembrane KKGK motif to interact with Rictor, the defining subunit of mTORC2. The formation of the CD146-Rictor/mTORC2 complex protects Rictor from ubiquitin-proteasome-mediated degradation, thereby specifically upregulating mTORC2 activity with no intervention of the PI3K and mTORC1 pathways. This CD146-mediated mTORC2 activation in response to GF stimulation promotes cell proliferation and survival. Therefore, our findings identify a molecular mechanism by which extracellular stimuli regulate mTORC2 activity, linking environmental cues with mTORC2 regulation.

CD146 interacts with galectin-3 to mediate endothelial cell migration.

Here, we investigated the role of the cell membrane protein CD146 in galectin-3-mediated endothelial cell migration at the molecular level. Our results show that knocking down CD146 significantly attenuates galectin-3-mediated cell migration. Pull-down assays, gel filtration, and biolayer interferometry further demonstrate that galectin-3 binds to the CD146 ectodomain (eFL) with a KD of ~1.1 µm. To identify the galectin-3-binding site, we used mass spectrometry to show that CD146 eFL has four N-glycosites, with PNGase F treatment indicating that N-glycans define the binding epitope. Galectin-3 likely interacts with Domain 5 on CD146 eFL, because it contains poly-N-acetyllactosamine sites, and deletion of this domain significantly reduces binding. Overall, our findings provide a better understanding of how galectin-3 interacts with cell membrane receptors to mediate endothelial cell migration.

ß-1,3-Galactosyl-O-Glycosyl-Glycoprotein ß-1,6-N-Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility.

We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) ß, that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of ß-1,3-galactosyl-O-glycosyl-glycoprotein ß-1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9-MCAM axis.

CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion.

BACKGROUND: We have previously validated three novel CD44-downstream positively regulated transcriptional targets, including Cortactin, Survivin and TGF-ß2, and further characterized the players underlying their separate signaling pathways. In the present study, we identified CD146 as a potential novel target, negatively regulated by CD44. While the exact function of CD146 in breast cancer (BC) is not completely understood, substantial evidence from our work and others support the hypothesis that CD146 is a suppressor of breast tumor progression. METHODS: Therefore, using molecular and pharmacological approaches both in vitro and in breast tissues of human samples, the present study validated CD146 as a novel target of CD44-signaling suppressed during BC progression. RESULTS: Our results revealed that CD44 activation could cause a substantial decrease of CD146 expression with an equally notable converse effect upon CD44-siRNA inhibition. More interestingly, activation of CD44 decreased cellular CD146 and increased soluble CD146 through CD44-dependent activation of MMP. CONCLUSION: Here, we provide a possible mechanism by which CD146 suppresses BC progression as a target of CD44-downstream signaling, regulating neovascularization and cancer cell motility.

Physical Binding of Endothelial MCAM and Neural Transmembrane Protease Matriptase-Novel Cell Adhesion in Neural Stem cell Vascular Niche.

Brain neural stem cells and transit amplifying cells in the subventricular zone (SVZ) of the lateral ventricles are in direct contact with the microvascular endothelium. The mechanisms/molecules of direct cell contact in the SVZ neurovascular niche are not fully understood. We previously showed that neural stem/progenitor (NS/P) cells induce brain endothelial signaling in direct cell contact through matriptase (MTP) on NS/P cell surface. In the present study, using pull-down and LC-MS/MS, we identified melanoma cell adhesion molecule (MCAM) the brain endothelial molecule that interacts with MTP. MCAM physically binds to the CUB domains of MTP and induces a chain of brain endothelial signaling including p38MAPK activation, GSK3ß inactivation and subsequently ß-catenin activation; none of these signaling events occurred when either MTP or MCAM is deleted. MTP-MCAM binding and induction of endothelial signaling were all sensitive to cholera toxin. Together, we identified key molecules that may represent a mechanism in neural stem cell vascular niche regulation.

The membrane protein melanoma cell adhesion molecule (MCAM) is a novel tumor marker that stimulates tumorigenesis in hepatocellular carcinoma.

Yes-associated protein (YAP) is overexpressed and has an oncogenic role in hepatocellular carcinoma (HCC). However, whether membrane protein can serve not only as a tumor marker that reflects YAP function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown. Here we report that the membrane protein melanoma cell adhesion molecule (MCAM) was under positive regulation by YAP and was highly elevated in HCC cells. Within the MCAM promoter, we found the presence of a cAMP Response Element (CRE; -32 to -25 nt), which is conserved among species and is essential for YAP- and CREB-dependent regulation. Moreover, the interaction between CREB and YAP at the CRE site was dependent on PTPIY-WW domain interactions. However, MCAM expression was low and could not be regulated by YAP in breast and colon cancer cells because of the low levels of the acetyltransferase p300. In HCC cells, high levels of p300 facilitated the binding of YAP to the MCAM promoter, which in turn enhanced histone acetylation and polymerase II recruitment through the dissociation of the deacetylase Sirt1. These results suggest that MCAM is an HCC-specific target of YAP. In clinical serum samples, we found that the serum levels of MCAM were highly elevated in patients with HCC compared with healthy controls and with patients with cirrhosis, hepatitis, colon cancer and breast cancer. MCAM levels were shown to be a slightly better indicator than serum alpha-fetoprotein for predicting HCC. We further demonstrated that MCAM is essential for the survival and transformation of HCC. Mechanistically, MCAM induced translation initiation and the transcriptional activities of c-Jun/c-Fos. In addition, AKT activation had an essential role in the MCAM-promoted binding of eukaryotic initiation factor 4E to c-Jun/c-Fos mRNA. In conclusion, we demonstrated that MCAM may be a potential tumor marker and therapeutic target for the diagnosis and treatment of HCC.

CD146, a multi-functional molecule beyond adhesion.

CD146 is a cell adhesion molecule (CAM) that is primarily expressed at the intercellular junction of endothelial cells. CD146 was originally identified as a tumor marker for melanoma (MCAM) due to its existence only in melanoma but not in the corresponding normal counterpart. However CD146 is not just a CAM for the inter-cellular and cell-matrix adhesion. Recent evidence indicates that CD146 is actively involved in miscellaneous processes, such as development, signaling transduction, cell migration, mesenchymal stem cells differentiation, angiogenesis and immune response. CD146 has increasingly become an important molecule, especially identified as a novel bio-marker for angiogenesis and for cancer. Here we have reviewed the dynamic research of CD146, particularly newly identified functions and the underlying mechanisms of CD146.

The involvement of CD146 and its novel ligand Galectin-1 in apoptotic regulation of endothelial cells.

CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.

Enrichment of mouse spermatogonial stem cells by melanoma cell adhesion molecule expression.

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis, but studies are hampered by their scarcity. Although the cryptorchid operation is often used to obtain an enriched SSC population, making cryptorchid testes is time-consuming and the technique is not applicable to many animal species. In the present study, we screened for a new surface antigen on SSCs using germline stem (GS) cells (i.e., cultured SSCs). Germ cell transplantation experiments showed that SSCs express melanoma cell adhesion molecule (MCAM), which belongs to the immunoglobulin superfamily and mediates cation-independent adhesion. Although MCAM overexpression in GS cells did not influence SSC colony formation frequency or subsequent spermatogenesis after transplantation, MCAM knockdown in GS cells by short-interfering RNA treatment reduced colony numbers, suggesting that MCAM plays a role in sustaining SSC potential. Multiparameter selection of wild-type adult testis cells with a CD9⁺EPCAM(low)MCAM⁺KIT⁻ phenotype resulted in a 561-fold enrichment of SSCs. Development of a new strategy for SSC enrichment from mature adult testes will facilitate analyses of SSCs in the normal testicular microenvironment.

CD146 is a coreceptor for VEGFR-2 in tumor angiogenesis.

CD146 is a novel endothelial biomarker and plays an essential role in angiogenesis; however, its role in the molecular mechanism underlying angiogenesis remains poorly understood. In the present study, we show that CD146 interacts directly with VEGFR-2 on endothelial cells and at the molecular level and identify the structural basis of CD146 binding to VEGFR-2. In addition, we show that CD146 is required in VEGF-induced VEGFR-2 phosphorylation, AKT/p38 MAPKs/NF-κB activation, and thus promotion of endothelial cell migration and microvascular formation. Furthermore, we show that anti-CD146 AA98 or CD146 siRNA abrogates all VEGFR-2 activation induced by VEGF. An in vivo angiogenesis assay showed that VEGF-promoted microvascular formation was impaired in the endothelial conditional knockout of CD146 (CD146(EC-KO)). Our animal experiments demonstrated that anti-CD146 (AA98) and anti-VEGF (bevacizumab) have an additive inhibitory effect on xenografted human pancreatic and melanoma tumors. The results of the present study suggest that CD146 is a new coreceptor for VEGFR-2 and is therefore a promising target for blocking tumor-related angiogenesis.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Endothelial CD146 is required for in vitro tumor-induced angiogenesis: the role of a disulfide bond in signaling and dimerization.

Tumor angiogenesis, induced by tumor-secreted pro-angiogenic factors, is an essential process for cancer development and metastasis. CD146 is identified as an endothelial cell adhesion molecule and implicated in blood vessel formation, however, its exact role in angiogenesis, particularly tumor angiogenesis, and its potential function of mediating downstream signaling are still unclear. In present study, we evidenced that silencing endogenous endothelial CD146 by RNAi significantly impaired hepatocarcinoma cell secretions-promoted tubular morphogenesis and -enhanced motility of endothelial cells. Biochemical studies revealed that CD146 was required for the activation of p38/IKK/NF kappaB signaling cascade and up-regulation of NF kappaB downstream pro-angiogenic genes, notably IL-8, ICAM-1 and MMP9, in response to tumor secretions. Interestingly, specific anti-CD146 mAb AA98, which bound a conformational epitope depending on C452-C499 disulfide bond, could abrogate NF kappaB activation and tumor angiogenesis, whereas another anti-CD146 mAb AA1 recognizing a linear epitope containing aa50-54 did not have such effects. Further structure-function analysis identified that C452-C499 disulfide bond within the fifth extracellular Ig domain was indispensible for CD146-mediated signaling and tube formation. Moreover, dimerization of CD146, which was enhanced by tumor secretions and suppressed by AA98 but not AA1, also relied on C452 and C499. Together, this study for the first time uncovered the pro-angiogenic role of CD146 and also pinpointed the key structural basis responsible for its signaling function and dimerization. These findings also suggested that CD146 might serve as not just a cell adhesion molecule but also a membrane signal receptor in tumor-induced angiogenesis.

Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146.

CD146, also known as melanoma cell adhesion molecule or MCAM, is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis. CD146 promotes tumor progression of many cancers including melanoma and prostate. Strikingly, its expression is frequently lost in breast carcinoma cells, and it may act as a suppressor of breast cancer progression. While upstream mechanisms regulating CD146 are well documented, our understanding of the downstream molecular events underlying its mode of action remains to be elucidated. This review aims to focus on the progress in understanding the signaling mechanisms and the functional relevance of CD146, a multifaceted molecule, in cancer with particular emphasis on its role in inhibiting breast cancer progression.

CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines.

INTRODUCTION: Metastasis is a complex process involving loss of adhesion, migration, invasion and proliferation of cancer cells. Cell adhesion molecules play a pivotal role in this phenomenon by regulating cell-cell and cell-matrix interactions. CD146 (MCAM) is associated with an advanced tumor stage in melanoma, prostate cancer and ovarian cancer. Studies of CD146 expression and function in breast cancer remain scarce except for a report concluding that CD146 could act as a tumor suppressor in breast carcinogenesis. METHODS: To resolve these apparent discrepancies in the role of CD146 in tumor cells, we looked at the association of CD146 expression with histoclinical features in human primary breast cancers using DNA and tissue microarrays. By flow cytometry, we characterized CD146 expression on different breast cancer cell lines. Using siRNA or shRNA technology, we studied functional consequences of CD146 downmodulation of MDA-MB-231 cells in migration assays. Wild-type, mock-transfected and downmodulated transfected cells were profiled using whole-genome DNA microarrays to identify genes whose expression was modified by CD146 downregulation. RESULTS: Microarray studies revealed the association of higher levels of CD146 with histoclinical features that belong to the basal cluster of human tumors. Expression of CD146 protein on epithelial cells was detected in a small subset of cancers with histoclinical features of basal tumors. CD146+ cell lines displayed a mesenchymal phenotype. Downmodulation of CD146 expression in the MDA-MB-231 cell line resulted in downmodulation of vimentin, as well as of a set of genes that include both genes associated with a poor prognosis in a variety of cancers and genes known to promote cell motility. In vitro functional assays revealed decreased migration abilities associated with decreased CD146 expression. CONCLUSIONS: In addition to its expression in the vascular compartment, CD146 is expressed on a subset of epithelial cells in malignant breast. CD146 may directly or indirectly contribute to tumor aggressiveness by promoting malignant cell motility. Changes in molecular signatures following downmodulation of CD146 expression suggest that CD146 downmodulation is associated with the reversal of several biological characteristics associated with epithelial to mesenchymal transition, and the phenomenon associated with the metastatic process.