Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

The metastatic microenvironment: Claudin-1 suppresses the malignant phenotype of melanoma brain metastasis.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.

Rewired ERK-JNK signaling pathways in melanoma.

Constitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype.

The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1-silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation.

Driving transcriptional regulators in melanoma metastasis.

The progression of melanoma toward the metastatic phenotype occurs in a defined stepwise manner. While many molecular changes take place early in melanoma development, progression toward the malignant phenotype, most notably during the transition from the radial growth phase (RGP) to the vertical growth phase (VGP) involves deregulated expression of several transcription factors. For example, the switch from RGP to VGP is associated with the loss of the transcription factor AP2α and gain of transcriptional activity of cAMP-responsive element binding protein. Together with the upregulation of microphthalmia-associated transcription factor, activating transcription factor 2, nuclear factor kappa B, and other transcription factors, these changes lead to dysregulated expression or function of important cellular adhesion molecules, matrix degrading enzymes, survival factors, as well as other factors leading to metastatic melanoma. Additionally, recent evidence suggests that microRNAs and RNA editing machinery influence the expression of transcription factors or are regulated themselves by transcription factors. Many of the downstream signaling molecules regulated by transcription factors, such as protease activated receptor-1, interleukin-8, and MCAM/MUC18 represent new treatment prospects.

Lysophosphatidic acid induces lymphangiogenesis and IL-8 production in vitro in human lymphatic endothelial cells.

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Transcriptional control of melanoma metastasis: the importance of the tumor microenvironment.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) and the metastatic phenotype are not very well defined. However, some of the genes involved in this process and their transcriptional regulation are beginning to be elucidated. For example, the switch from RGP to VGP and the metastatic phenotype is associated with loss of the AP-2α transcription factor. AP-2α regulates the expression of c-KIT, MMP-2, VEGF, and the adhesion molecule MCAM/MUC18. Recently, we reported that AP-2α also regulates two G-protein coupled receptors (GPCRs) PAR-1 and PAFR. In turn, the thrombin receptor, PAR-1, regulates the expression of the gap junction protein Connexin-43 and the tumor suppressor gene Maspin. Activation of PAR-1 also leads to overexpression and secretion of proangiogenic factors such as IL-8, uPA, VEGF, PDGF, as well certain integrins. PAR-1 also cooperates with PAFR to regulate the expression of the MCAM/MUC18 via phosphorylation of CREB. The ligands for these GPCRs, thrombin and PAF, are secreted by stromal cells, emphasizing the importance of the tumor microenvironment in melanoma metastasis. The metastatic phenotype of melanoma is also associated with overexpression and function of CREB/ATF-1. Loss of AP-2α and overexpression of CREB/ATF-1 results in the overexpression of MCAM/MUC18 which by itself contributes to melanoma metastasis by regulating the inhibitor of DNA binding-1 (Id-1). CREB/ATF-1 also regulates the angiogenic factor CYR-61. Our recent data indicate that CREB/ATF-1 regulates the expression of AP-2α, thus, supporting the notion that CREB is an important "master switch" in melanoma progression.

Back to the embryonic stage: Nodal as a biomarker for breast cancer progression.

The embryonic morphogen Nodal, a member of the transforming growth factor-ß superfamily, is not expressed in the majority of normal adult tissues. However, a growing body of evidence indicates that Nodal expression re-emerges in a number of human cancers, including melanoma, glioma, endometrial, and prostate cancers. Reactivation of Nodal signaling in these tumors contributes to their aggressiveness. Strizzi and colleagues, in a paper published in this issue of Breast Cancer Research, investigate the clinical significance of Nodal expression in breast cancer. They report that Nodal expression is significantly greater in malignant versus benign breast disease. More importantly, Nodal levels correlated with grading, staging, and lymph node involvement, independent of the estrogen receptor/progesterone receptor or HER2 status. Collectively, these data suggest that Nodal could serve as a potential biomarker for invasive disease and a potential therapeutic target in breast cancer.

Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis.

A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.

Dicer, Drosha, and outcomes in patients with ovarian cancer.

BACKGROUND: We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer. METHODS: We measured messenger RNA (mRNA) levels of Dicer and Drosha in specimens of invasive epithelial ovarian cancer from 111 patients, using a quantitative reverse-transcriptase-polymerase-chain-reaction assay, and compared the results with clinical outcomes. Validation was performed with the use of published microarray data from cohorts of patients with ovarian, breast, and lung cancer. Mutational analyses of genomic DNA from the Dicer and Drosha genes were performed in a subgroup of ovarian-cancer specimens. Dicer-dependent functional assays were performed by means of in vitro transfection with small interfering RNA (siRNA) and short hairpin RNA (shRNA). RESULTS: Levels of Dicer and Drosha mRNA correlated with the levels of expression of the corresponding protein and were decreased in 60% and 51% of ovarian-cancer specimens, respectively. Low Dicer expression was significantly associated with advanced tumor stage (P=0.007), and low Drosha expression with suboptimal surgical cytoreduction (P=0.02). Cancer specimens with both high Dicer expression and high Drosha expression were associated with increased median survival (>11 years, vs. 2.66 years for other subgroups; P<0.001). We found three independent predictors of reduced disease-specific survival in multivariate analyses: low Dicer expression (hazard ratio, 2.10; P=0.02), high-grade histologic features (hazard ratio, 2.46; P=0.03), and poor response to chemotherapy (hazard ratio, 3.95; P<0.001). Poor clinical outcomes among patients with low Dicer expression were validated in additional cohorts of patients. Rare missense mutations were found in the Dicer and Drosha genes, but their presence or absence did not correlate with the level of expression. Functional assays indicated that gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression. CONCLUSIONS: Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.

Receptor heterodimerization: a new mechanism for platelet-derived growth factor induced resistance to anti-epidermal growth factor receptor therapy for bladder cancer.

PURPOSE: Human bladder cancer cells resistant to anti-epidermal growth factor receptor therapy often co-express platelet-derived growth factor receptor-ß. We determined whether there is functional crosstalk between epidermal growth factor receptor and platelet-derived growth factor receptor-ß, and how this regulates biological functions in bladder cancer cases. MATERIALS AND METHODS: We determined heterodimerization and co-localization of epidermal growth factor receptor and platelet-derived growth factor receptor-ß by immunoprecipitation and confocal microscopy, respectively. We tested the antiproliferative effects of specific inhibitory monoclonal antibodies to each receptor by (3)H-thymidine uptake assay. We transfected the nonplatelet-derived growth factor receptor-ß expressing bladder cancer cell line UMUC5 with the platelet-derived growth factor receptor-ß gene. These cells were analyzed in vitro by (3)H-thymidine uptake and by Matrigel™ invasion assay, and in vivo for tumorigenicity, metastatic potential and orthotopic growth. In a treatment study nude mice were inoculated with orthotopic tumors and treated with the inhibitory antibodies alone and in combination. RESULTS: Immunoprecipitation revealed epidermal growth factor receptor/platelet-derived growth factor receptor-ß heterodimers in all platelet-derived growth factor receptor-ß expressing cell lines. Forced expression of platelet-derived growth factor receptor-ß in epidermal growth factor receptor sensitive UMUC5 cells (50% inhibitory concentration less than 10 nM) significantly decreased responsiveness to epidermal growth factor receptor inhibition (50% inhibitory concentration greater than 100 nM) and increased invasive potential 3-fold as well as tumorigenicity. Increased invasiveness was associated with epidermal growth factor triggered platelet-derived growth factor receptor-ß transactivation, increased mitogen activated protein kinase and glycogen synthase kinase-3ß phosphorylation, and decreased E-cadherin. Inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-ß receptors blocked cell invasion, decreased cell proliferation, reduced xenograft tumor growth and increased E-cadherin expression. CONCLUSIONS: In epidermal growth factor receptor expressing bladder cancer co-expression of platelet-derived growth factor receptor-ß has implications for tumor biology. Thus, it should be further evaluated as a strategy involving dual receptor targeting.

Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression. IMPLICATIONS: Our study describes a novel regulatory role of gene body DNA methylation-dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Silencing cAMP-response element-binding protein (CREB) identifies CYR61 as a tumor suppressor gene in melanoma.

Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis.

Crosstalk between protease-activated receptor 1 and platelet-activating factor receptor regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and melanoma metastasis.

The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis.

MIF inhibition as a strategy for overcoming resistance to immune checkpoint blockade therapy in melanoma.

Immune checkpoint blockade (ICB) has demonstrated an impressive outcome in patients with metastatic melanoma, yet, durable complete response; even with Ipilimumab/Nivolumab combo are under 30%. Primary and acquired resistance in response to ICB is commonly due to a tumor immune escape mechanism dictated by the tumor microenvironment (TME). Macrophage Migratory Inhibition Factor (MIF) has emerged as an immunosuppressive factor secreted in the TME. We have previously demonstrated that blockade of the MIF-CD74 signaling on macrophages and dendritic cells restored the anti-tumor immune response against melanoma. Here, we report that inhibition of the MIF-CD74 axis combined with ipilimumab could render resistant melanoma to better respond to anti-CTLA-4 treatment. We provide evidence that blocking the MIF-CD74 signaling potentiates CD8+ T-cells infiltration and drives pro-inflammatory M1 conversion of macrophages in the TME. Furthermore, MIF inhibition resulted in reprogramming the metabolic pathway by reducing lactate production, HIF-1α and PD-L1 expression in the resistant melanoma cells. Melanoma patient data extracted from the TCGA database supports the hypothesis that high MIF expression strongly correlates with poor response to ICB therapy. Our findings provide a rationale for combining anti-CTLA-4 with MIF inhibitors as a potential strategy to overcome resistance to ICB therapy in melanoma, turning a "cold" tumor into a "hot" one mediated by the activation of innate immunity and reprogramming of tumor metabolism and reduced PD-L1 expression in melanoma cells.

Author Correction: Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Prognostic Gene Expression Profiling in Cutaneous Melanoma: Identifying the Knowledge Gaps and Assessing the Clinical Benefit.

Importance: Use of prognostic gene expression profile (GEP) testing in cutaneous melanoma (CM) is rising despite a lack of endorsement as standard of care. Objective: To develop guidelines within the national Melanoma Prevention Working Group (MPWG) on integration of GEP testing into the management of patients with CM, including (1) review of published data using GEP tests, (2) definition of acceptable performance criteria, (3) current recommendations for use of GEP testing in clinical practice, and (4) considerations for future studies. Evidence Review: The MPWG members and other international melanoma specialists participated in 2 online surveys and then convened a summit meeting. Published data and meeting abstracts from 2015 to 2019 were reviewed. Findings: The MPWG members are optimistic about the future use of prognostic GEP testing to improve risk stratification and enhance clinical decision-making but acknowledge that current utility is limited by test performance in patients with stage I disease. Published studies of GEP testing have not evaluated results in the context of all relevant clinicopathologic factors or as predictors of regional nodal metastasis to replace sentinel lymph node biopsy (SLNB). The performance of GEP tests has generally been reported for small groups of patients representing particular tumor stages or in aggregate form, such that stage-specific performance cannot be ascertained, and without survival outcomes compared with data from the American Joint Committee on Cancer 8th edition melanoma staging system international database. There are significant challenges to performing clinical trials incorporating GEP testing with SLNB and adjuvant therapy. The MPWG members favor conducting retrospective studies that evaluate multiple GEP testing platforms on fully annotated archived samples before embarking on costly prospective studies and recommend avoiding routine use of GEP testing to direct patient management until prospective studies support their clinical utility. Conclusions and Relevance: More evidence is needed to support using GEP testing to inform recommendations regarding SLNB, intensity of follow-up or imaging surveillance, and postoperative adjuvant therapy. The MPWG recommends further research to assess the validity and clinical applicability of existing and emerging GEP tests. Decisions on performing GEP testing and patient management based on these results should only be made in the context of discussion of testing limitations with the patient or within a multidisciplinary group.

Expression profiling of Galectin-3-depleted melanoma cells reveals its major role in melanoma cell plasticity and vasculogenic mimicry.

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro, Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes, including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells, causing melanoma cell plasticity. These genes included vascular endothelial-cadherin, which plays a pivotal role in vasculogenic mimicry, as well as interleukin-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1, and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover, transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus, Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.