Evidence and impact of neutrophil extracellular traps in malignant melanoma

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non‐ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non‐ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin‐mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co‐culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma.     

Fli-1 expression in malignant melanoma

Friend leukemia integration site 1 (Fli-1) has been reported as the first nuclear marker of endothelial differentiation; it is expressed in leukocytes and recently demonstrated in melanomas. Formalin-fixed, paraffin-embedded tissue sections from 97 melanomas including 69 cases of primary and 28 metastatic melanomas were evaluated by immunohistochemistry. Five melanoma cell lines were evaluated by Western blot and immunocytochemistry. Fli-1 expression was observed in all cell lines. Fli-1 expression was higher in metastatic than in primary tumors (r=0.208, p=0.041, Spearman correlation), it positively correlated with Ki-67 expression (r=0.233, p=0.022, Spearman correlation), and the presence of an ulcer in the primary tumor (r=0.267, p=0.030, Spearman correlation). Therefore, the expression of Fli-1 in malignant melanoma appears to be associated with biologically more aggressive tumors.     

Tumor-infiltrating lymphocytes: apparently good for melanoma patients. But why?

Tumor-infiltrating T lymphocytes (TILs) are observed in a number of human primary or metastatic tumors. Recently, gene expression profiling experiments suggested that the presence of T cells in metastatic melanomas before vaccinating the patients with tumor antigens could be a biomarker for clinical benefit from the vaccines. In this context, we review results pertaining to TILs in human melanomas, their prognostic value, and some possible reasons why their presence could help in selecting melanoma patients for vaccination against tumor-specific antigens.     

Copy Number Loss of the Interferon Gene Cluster in Melanomas Is Linked to Reduced T Cell Infiltrate and Poor Patient Prognosis

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination. Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes. We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes. As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3. Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial.     

DNA methylation profiles in primary cutaneous melanomas are associated with clinically significant pathologic features

DNA methylation studies have elucidated a methylation signature distinguishing primary melanomas from benign nevi and provided new insights about genes that may be important in melanoma development. However, it is unclear whether methylation differences among primary melanomas are related to tumor pathologic features with known clinical significance. We utilized the Illumina GoldenGate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Arraywide methylation patterns revealed a positive association of methylation with Breslow thickness and mutated BRAF, a negative association with mitotic rate, and a weak association with ulceration. Hierarchical clustering on CpG sites exhibiting the most variable methylation (n = 235) divided the melanoma samples into three clusters, including a highly methylated cluster that was positively associated with Breslow thickness and an intermediately methylated cluster associated with Breslow thickness and mitotic rate. Our findings provide support for the existence of methylation‐defined subsets in melanomas with increased methylation associated with Breslow thickness.     

Constitutive aberrant endogenous interleukin-1 facilitates inflammation and growth in human melanoma

Interleukin (IL)-1-mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH(2)-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway-specific siRNAs led to growth arrest in IL-1-positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1-positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1-positive melanoma cells.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

Genomic analysis reveals low tumor mutation burden which may be associated with GNAQ/11 alteration in a series of primary leptomeningeal melanomas

Primary central nervous system melanoma is rare and characterized by a variable prognosis, and no current treatment guidelines exist. We describe the clinical course of a 70‐year‐old female patient diagnosed with primary leptomeningeal melanoma (LMN) whose case represents the diagnostic and management challenges of this tumor. Targeted genomic sequencing of 315 genes from this tumor revealed GNAQ Q209L mutation and low (4 mutations/Megabase) tumor mutation burden (TMB). Wild‐type NRAS, KIT, and BRAF were also observed. A cohort of 4,787 melanomas was subsequently analyzed to identify additional primary central nervous system melanomas, of which 10 additional tumors met pathologic criteria (0.21% of total melanoma cohort). These tumors were genomically assessed according to the same targeted sequencing panel, and 6 of the tumors were also found to harbor a GNAQ mutation. All 10 tumors had low (less than or equal to 2 mutations/Megabase) TMB indicating a potential trend between G‐protein‐coupled receptor (GPCR) alterations and low TMB in LMNs. GPCR alterations were found to significantly correlate with TMB across the cohort of 4,787 melanomas, supporting this potential finding in the limited LMN subset.     

MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.     

Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes

Recently developed lymphatic‐specific immunohistochemical markers can now be utilized to assess intratumoral and/or peritumoral lymphatic vessel density (LVD), to detect lymphatic vessel invasion (LVI) by melanoma cells and to identify lymphatic marker expression in melanoma cells themselves. We systematically reviewed the available evidence for the expression of lymphatic markers as predictors of regional node metastasis and survival in melanoma patients. The currently available evidence suggests that LVD (particularly in a peritumoral location) and LVI are predictors of sentinel node metastasis and poorer survival. Nevertheless, adherence to international guidelines in the conduct and reporting of the studies was generally poor, with wide methodologic variations and heterogeneous findings. Larger, carefully conducted and well‐reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time‐consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas.     

Enhancement of human melanoma antigen expression by IFN-beta

Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines. We have now identified IFN-beta as an additional stimulus to TAA expression, including Melan-A/MART-1, gp100, and MAGE-A1. IFN-beta (but neither IFN-alpha nor IFN-gamma) augmented both protein and mRNA expression of melanocytic TAA in 15 melanoma lines (irrespective of initial Ag-expression levels). Treatment of low Ag melanoma lines with IFN-beta increased expression of melanocyte-lineage Ags, inducing susceptibility to lysis by specific CTLs. Treatment with IFN-beta also enhances expression of class I HLA molecules, thereby inducing both nominal TAA and the presenting HLA molecule. Data from fluorescent cellular reporter systems demonstrated that IFN-beta triggers promoter activation, resulting in augmentation of Ag expression. In addition to enhancing TAA expression in melanomas, IFN-beta also stimulated expression of the melanocytic Ag gp100 in cells of other neural crest-derived tumor lines (gliomas) and certain unrelated tumors. Because IFN-beta is already approved for human clinical use in other contexts, it may prove useful as a cotreatment for augmenting tumor Ag expression during immunotherapy.     

Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-3 and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.     

Screening for therapeutic targets of tumor angiogenesis signatures in 31 cancer types and potential insights

Tumor vessel normalization can increase pericyte coverage, perfusion efficiency and immune infiltration, while reducing hypoxia, vessel leakage, CTC and metastasis. In this study, we systemically presented the expression pattern of tumor angiogenesis gene signatures in 31 cancer types and its association with immune infiltration and cancer metastasis. Specifically, READ, COAD etc. have relatively similar expression patterns with low GPAGs and high PPAGs. Patients with this expression pattern may benefit from tumor vessel normalization. COAD was selected for further investigation and we found GPAG CXCL12 was downregulated while PPAG EPHB3 was overexpressed in COAD, which were further validated using two independent colon cancer dataset. Further study indicated that CXCL12 expression was positively correlated innate inflammation pathways such as NFκB and negatively correlated with metastasis, while EPHB3 had a reverse result. Moreover, CXCL12 was positively correlated with cancer immune infiltration while EPHB3 was negatively correlated with cancer immune infiltration. Besides, the association between CXCL12/EPHB3 and mutation/CNA landscape were also explored. We also discussed the potential application of gut microbiota in cancer treatment. In summary, blood vessel normalization could promote immune infiltration and repress cancer metastasis while immune cell infiltration can promote blood vessel normalization through a positive feedback loop.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

ABSTRACT: BACKGROUND: Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. METHODS: To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. RESULTS: We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer. CONCLUSIONS: These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.     

Menin represses malignant phenotypes of melanoma through regulating multiple pathways

Substantial genetic evidence suggests that chromosome 11q is involved in regulating initiation and progression of malignant melanomas. Mutations of the MEN1 gene, located in chromosome 11q13, predispose individuals to the multiple endocrine neoplasia type 1 (MEN1) familial syndrome. MEN1 patients develop primary malignant melanoma, suggesting a potential link between MEN1 syndrome and development of melanomas, but the precise molecular mechanism is poorly understood. Here we show that the MEN1 gene suppresses malignant phenotypes of melanoma cells through multiple signalling pathways. Ectopic expression of menin, the product of MEN1 gene, significantly inhibited melanoma cell proliferation and migration in vitro and in vivo. The inhibition was partly achieved through suppressing expression of growth factor pleiotrophin (PTN) and receptor protein tyrosine phosphatase (RPTP) β/ζ, accompanied with the reduced expression of phosphatidylinositol 3-kinase (pI3K) and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2). Interestingly, reduced expression of menin was associated with hypermethylation of the CpG islands of the MEN1 promoter in melanoma cells. Taken together, these findings suggest a previously unappreciated function for menin in suppressing malignant phenotypes of melanomas and unravel a novel mechanism involving in regulating PTN signalling by menin in development and progression of melanomas.