Melanoma Cell Adhesion Molecule Research Articles

Negative Expression of Melanoma Cell Adhesion Molecule (MCAM) Correlated with Axillary Lymph Node Metastasis in Triple Negative Breast Cancer

Pengetahuan sangat penting dalam membentuk perilaku seseorang. Salah satu upaya memotivasi akseptor kontrasepsi suntikan progestin agar tercapai program penundaan kehamilannya yaitu dengan pemberian informasi melalui leaflet. Tujuan penelitian ini untuk menentukan pengaruh pemberian leaflet terhadap pengetahuan akseptor mengenai kontrasepsi suntikan progestin di Puskesmas Sungai Kakap Kabupaten Kubu Raya tahun 2015. Penelitian ini menggunakan metode quasi experiment , dengan rancangan non‑randomized control group pretest posttest design pada 62 akseptor yang ditentukan secara purposive sampling. Akseptor dibagi menjadi dua kelompok yaitu kelompok kontrol dan kelompok leaflet dengan 31 akseptor tiap kelompok. Pemberian leaflet hanya dilakukan pada kelompok leaflet setelah pretest . Setelah 1 bulan dilakukan posttest pada kedua kelompok. Data yang diperoleh dianalisis dengan uji Chi-Square . Hasil penelitian menunjukkan bahwa jumlah responden kelompok kontrol dengan pengetahuan baik sebesar 58,06% saat pretest , dan menurun sebesar 54,84% pada saat posttest . Responden kelompok leaflet yang memiliki pengetahuan baik pada saat pretest sebanyak 58,06% dan meningkat sebesar 74,19% pada saat posttest . Terdapat hubungan yang signifikan antara pemberian leaflet dengan pengetahuan responden mengenai kontrasepsi suntikan progestin ( p value = 0,000). Sosialisasi informasi secara luas mengenai kontrasepsi suntikan progestin harus terus dilanjutkan untuk meningkatkan penggunaannya meskipun pengetahuan baik terlihat pada akseptor. Kata Kunci: Leaflet, kontrasepsi suntikan progestin, pengetahuan Effect of Educational Leaflet on The Acceptor’s Knowledge About Progestogen-Only Injectable Contraceptive at Sungai Kakap Community Health Center Kubu Raya Regency in 2015 The knowledge is very important in shaping a person’s behavior. One of the ways motivating the progestogen-only injectable contraceptive acceptors to achieve the program of delayed pregnancy is given information by leaflet. The aim of this study was to determine the effect of the leaflet on the acceptor’s knowledge about progestogen-only injectable contraceptive at Sungai Kakap community health center Kubu Raya regency in 2015. This study was a quasi-experiment with non-randomized control group pretest-posttest design on 62 selected acceptors who were determined by purposive sampling. The acceptors were divided into two groups, both leaflet and control groups had 31 acceptors. Only the leaflet group was given leaflets after the pretest. After one month, both leaflet and control groups were done the posttest. Data was analyzed using Chi-Square. The results showed that 58,06% of acceptors in the control group had good knowledge on pretest and decreased by 54,84% on the posttest. The acceptors in the leaflet group as much as 58,06% had good knowledge on the pretest and increased by 74,19% on posttest. There was a significant correlation between giving leaflet and respondent’s knowledge about injectable progestogen‑only contraceptive (p-value = 0.000). Although most of the acceptors had good knowledge, widespread information regarding progestogen-only injectable contraceptive should be continued constantly. Keywords: Knowledge, leaflet, progestogen-only injectable contraceptive

Abstract 1635: The histone demethylase KDM3A in Ewing Sarcoma metastasis

Abstract This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit growth and metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult. Citation Format: Marybeth Sechler, Janet Parrish, Paul Jedlicka. The histone demethylase KDM3A in Ewing Sarcoma metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1635.

Beneficial roles of melanoma cell adhesion molecule in spinal cord transection recovery in adult zebrafish.

Melanoma cell adhesion molecule (MCAM) is a multifunctional protein involved in miscellaneous processes, including development and tumor angiogenesis. Here, spinal cord transection in adult zebrafish was used to investigate the effects of MCAM on spinal cord injury (SCI) and subsequent recovery. Expression of MCAM mRNA increased and co-localized with motoneurons in the spinal cord after SCI. With MCAM morpholino treatment, inhibition of MCAM retarded both axon regrowth and locomotor recovery in the spinal cord injured zebrafish. Furthermore, MCAM mRNA expression was also observed in fli1a:EGFP transgenic zebrafish, which specifically show labeled blood vessels. Inhibition of MCAM down-regulated the expression of angiogenesis-related factors, such as VEGFR-2, p-p38 and p-AKT, and the inflammatory factors TNF-α, IL-1β and IL-8. Taken together, these data suggest that MCAM may have a beneficial role in the recovery from SCI, via the promotion of neurogenesis and angiogenesis. In the context of adult zebrafish spinal cord injury, we proved that Melanoma cell adhesion molecule (MCAM) is beneficial to the recovery, possibly via mechanisms of angiogenensis and inflammation. MCAM promotes angiogenesis by adjusting VEGFR-2, p-p38 and p-AKT. MCAM affects inflammatory factors such as TNF-α, IL-1β and IL-8. Our results extend the beneficial role of MCAM in the regeneration of central nervous system.

Melanoma cell adhesion molecule as an emerging biomarker with prognostic significance in systolic heart failure.

Melanoma cell adhesion molecule (MCAM) is a marker of endothelial damage. MCAM diagnostic and prognostic value was assessed in chronic heart failure (CHF). 130 CHF patients and 32 controls were included in the study. Telephone follow-up lasted one year. End points were: death from all causes, and hospitalization with CHF exacerbation. MCAM was higher in patients than in controls (p = 0.01). Receiver operator curve analysis revealed that MCAM may serve as a predictor of death (area under the curve: 0.8404; p < 0.002). Patients with MCAM above 500 ng/ml had worse prognosis (p = 0.03). NT-proBNP and age were independent predictors of death in multivariate analysis. The increased MCAM indicates endothelial damage in CHF and may serve as a marker of worse prognosis in these patients.

Issue highlights - May 2016.

Issue highlights - May 2016.

Melanoma cell adhesion molecule stimulates yes-associated protein transcription by enhancing CREB activity via c-Jun/c-Fos in hepatocellular carcinoma cells.

Dysfunction of the yes-associated protein (YAP) signaling pathway has previously been associated with liver tumorigenesis. Recently, the membrane protein melanoma cell adhesion molecule (MCAM) was identified as a novel, hepatocellular carcinoma (HCC)-specific YAP target protein that promotes carcinogenesis in HCC. However, whether MCAM conversely regulates YAP remains unknown. The aim of the current study was to demonstrate whether and how MCAM regulates YAP in HCC cells. The present study demonstrated that MCAM has a positive effect on the regulation of YAP activity and expression. Mechanistically, MCAM stimulated YAP transcription through its downstream effector c-Jun/c-Fos heterodimer. Gain and loss of function analysis by the present study indicated that c-Jun/c-Fos is capable of inducing cAMP response element-binding protein activation, which is a transcription factor that directly binds to the YAP promoter. Finally, it was identified that an impaired transformative phenotype in MCAM- or c-Jun/c-Fos-depleted HCC cells could be partially rescued by simultaneous overexpression of YAP, suggesting that YAP may function as a downstream effector of the MCAM-c-Jun/c-Fos signaling pathway. Collectively, a complete, positive, auto-regulatory loop was established by the present study, in which YAP is not only an upstream regulator, but also a downstream target of MCAM in HCC cells.

MCAM and LAMA4 Are Highly Enriched in Tumor Blood Vessels of Renal Cell Carcinoma and Predict Patient Outcome.

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma, or colorectal liver metastasis. We identified a panel of genes consistently upregulated by tumor blood vessels, of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes. This result was subsequently confirmed by immunohistochemical analysis of MCAM and LAMA4 expression in RCC and colorectal carcinoma blood vessels. Strong MCAM and LAMA4 expression was also shown to predict poor survival in RCC, but not in colorectal carcinoma. Notably, MCAM and LAMA4 were enhanced in locally advanced tumors as well as both the primary tumor and secondary metastases. Expression analysis in 18 different cancers and matched healthy tissues revealed vascular MCAM as highly specific in RCC, where it was induced strongly by VEGF, which is highly abundant in this disease. Lastly, MCAM monoclonal antibodies specifically localized to vessels in a murine model of RCC, offering an opportunity for endothelial-specific targeting of anticancer agents. Overall, our findings highlight MCAM and LAMA4 as prime candidates for RCC prognosis and therapeutic targeting. Cancer Res; 76(8); 2314-26. ©2016 AACR.

CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

Abstract B19: The histone demethylase KDM3A in Ewing sarcoma metastasis

Abstract This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult. Citation Format: Marybeth Sechler, Janet Parrish, Paul Jedlicka. The histone demethylase KDM3A in Ewing sarcoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B19.

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy.

Abstract A22: The histone demethylase KDM3A in Ewing sarcoma metastasis

Abstract This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit growth and metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult. Citation Format: Marybeth Sechler, Janet Parrish, Paul Jedlicka. The histone demethylase KDM3A in Ewing sarcoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A22.

Altered Expression of Angiotensinogen and Mediators of Angiogenesis in Ileal Crohn's Disease.

Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease. Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting. Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings. Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.

Expression of pro-angiogenic genes in mesenchymal stem cells derived from dermis of patients with psoriasis.

Recent experimental studies revealed that angiogenesis and lymphangiogenesis are closely related to psoriasis. Our microarray analysis suggested that the pro-angiogenic genes platelet endothelial cell adhesion molecule-1 (PECAM1), facio-genital dysplasia-5 (FGD5), prostaglandin-endoperoxide synthase-1 (PTGS1), melanoma cell adhesion molecule (MCAM), vasohibin-2 (VASH2), and stabilin-1 (STAB1) are differentially expressed in dermal mesenchymal stem cells in psoriasis. The aim of this study was to investigate the mRNA and protein expression of PECAM1, FGD5, PTGS1, MCAM, VASH2, and STAB1 for angiogenesis and the possible mechanisms in psoriasis. We studied 12 patients with plaque psoriasis and 14 healthy controls matched for age and sex. Dermal mesenchymal stem cells were expanded, passaged, and identified by cellular morphology, immunophenotyping, and multipotential differentiation. The mRNA and protein expression of the above-mentioned six genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. The significantly decreased expression of PECAM1, PTGS1, FGD5, and MCAM at both mRNA and protein level (except VASH2 and STAB1) were demonstrated in mesenchymal stem cells from psoriatic skin lesions compared with non-lesional from healthy controls. We provide the first report that pro-angiogenic genes PECAM1, PTGS1, FGD5, and MCAM rather than VASH2 and STAB1 may be play a vital role in pathological dermal angiogenesis disorders of psoriasis. Therefore, anti-angiogenesis is attractive and offers future potential for application in patients with psoriasis.

Electrotransfer parameters as a tool for controlled and targeted gene expression in skin

Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

Human Cementoblastoma- Derived Cells Express Cartilage- Like Phenotype In vitro and In vivo and Induce Bone Formation

The molecular mechanisms that regulate proliferation and differentiation of cementoblasts, have not been elucidated to date. In this paper, it is shown that human cementoblastoma-derived cells (HCDC) express greater levels of cartilage markers such as type II and X collagens, aggrecan (ACAN) and SRY-box 9 (SOX9) stem cell markers; MCAM (melanoma cell adhesion molecule; synonym: CD146) and STRO-1 than human gingival fibroblasts (HGF). Our in vivo studies demonstrate that HCDC induce bone formation through endochondral ossification as observed 14 days after HCDC were implanted in rat critical-size calvarial defects. At 30 and 60 days post-implantation, the defects treated with HCDC were filled with 70 ± 1.6 and 91 ± 1.3% of newly formed bone. To confirm the identity of this tissue, we analyzed the newly formed bone using histomorphology and immunostaining. The results showed the expression of bone sialoprotein (BSP) and osteocalcin (OCN). Immmunostaining of human periodontal structures showed that cementoblasts and periodontal ligament cells express cementum protein 1 (CEMP1), cartilage markers, type II and X collagens and CD146 which has been identified as a marker for chondroprogenitor cells. Altogether these results indicate that HCDC has the capacity of multilineage differentiation and induces regeneration of mineralized tissues other than cementum.

Brief report: enrichment of activated group 3 innate lymphoid cells in psoriatic arthritis synovial fluid.

Innate lymphoid cells (ILCs) are a recently discovered group of cells that are essential to epithelial homeostasis and are implicated in psoriasis pathogenesis, yet they have never been reported in psoriatic arthritis (PsA). ILC classes and subsets were characterized in the peripheral blood (PB) of healthy controls, patients with psoriasis, and patients with PsA and in the synovial fluid (SF) of patients with PsA and patients with rheumatoid arthritis (RA). Cell surface marker expression and intracellular cytokine production following stimulation were analyzed using flow cytometry. ILCs were identified in the SF and were 4-fold more abundant in PsA SF than in PsA PB. Fewer CCR6+ ILCs were found in PsA PB than in healthy control PB, while PsA SF was enriched for CCR6+ ILCs compared to PsA PB and RA SF. Natural cytotoxicity receptor NKp44+ group 3 ILCs were rare in PB and RA SF, but abundant in PsA SF. Increased numbers of interleukin-17A (IL-17A)-producing ILCs were present in PsA SF compared to RA SF. CCR6, NKp44, and melanoma cell adhesion molecule (MCAM) were expressed on the cell surface of SF ILCs that produced IL-17A. The number of circulating NKp44+, CCR6+, and MCAM+ ILCs in blood was inversely correlated with PsA disease activity. Our findings indicate that PsA SF is enriched for group 3 ILCs that express CCR6 and NKp44, which distinguishes the synovial compartment from RA. The increased IL-17A production by SF ILCs indicates a novel role for ILCs in PsA.

Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III–IV) and 16 early-stage (I–II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6–13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82–41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

Melanoma Cell Galectin-1 Ligands Functionally Correlate with Malignant Potential

Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening antitumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity, and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely dysplastic nevi, as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAM(KD)) or ST6GalNAc2-overexpressing (ST6(O/E)) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAM(KD) or ST6(O/E) melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1/melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy.

Protein Depalmitoylation Is Induced by Wnt5a and Promotes Polarized Cell Behavior

Wnt5a signaling regulates polarized cell behavior, but the downstream signaling events that promote cell polarity are not well understood. Our results show that Wnt5a promotes depalmitoylation of the melanoma cell adhesion molecule (MCAM) at cysteine 590. Mutation of Cys-590 to glycine is sufficient to polarize MCAM localization, similar to what is observed with Wnt5a stimulation. Inhibition of the depalmitoylating enzyme APT1 blocks Wnt5a-induced depalmitoylation, asymmetric MCAM localization, and cell invasion. Directly altering expression of the basal protein palmitoylation machinery is sufficient to promote cell invasion. Additionally, cancer mutations in palmitoyltransferases decrease MCAM palmitoylation and have impaired ability to suppress cell invasion. Our results provide evidence that Wnt5a induces protein depalmitoylation, which promotes polarized protein localization and cell invasion.

CD166 positively regulates MCAM via inhibition to ubiquitin E3 ligases Smurf1 and βTrCP through PI3K/AKT and c-Raf/MEK/ERK signaling in Bel-7402 hepatocellular carcinoma cells

Both Cluster of Differentiation 166 (CD166) and Melanoma Cell Adhesion Molecule (MCAM) play critical roles in maintaining transformative phenotype of Hepatocellular Carcinoma (HCC) cells. However, the relationship between these two membrane proteins remains unknown. Here, we found that CD166 has a positive impact on the expression of MCAM, while MCAM has no feedback on CD166. Tissue microarray analysis (TMA) also showed a positive correlation between CD166 and MCAM. Depletion of CD166-induced anti-carcinogenic phenotype could be reversed by overexpression of MCAM, suggesting MCAM is functional important in the CD166-induced liver tumorigenesis. Furthermore, we found CD166 regulates MCAM mainly through protecting MCAM from ubiquitin-mediated protein degradation. Mechanically, CD166 down-regulated two ubiquitin E3 ligases, βTrCP and Smurf1, which play critical roles in the destability of MCAM protein. In addition, overexpression of βTrCP and Smurf1-reduced transformative phenotype could be partially reversed by MCAM, providing evidence that MCAM is a target of βTrCP and Smurf1. Moreover, we identified c-Raf/MEK/ERK signaling acts as a downstream effecter of CD166/PI3K/AKT axis to stimulate ubiquitination and destability of βTrCP and Smurf1. Taken together, we establish a model that CD166 regulates MCAM through a signaling flow from activation of PI3K/AKT and c-Raf/MEK/ERK signaling to the inhibition of potential MCAM ubiquitin E3 ligases, βTrCP and Smurf1, blockage of this signaling cascade may be useful in the treatment of CD166 and MCAM-dependent HCC.