Aggressive Melanoma Phenotype Research Articles

Abstract IA014: The role of aging in reactivation from metastatic melanoma dormancy

Abstract Disseminated cancer cells (DCCs) that escape the primary site can seed in distal tissues, but may take several years, or even decades to grow out into overt metastases, a phenomenon termed tumor dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully model or characterize dormancy within melanoma. Our previous data has indicated that while tumors grow more slowly in aged mouse skin, they form more aged lung metastases. We have previously shown that dermal fibroblasts were key drivers of age-related changes in the skin to promote a more aggressive melanoma phenotype. A model of melanoma progression that addresses these microenvironmental complexities is the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state. We queried whether the lung microenvironment undergoes similar age-related changes that provide a permissive niche for metastatic outgrowth. Our new data identified that while Wnt5A and AXL are required for efficient dissemination from the primary tumor towards metastatic tissues, they act as master regulators of activating melanoma dormancy within the lung. The young lung microenvironment maintains this dormant melanoma state; however, age induced reprograming of lung fibroblasts increases their secretion of the soluble Wnt antagonist sFRP1, which inhibits both Wnt5A and AXL to enable reactivation from dormancy and the formation of larger metastatic colonies in the aged lung. We find that downregulation of AXL in melanoma cells leads to an increase in the related TAM family receptor MERTK, which promotes a proliferative melanoma phenotype, enables reactivation from dormancy and induces efficient metastatic outgrowth in aged models. Follow up data from these models highlights decreases in immune infiltration of CD4+ and CD8+ T-cells, which appears to be partially mediated by an increase in secretion of Arginase-1 (ARG1) from aged lung fibroblasts. Further, we find that aged lung fibroblasts secrete CXCL1 which promotes infiltration of immunosuppressive T-regulatory cells and Myeloid Derived Suppressive Cells. Finally, we see dramatic changes in extracellular matrix secretion and remodeling from young vs aged lung fibroblasts, which alters in vitro melanoma growth to promote dormancy in melanoma cells grown on young lung fibroblast ECM. Overall, we find that a plethora of age-related changes in metastatic microenvironments such as the lung contribute to an increase in melanoma progression relative to younger niches. This not only provides novel tools to assess metastatic dormancy in melanoma, which is critically understudied due to a lack of pre-clincal in vitro and in vivo models, but also necessitates the idea that age needs to be considered in pre-clinical modelling of age-related cancers such as melanoma, where the median age of diagnosis is 63, and when there is a clear increase in incidence, mortality and metastasis within geriatric patients. Citation Format: Mitchell Fane, Yash Chhabra, Gretchen Alicea, Daniel Zabransky, Laura Hüser, Devin Maranto, Julio Aguirre-Ghiso, Ashani Weeraratna. The role of aging in reactivation from metastatic melanoma dormancy [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA014.

Fra-2/AP-1 regulates melanoma cell metastasis by downregulating Fam212b.

Metastatic melanoma remains a challenging disease. Understanding the molecular mechanisms how melanoma becomes metastatic is therefore of interest. Herein we show that downregulation of the AP-1 transcription factor member Fra-2 in melanoma cells is associated with an aggressive melanoma phenotype in vitro and in vivo. In vitro, Fra-2 knockdown in melanoma cells promoted cell migration and invasion associated with increased Snail-1, Twist-1/2, and matrix metalloproteinase-2 (MMP-2) expression. In vivo, Fra-2 knockdown in a melanoma cell line led to increased metastasis into the lungs and liver. The increased metastatic potential of Fra-2 knockdown melanoma cells was likely due to an accelerated cell cycle transition and increased tissue angiogenesis. Using Fra-2 knockdown cell lines microarray analysis, we identified the protein Fam212b (family with sequence similarity 212 member B) as a downstream target of Fra-2. By additional knockdown of Fam212b in Fra-2 mutant cells, we mitigated the cell migration, invasion, and cell cycle transition phenotype induced by Fra-2 knockdown. Furthermore, Fam212b overexpression enhanced β-catenin pathway. Finally, Fam212b expression is correlated with increased melanoma metastasis and poor clinical outcomes in human patients. In summary, these findings reveal the Fra-2-Fam212b axis as a new pathway of melanoma metastasis, which can be in the future used as potential marker of the metastatic properties of melanoma.

Heterogeneity of Melanoma with Stem Cell Properties.

Metastatic melanoma continues to present a significant challenge-with a cure rate of less than 10% and a median survival of 6-9months. Despite noteworthy advances in the field, the heterogeneity of melanoma tumors, comprised of cell subpopulations expressing a cancer stem cell (CSC) phenotype concomitant with drug resistance markers presents a formidable challenge in the design of current therapies. Particularly vexing is the ability of distinct subpopulations of melanoma cells to resist standard-of-care treatments, resulting in relapse and progression to metastasis. Recent studies have provided new information and insights into the expression and function of CSC markers associated with the aggressive melanoma phenotype, such as the embryonic morphogen Nodal and CD133, together with a drug resistance marker ABCA1. This chapter highlights major findings that demonstrate the promise of targeting Nodal as a viable option to pursue in combination with standard-of-care therapy. In recognizing that aggressive melanoma tumors utilize multiple mechanisms to survive, we must consider a more strategic approach to effectively target heterogeneity, tumor cell plasticity, and functional adaptation and resistance to current therapies-to eliminate relapse, disease progression, and metastasis.

Identification of microRNAs associated with invasive and aggressive phenotype in cutaneous melanoma by next-generation sequencing

A comprehensive repertoire of human microRNAs (miRNAs) that could be involved in early melanoma invasion into the dermis remains unknown. To this end, we sequenced small RNAs (18–30 nucleotides) isolated from an annotated series of invasive melanomas (average invasive depth, 2.0 mm), common melanocytic nevi, and matched normal skin (n=28). Our previously established bioinformatics pipeline identified 765 distinct mature known miRNAs and defined a set of top 40 list that clearly segregated melanomas into thin (0.75 mm) and thick (2.7 mm) groups. Among the top, miR-21-5p, let-7b-5p, let-7a-5p, miR-424-5p, miR-423-5p, miR-21-3p, miR-199b-5p, miR-182-5p, and miR-205-5p were differentially expressed between thin and thick melanomas. In a validation cohort (n=167), measured expression of miR-21-5p and miR-424-5p, not previously reported in melanoma, were significantly increased in invasive compared with in situ melanomas (P<0.0001). Increased miR-21-5p levels were significantly associated with invasive depth (P=0.038), tumor mitotic index (P=0.038), lymphovascular invasion (P=0.0036), and AJCC stage (P=0.038). In contrast, let-7b levels were significantly decreased in invasive and in situ melanomas compared with common and dysplastic nevi (P<0.0001). Decreased let-7b levels were significantly associated with invasive depth (P=0.011), Clark's level (P=0.013), ulceration (P=0.0043), and AJCC stage (P=0.011). These results define a distinct set of miRNAs associated with invasive and aggressive melanoma phenotype.

ITOC2 – 009. Role of signal transduction and microRNAs on the immunogenicity of melanoma cells

Melanoma is a heterogeneous malignancy with a distinct clinical outcome of patients due to different genetic features and various strategies of melanoma cells leading to immune evasion. For a better understanding of the aggressive melanoma phenotype the molecular mechanisms of aberrant expression of the classical HLA class I antigen processing and presentation machinery (APM) and of the non-classical HLA-G antigen is analysed. A reduced mRNA and/or protein expression of various APM components was identified in a large series of melanoma cell lines and lesions, which correlated with an increased tumour grading and lack of immune therapy response. In addition, microRNAs (miRs) targeting HLA class I APM components as well as HLA-G thereby affecting the immune response have been identified suggesting an important role for posttranscriptional control in this process. These miRs might be used as novel targets for the treatment of melanoma or for selection of melanoma patients undergoing the most effective (immuno)therapy. Furthermore, a direct link between the interferon (IFN) signalling pathway and the constitutive HLA class I expression was found. Altered expression or loss of single components of the IFN-<i>γ</i> signal cascade was detected, which was accompanied by deficient IFN-<i>γ</i> response due to structural alterations, epigenetic control or transcriptional/post-transcriptional deregulation. These were directly associated with a downregulation of basal HLA class I APM component expression in melanoma lesions and melanoma cell lines and confirmed by silencing of STAT1, JAK1 and JAK2. Thus these data provide novel insights into the complex regulation of HLA class I expression and the rational for selecting patients for specific immunotherapies.

Absolute quantitative PCR for detection of molecular biomarkers in melanoma patients: A preliminary report

BackgroundMalignant melanoma is the most malignant tumours of skin and mucous membranes mainly due to its aggressive biological behaviour and tendency to generate early metastases. Unfortunately, the mechanisms underlying the development, progression and the expression of an aggressive melanoma phenotype still remain largely unknown. ObjectivesThe purpose of this study was to determine whether a multi-panel of molecular transcripts can be predictive for risk of recurrent disease in malignant melanoma patients. ResultsPeripheral blood was collected from 31 malignant melanoma patients in follow-up for melanoma and from 30 healthy volunteers randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of six markers: PAX3d, TYR, MITFm, MCAM, TGFβ2 and ABCB5. Malignant melanoma patients expressed an important number of markers, with a median value of four markers. Only PAX3d displayed a trend in terms of differences when the levels of gene expression were made in function of Breslow index. Furthermore, PAX3d showed the best diagnostic capacity among the remaining residual markers or in combination with TGFβ2 and MTIF. ConclusionsWe demonstrated the usefulness of multimarker qRT-PCR to detect circulating melanoma cells in blood and to potentially assessing patient disease status or progression, especially when PAX3d was used in combination with MTIFm and TGFβ2.

Role of signal transduction and microRNAs on the immunogenicity of melanoma cells

Melanoma tumors are heterogeneous and involve different processes generating cells with variable metastatic capacity, which also results in distinct clinical outcome of patients and variations in therapy responses. This might be due to different strategies leading to evasion of T and or NK cell-mediated surveillance, which is accompanied by disease progression and a poor survival of melanoma patients. Therefore, the characterization of immune escape mechanisms might contribute to a better understanding of the development of the aggressive melanoma phenotype. This study analysed the underlying molecular mechanisms of HLA class I abnormalities and aberrant HLA-G expression in melanoma. A reduced mRNA and/or protein expression of various components of the MHC class I antigen processing machinery (APM) was identified in a large series of melanoma cell lines and lesions, which could be correlated with an increased tumor grading. In addition, adoptive T cell therapy also caused an escape from immune cell recognition by down-regulation components of the antigen processing machinery (APM). Recently, microRNAs (miRs) targeting HLA class I APM components were identified suggesting an important role for posttranscriptional control in this process. These miRs have functional activities on APM components, thereby also affecting HLA class I surface expression. Thus, these miRs might be used as novel targets for the treatment of melanoma or for selection of melanoma patients undergoing the most effective (immuno)therapy. In addition, a direct link of the IFN signalling pathway and the constitutive HLA class I APM component expression was found in melanoma. Down-regulation or loss of single components of the IFN-γ signal cascade was detected, which could be due to structural alterations, epigenetic mechanisms, such as methylation and/or histone deacetylation, or transcriptional/post-transcriptional deregulation. This was accompanied by a heterogeneous response of melanoma cells to IFN-γ treatment. Alterations in the IFN-γ signalling pathway were directly associated with a down-regulation of constitutive HLA class I components as demonstrated in melanoma lesions as well as in melanoma cell lines. Using shRNA-mediated silencing a direct effect of STAT1, JAK1 and JAK2 on MHC class I surface expression was confirmed. These data may provide novel insights into the complex regulation of HLA class I expression in melanoma and gives a rational for excluding patients for specific immunotherapies. Furthermore, strategies will be developed for restoration of the HLA class I positive phenotype.

Abstract 5204: Mechanisms of Notch4-Nodal regulation of the aggressive melanoma phenotype

Abstract Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. The current lack of effective therapies for treating metastatic melanoma exemplifies the desperate need for the identification of new targets and the development of new therapies. Nodal is an embryonic signaling molecule from the TGFβ superfamily that is not typically expressed in adult tissues but is reactivated in a number of aggressive cancers including melanoma, and breast and prostate carcinomas. In a study recently published by our group, we determined a connection between the Notch and Nodal pathways in aggressive melanoma. We identified a specific correlation between the expression of Notch4 and Nodal in aggressive melanoma cell lines and in advanced stage human melanomas. By inhibiting Notch4 expression or function, we established a link between Notch4 and Nodal signaling in promoting the aggressive phenotype of metastatic melanoma cells in vitro. Notch4 regulation of Nodal expression was identified as important in cellular plasticity, since inhibition of Notch4 function impaired the ability of aggressive cells to engage in vasculogenic mimicry (de novo formation of vascular-like networks by non-endothelial tumor cells). Notably, vascular-like network formation could be partially rescued by recombinant human Nodal, suggesting both pathways are important for this phenomenon. To extend our published observations, we utilized neutralizing antibodies to Notch4 and to Nodal, and compared gene expression profiles. From this, we identified a mechanism of feedback between signaling pathways that controls the expression of components of both pathways, including Nodal, Notch4, and the Notch ligand, Jagged2. Since Notch4 is enriched in the subpopulation of cells that form vascular-like networks in melanoma, we specifically analyzed the expression of angiogenesis signature genes previously associated with vascular-like network formation, and determined that inhibition of Notch4 or Nodal function downregulates a large group of these genes including members of the VEGF, angiopoietin, MMP, and cadherin families. We are currently investigating the hypothesis that the Notch4-Nodal signaling axis may be a master regulator of the vascular-like phenotype in aggressive melanoma cells. We suggest that inhibition of Notch4 and/or Nodal function may offer a potential molecular targeting strategy for metastatic melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5204. doi:1538-7445.AM2012-5204

Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Metastatic melanoma is an aggressive skin cancer associated with poor prognosis. The reactivation of the embryonic morphogen Nodal in metastatic melanoma has previously been shown to regulate the aggressive behavior of these tumor cells. During the establishment of left-right asymmetry in early vertebrate development, Nodal expression is specifically regulated by a Notch signaling pathway. We hypothesize that a similar relationship between Notch and Nodal may be reestablished in melanoma. In this study, we investigate whether cross talk between the Notch and Nodal pathways can explain the reactivation of Nodal in aggressive metastatic melanoma cells. We show a molecular link between Notch and Nodal signaling in the aggressive melanoma cell line MV3 via the activity of an RBPJ-dependent Nodal enhancer element. We show a precise correlation between Notch4 and Nodal expression in multiple aggressive cell lines but not poorly aggressive cell lines. Surprisingly, Notch4 is specifically required for expression of Nodal in aggressive cells and plays a vital role both in the balance of cell growth and in the regulation of the aggressive phenotype. In addition, Notch4 function in vasculogenic mimicry and anchorage-independent growth in vitro is due in part to Notch4 regulation of Nodal. This study identifies an important role for cross talk between Notch4 and Nodal in metastatic melanoma, placing Notch4 upstream of Nodal, and offers a potential molecular target for melanoma therapy.

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, alpha-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by alpha-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.

Focal Adhesion Kinase Promotes the Aggressive Melanoma Phenotype

Malignant melanoma continues to remain a significant health threat, with death often occurring as a result of metastasis. The metastatic phenotype typically is characterized by augmented tumor cell invasion and migration in addition to tumor cell plasticity as shown by vasculogenic mimicry. Therefore, understanding the molecular mechanisms that promote an aggressive phenotype is essential to predicting the likelihood of metastasis at a stage when intervention may be possible. This study focuses on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, including cell survival, invasion, and migration. We found FAK to be phosphorylated on its key tyrosine residues, Tyr397 and Tyr576, in only aggressive uveal and cutaneous melanoma cells, which correlates with their increased invasion, migration, and vasculogenic mimicry plasticity. Additionally, we confirmed the presence of FAK phosphorylated on Tyr397 and Tyr576 in both cutaneous and uveal melanoma tumors in situ. Examination of a functional role for FAK in aggressive melanoma revealed that disruption of FAK-mediated signal transduction pathways, through the expression of FAK-related nonkinase (FRNK), results in a decrease in melanoma cell invasion, migration, and inhibition of vasculogenic mimicry. Moreover, we found that FRNK expression resulted in a down-regulation of Erk1/2 phosphorylation resulting in a decrease in urokinase activity. Collectively, these data suggest a new mechanism involved in promoting the aggressive melanoma phenotype through FAK-mediated signal transduction pathways, thus providing new insights into possible therapeutic intervention strategies.

Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: Establishment of KT293, A novel G-CSF-secreting melanoma cell line

Paraneoplastic leukemoid reaction (PLR) is a rare condition of leucocytosis in cancer patients. Here we report the rapid progression of a patient suffering from a metastasized malignant melanoma and PLR. The patient’s white blood cell count exceeded 200,000 cells per µl and the serum level of Granulocyte Colony-Stimulating Factor (G-CSF) was elevated up to 780 pg/µl. A Tc-m99-labeled anti-NCA90/95 based granulocyte scan demonstrated reactive bone marrow expansion, splenomegaly and granulocyte infiltration into the tumor. KT293, a S100, gp100 and CD68 positive melanoma cell line derived from an axillary metastasis, produced large amounts of G-CSF in vitro and induced rapidly growing tumors and PLR after subcutaneous inoculation in SCID mice. In contrast to G-CSF-secreting cancer cells of other tissue origin, G-CSF-neutralizing antibodies failed to inhibit the growth of KT293 cells. In addition, KT293 cells did not express G-CSF-receptor. These observations suggest that paracrine effects of G-CSF-secretion and PLR might promote an aggressive melanoma phenotype, as seen in this patient.