Melanoma-derived Cell Lines Research Articles

Growth requirement for methionine in human melanoma-derived cell lines with different levels of MMACHC expression and methylation

Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.

Broad-spectrum metastasis suppressing compounds and therapeutic uses thereof in human tumors

Previously, we have identified a novel human metastasis-inducing lncRNA (named SKAI1BC), that suppresses the KAI1/CD82 metastasis-suppressing gene and is upregulated in triple negative breast cancer and melanoma derived cell lines. Modeling of the SKAI1BC lncRNA secondary structure and its potential interaction with Inforna compounds, led us to identify several compounds that might bind the SKAI1BC lncRNA. We found that these compounds inhibit metastasis invasion and cell migration in culture, in all eight types of solid human cancers tested: several of which are the most lethal and/or frequent human malignancies. Moreover, in most cases, the mechanism of action of several of our compounds involves enhancement of KAI1/CD82 RNA level depending on the specific compound and the human tumor type. With the epigenetic inactivation of KAI1/CD82 in at least ten additional solid human cancers, this implies a very good chance to broaden the spectrum of human cancers affected by our compounds. This is the first time that modeling of a large lncRNA (> 700 bp) secondary structure followed by its potential interaction with Inforna like compounds database has led to the identification of potential biologically active small molecule drugs.

Overexpression of Krüppel-Like Factor 9 Enhances the Antitumor Properties of Paclitaxel in Malignant Melanoma-Derived Cell Lines.

Over the past decade, the treatment of metastatic melanoma has improved significantly due to the development of innovative therapies, such as drugs that target the BRAF/MAPK kinase pathway and the PD-1 pathway. However, these therapies do not work for all patients, highlighting the need for additional research on the pathophysiology of melanoma. Paclitaxel is a chemotherapeutic agent used when first-line treatments are unsuccessful; however, its efficacy is limited. Since Krüppel-like factor 9 (KLF9) (antioxidant repressor) is downregulated in melanoma, we propose that restoring KLF9 levels may sensitize malignant melanoma to chemotherapeutic agents, such as paclitaxel. We used adenovirus overexpression and siRNA technologies to assess the role of KLF9 in mediating the response of malignant melanoma-derived cell lines RPMI-7951 and A375 to paclitaxel treatment. We found that increasing KLF9 levels potentiates the effectiveness of paclitaxel, as shown by apoptotic parameters such as decreased cell viability, pro-caspase-3 activation, increased number of annexin V-positive cells, and reduction in nuclear proliferation marker (KI67). These results suggest that KLF9 may be a potential target for improving chemotherapeutic response in melanoma.

Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients’ metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2′-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHβ genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness.

Tumor-specific radiosensitizing effect of the ATM inhibitor AZD0156 in melanoma cells with low toxicity to healthy fibroblasts

PurposeDespite new treatment options, melanoma continues to have an unfavorable prognosis. DNA damage response (DDR) inhibitors are a promising drug class, especially in combination with chemotherapy (CT) or radiotherapy (RT). Manipulating DNA damage repair during RT is an opportunity to exploit the genomic instability of cancer cells and may lead to radiosensitizing effects in tumors that could improve cancer therapy.MethodsA panel of melanoma-derived cell lines of different origin were used to investigate toxicity-related clonogenic survival, cell death, and cell cycle distribution after treatment with a kinase inhibitor (KI) against ATM (AZD0156) or ATR (VE-822, berzosertib), irradiation with 2 Gy, or a combination of KI plus ionizing radiation (IR). Two fibroblast cell lines generated from healthy skin tissue were used as controls.ResultsClonogenic survival indicated a clear radiosensitizing effect of the ATM inhibitor (ATMi) AZD0156 in all melanoma cells in a synergistic manner, but not in healthy tissue fibroblasts. In contrast, the ATR inhibitor (ATRi) VE-822 led to additive enhancement of IR-related toxicity in most of the melanoma cells. Both inhibitors mainly increased cell death induction in combination with IR. In healthy fibroblasts, VE-822 plus IR led to higher cell death rates compared to AZD0156. A significant G2/M block was particularly induced in cancer cells when combining AZD0156 with IR.ConclusionATMi, in contrast to ATRi, resulted in synergistic radiosensitization regarding colony formation in melanoma cancer cells, while healthy tissue fibroblasts were merely affected with respect to cell death induction. In connection with an increased number of melanoma cells in the G2/M phase after ATMi plus IR treatment, ATMi seems to be superior to ATRi in melanoma cancer cell treatments when combined with RT.

Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics.

Abstract 348: Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma cells

Abstract Malignant melanoma is the most aggressive form of skin cancer. Although kinase inhibitors have proven to be efficient with promising clinical results, the development of drug resistance is one of the major issues in targeted therapy. CD133, also known asProminin-1, is a membrane glycoprotein studied extensively as a cell surface marker for cancer stem cells. In our previous studies, mebendazole, a repurposed broad spectrum anti-helminthic agent, inhibited the growth of NRAS mutant melanoma cells in combination with trametinib, a MEK inhibitor. However, a CD133-positive cancer stem cell population remain resistant to this targeted therapy. Use of RNA aptamers in diagnostic as well as therapeutics has been studied recently in different types of cancer cells. Drug delivery of aCD133 aptamer coupled with doxorubicin has been found to inhibit the growth of liver cancer cells. In this study, we targeted the cancer stem cells expressing CD133 with RNA aptamers, in combination with trametinib and mebendazole to sensitize the cells to the combination treatment. Patient derived melanoma cell lines, BAKP and POT, harboring BRAFWT/NRASQ61K and BRAFWT/NRASQ61R driver mutations, were co-transduced with aTet-on vector expressing CD133 and a Tet activator. The doxycycline-induced and uninduced cells were subjected to single agent treatment with 100 nM trametinib, 100 nM mebendazole or 100 nM CD133 RNA aptamer, as well as dual and triple agent combinations for 24 or 48 hours. XTT cell viability and apoptotic assays such as AnnexinV-PI flow cytometry analysis revealed that the triple combination with trametinib, mebendazole and the CD133 aptamer was the most effective in inducing apoptotic cell death, even in the doxycycline-induced CD133-overexpressing melanoma cells. Further studies will use preclinical in vivo mouse xenograft models to investigate the effects on tumor growth of CD133 RNA aptamers in combination treatments, potentially making it available for clinical trials aiming to improve patient response to therapeutics against drug-resistant melanoma cells. Citation Format: Yoga Haribabu, Raymond Chien, Ryyan Alobaidi, Li-Wei Kuo, Nusrat Islam, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal. Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 348.

Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness.

Simple SummaryTP53 is one of the most important tumor suppressor genes, which has been found to be mutated in more than half of human cancers and is considered the “Guardian of the genome”. However, it is rarely mutated in melanoma (less than 20% of cases). Although several cancer-oriented studies focus on p53 biology, only recently have researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of p53-dependent responses. In this study, we showed that melanoma-derived cell lines express a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable. For the first time, we reported that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and, potentially, migration. Lastly, we showed an increased expression of the potentially pro-oncogenic Δ40p53β isoform and a decrease in the tumor-suppressive TAp73β isoform in melanoma cells resistant to vemurafenib (BRAF inhibitor). With this study, we suggest that p53 family isoforms play a significant role in melanoma cells’ aggressiveness.Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells’ aggressiveness.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma

Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.

Heme oxygenase promotes B-Raf-dependent melanosphere formation.

Biosynthesis and degradation of heme, an iron-bound protoporphyrin molecule utilized by a wide variety of metabolic processes, are tightly regulated. Two closely related enzymes, heme oxygenase 1 (HMOX1) and heme oxygenase 2 (HMOX2), degrade free heme to produce carbon monoxide, Fe2+ , and biliverdin. HMOX1 expression is controlled via the transcriptional activator, NFE2L2, and the transcriptional repressor, Bach1. Transcription of HMOX1 and other NFE2L2-dependent genes is increased in response to electrophilic and reactive oxygen species. Many tumor-derived cell lines have elevated levels of NFE2L2. Elevated expression of NFE2L2-dependent genes contributes to tumor growth and acquired resistance to therapies. Here, we report a novel role for heme oxygenase activity in melanosphere formation by human melanoma-derived cell lines. Transcriptional induction of HMOX1 through derepression of Bach1 or transcriptional activation of HMOX2 by oncogenic B-RafV600E results in increased melanosphere formation. Genetic ablation of HMOX1 diminishes melanosphere formation. Further, inhibition of heme oxygenase activity with tin protoporphyrin markedly reduces melanosphere formation driven by either Bach1 derepression or B-RafV600E expression. Global transcriptome analyses implicate genes involved in focal adhesion and extracellular matrix interactions in melanosphere formation.

Exhaustion of CD4+ T-cells mediated by the Kynurenine Pathway in Melanoma

Kynurenine pathway (KP) activation by the enzymatic activity of indoleamine 2,3-dioxygenase1 (IDO1) and kynurenine (KYN) production represents an attractive target for reducing tumour progression and improving anti-tumour immunity in multiple cancers. However, immunomodulatory properties of other KP metabolites such as 3-hydroxy kynurenine (3-HK) and kynurenic acid (KYNA) are poorly understood. The association of the kynurenine metabolic pathway with T-cell status in the tumour microenvironment were characterized, using gene expression data of 368 cutaneous skin melanoma (SKCM) patients from the TCGA cohort. Based on the identified correlations, we characterized the production of KYN, 3-HK, and KYNA in vitro using melanoma-derived cell lines and primary CD4+ CD25− T-cells. Activation of the CD4+ T-cells produced IFNγ, which yielded increased levels of KYN and KYNA. Concurrently, kynurenine 3-monooxygenase (KMO) expression and proliferation of CD4+ T-cells were reduced, whereas exhaustion markers such as PD-L1, AHR, FOXP3, and CTLA4 were increased. Additionally, an analysis of the correlation network reconstructed using TCGA-SKCM emphasized KMO and KYNU with high variability among BRAF wild-type compared with V600E, which underscored their role in distinct CD4+ T-cell behavior in tumour immunity. Our results suggest that, in addition to IDO1, there is an alternative immune regulatory mechanism associated with the lower KMO expression and the higher KYNA production, which contributes to dysfunctional effector CD4+ T-cell response.

Abstract 826: Global DNA methylation levels regulate PD-L1 expression in melanoma

Abstract The programmed death-ligand 1 (PD-L1) receptor is an important immune checkpoint and is often upregulated in cancer cells to allow immune evasion. In melanoma, the patients with PD-L1 expression and absense of tumour infiltrating lymphocytes (TILs) (i.e. “constitutive PD-L1 or PD-L1CON”) show worse response rates and prognosis than patients with PD-L1 expression and the presence of TILs (i.e. “inducible PD-L1 or PD-L1IND”). However, how PD-L1 expression is regulated in melanoma cells remains elusive. Understanding the mechanisms of how PD-L1 is regulated is important for predicting responses for anti-PD-L1 treatment and for developing new combinatorial therapies. We hypothesised that epigenetic state regulates constitutive and inducible PD-L1 expression in melanoma. To address the hypothesis, we have generated whole-genome scale DNA methylomes (using reduced representation bisulfite sequencing) and transcriptomes (RNA-Seq) for patient derived melanoma cell lines (in PD-L1IND and PD-L1CON groups). We discovered extensive global hypomethylation in the constitutive lines, particularly pronounced in intergenic repeat regions and gene bodies. A high proportion of hypomethylated regions exhibited dichotomous methylation patterns indicating a common regulatory mechanism between the PD-L1IND and PD-L1CON lines. RNA-Sequening data indicated that the hypomethylated state of the PD-L1CON cells was correlated with higher upregulation of the differentially expressed genes at a global-scale and the upregulated genes were associated with cancer hallmark properties. The upregulated genes exhibited signatures of viral mimicry and cytosolic sensing of dsRNA similar to what has been observed after DNA methyltransferase inhibitor (DNMTi) treatment in cancers. DNMTi treatment increased PD-L1 transcription in the PD-L1IND cell lines. PD-L1CON samples showed greater resistance to DNMTi compared to PD-L1 negative samples. This was observed at protein-coding genes, transposable elements and long non-coding RNAs. Moreover, genes involved in the innate immune pathway was strongly increased upon DNMTi mediated hypomethylation in the PD-L1IND cell lines (FDR-adjusted P-value = 0.02), however there was no expression change in the PD-L1 positive cells (Adjusted P-value = 1.0). Furthermore, we found that the IRF1 transcription factor, which plays a critical role in the innate immune pathway and also binds to the PD-L1 promoter, is significantly upregulated upon DNMTi in the PD-L1 negative cell lines (Adjusted P-value = 0.01) but not in the PD-L1 positive cells (Adjusted P-value = 0.9). These results show that global hypomethylation levels regulate PD-L1 expression in melanoma. We believe these results are the first to show that DNA methylation levels play a role in regulating PD-L1 on melanoma and suggest they may have important implications for combined treatments targeting methylation (DNMTi) and PD1/PD-L1 (anti-PD1 antibodies). Citation Format: Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger, Peter A. Stockwell, Matthew Parry, Jyoti Motwani, Stuart J. Gallagher, Elena Shklovskaya, Jessamy Tiffen, Peter Hersey, Michael R. Eccles. Global DNA methylation levels regulate PD-L1 expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 826.

The X-linked inhibitor of apoptosis protein (XIAP) is involved in melanoma invasion by regulating cell migration and survival.

The X-linked inhibitor of apoptosis (XIAP) is a potent cellular inhibitor of apoptosis, based on its unique capability to bind and to inhibit caspases. However, XIAP is also involved in a number of additional cellular activities independent of its caspase inhibitory function. The aim of this study was to investigate whether modulation of XIAP expression affects apoptosis-independent functions of XIAP in melanoma cells, restores their sensitivity to apoptosis and/or affects their invasive and metastatic capacities. XIAP protein levels were analyzed by immunohistochemical staining of human tissues and by Western blotting of melanoma cell lysates. The effects of pharmacological inhibition or of XIAP down-regulation were investigated using ex-vivo and transwell invasion assays. The biological effects of XIAP down-regulation on melanoma cells were analyzed in vitro using BrdU/PI, nucleosome quantification, adhesion and migration assays. In addition, new XIAP binding partners were identified by co-immunoprecipitation followed by mass spectrometry. Here we found that the expression of XIAP is increased in metastatic melanomas and in invasive melanoma-derived cell lines. We also found that the bivalent IAP antagonist birinapant significantly reduced the invasive capability of melanoma cells. This reduction could be reproduced by downregulating XIAP in melanoma cells. Furthermore, we found that the migration of melanoma cells and the formation of focal adhesions at cellular borders on fibronectin-coated surfaces were significantly reduced upon XIAP knockdown. This reduction may depend on an altered vimentin-XIAP association, since we identified vimentin as a new binding partner of XIAP. As a corollary of these molecular alterations, we found that XIAP down-regulation in melanoma cells led to a significant decrease in invasion of dermal skin equivalents. From our data we conclude that XIAP acts as a multifunctional pro-metastatic protein in skin melanomas and, as a consequence, that XIAP may serve as a therapeutic target for these melanomas.

Abstract 3684: HDAC inhibitors restore BRAF inhibitor sensitivity by altering PI3K and survival signaling in melanoma

Abstract Mutations in BRAF activate oncogenic MAPK signaling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signaling.Here we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo- derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat synergistically induced caspase-dependent apoptotic cell death. HDAC inhibition decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases including AXL, YAP, ERBB2 and ERBB3 but not EGFR or ERBB4. Both NOXA and BIM proteins were implicated as causing cell death, but in a cell line specific manner. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance is due to activation of MAPK-independent pathways. This work identifies patients that may respond well to this drug combination and demonstrates the necessity of disrupting multiple cell survival pathways in melanoma. Citation Format: Peter Hersey, Stuart J. Gallagher. HDAC inhibitors restore BRAF inhibitor sensitivity by altering PI3K and survival signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3684.

Abstract 714: A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells

Abstract Metastatic melanoma, leads to the highest number of skin cancer related deaths. Checkpoint inhibitor therapy has witnessed a high success rate in melanoma patients with anti-CTLA-4 and anti-PD-1. However, checkpoint inhibitor molecules and their compensatory stimulatory counterparts are widely expressed on T cells and antigen presenting cells suggesting a robust redundancy in these molecules as clinical targets. Some of these molecules that include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are also expressed on tumor cells. Their role in regulating an immune response whether it is cell killing or immune evasion remains to be elucidated. We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2. We screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR which revealed significant heterogeneity in expression of these molecules compared to normal adult melanocytes under normal conditions; underscoring the importance of understanding tumor tissue pleiotropy prior to designing a therapeutic regimen. Surprisingly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were upregulated differentially in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression on treatment with 10μM vemurafenib (PLX4032), a BRAFV600E inhibitor, for 24 hours. However the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma adaptation and escape from immune recognition. Exposing melanoma cells to classical activated dendritic cell cytokines, IL-6 and IL-12, led to a differential expression of these molecules. Additionally, experiments using tumor lysate loaded dendritic cells to study activation revealed an ability to modulate immune activation correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line. Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed in tumor cells. With eighty percent of melanoma patients being positive for the BRAFV600E lesion, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Marc Wallack, Jan Geliebter, Raj K. Tiwari. A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 714.

Abstract 3656: Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells

Abstract Melanoma is one of the deadliest forms of skin cancer, with a dim prognosis when metastasized, leading to the highest number of skin cancer related deaths. In recent years there has been a focus on the use of checkpoint inhibitor therapies like anti-CTLA-4, anti-PD-1 and anti-PD-L1 to treat melanoma. PD-L1, HVEM and VISTA expression on cancer cells has been shown to promote immune evasion and tumor survival. CTLA-4, when engaged on tumor cells leads to their apoptosis and LIGHT signaling leads to recruitment of T cells and effective tumor clearance. Thus, since these molecules do not function in isolation, we need to consider tissue pleiotropy and the expression of co-stimulatory and co-inhibitory molecules on tumor tissue. Our laboratory has characterized and established five primary patient derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM and GLM-2. In an effort to understand tumor tissue pleiotropy, we conducted a comprehensive expression pattern screening of eight co-inhibitory and ten co-stimulatory molecules by RT-PCR. Among others, VISTA, a CD4+ T cell suppressor, HVEM, an immune evasion regulator, LAG3 and TIM3, potential immune checkpoint targets, were seen to be differentially expressed in these primary cell lines. Current therapies target CTLA-4, PD-1 and PD-L1, which also exhibit varying expression in our primary cell lines, pointing to the importance of considering tissue expression of these molecules when administering these novel immunotherapies. Additionally, 80% of melanoma patients are positive for the BRAFV600E genetic lesion and are administered vemurafenib (PLX4032), an inhibitor of the overactive mutated BRAF. Our in vitro studies show that treatment with PLX4032, changed the expression of CTLA-4 and PD-L1 in these primary cells. We observed a decrease in PD-L1 protein expression in MEL-V and GLM-2 and an increase in PD-L1 protein expression in MEL-2 and KFM, the four BRAFV600E positive cell lines. CTLA-4 protein expression demonstrated an upward trend on treatment with PLX4032. This synergy observed between treatment with drugs targeting genetic lesions and the expression of immunomodulatory molecules warrants characterization of tumor biopsies prior to designing an effective combinatorial therapy regime. Our long term goal is to optimize combinatorial immune and drug therapies directed against both co-stimulators and checkpoint inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Tuli, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2017-3656

Altered integrin expression patterns shown by microarray in human cutaneous melanoma.

A large variety of molecular pathways in melanoma progression suggests that no individual molecular alteration is crucial in itself. Our aim was to define the molecular alterations underlying metastasis formation. Gene expression profiling was performed using microarray and qRT-PCR to define alterations between matched primary and metastatic melanoma cell lines. These data were integrated with publicly available unmatched tissue data. The invasiveness of cell lines was determined by Matrigel invasion assays and invasive clones from primary melanoma-derived cell lines were also selected. Two metastatic cell line models were created: the regional lymph node WM983A-WM983A-WM983B and the distant lung WM793B-WM793B-1205Lu metastatic models. The majority of metastasis genes were downregulated and enriched in adhesion and ITGA6-B4 pathways. Upregulation of immune pathways was characteristic of distant metastases, whereas increased Rap1 signaling was specific for regional (sub)cutaneous metastases. qRT-PCR analysis of selected integrins (A2, A3, A4, A9, B5, B8, A6, B1, and B3) highlighted the possible importance of ITGA3/4 and B8 in the metastatic process, distinguishing regional and distant metastases. We identified functionally relevant gene clusters that influenced metastasis formation. Our data provide further evidence that integrin expression patterns may be important in distant metastasis formation.

Tenascin C to regulate the function of sHER3, an endogenous inhibitor of neuregulin signaling.

e23002 Background: HER3 expression is associated with poor prognosis in melanoma patients but the mechanistic basis has not been determined. An endogenous secreted isoform of HER3, p85-sHER3, has been shown by us to inhibit neuregulin-1 mediated, Akt-dependent breast and ovarian cancer cell growth in vitro. Here we demonstrate that p85-sHER3 also regulates neuregulin-dependent melanoma cell migration via an Akt- independent, tenascin C-dependent mechanism. Methods: Immunoblot analysis and RT-PCR were used to study HER3 and p85-sHER expression. Cell proliferation and chamber assays were conducted to determine the effect of p85-sHER3 on melanoma cell growth and migration. Mass spectrometry (MS) was performed to identify sHER3-interacting proteins. CRSPR in vitro editing methods were used to knock out tenascin C (TNC) expression. Results: HER3 and p85-sHER3 were expressed in 5 of 6 human melanoma-derived cell lines tested, with exception of SK-MEL-103 which did not express either gene product. Exogenous addition of 10 nM p85-sHER3 inhibited growth in 3 cells lines, although Akt phosphorylation was not reduced. Protein extracts from M14-MEL cell conditioned media were characterized by MS, and identified p85-sHER3 and TNC. Their association was confirmed by co-immunoprecipitation. sHER3 was further shown to inhibit neuregulin stimulated cell migration in 2 cell lines which exhibited high levels of TNC. CRSPR knockout of TNC in these cell lines (but not in control lines) eliminated sHER3 inhibition of neuregulin stimulated melanoma cell migration. Conclusions: Our results suggest that p85-sHER3, predominantly secreted by HER3-expressing melanoma cells, inhibits melanoma cell proliferation and migration via an Akt-independent mechanism, which is likely achieved through interaction(s) with TNC. TNC promotes an intermediate adhesive state favoring cell migration and this state appears to be mediated by Rho-associated kinase signaling. The Rho inhibitor, CCG-203971 has been shown to inhibit melanoma cell migration. Together, these results suggest the existence of a TNC and HER3-dependent signaling pathway in melanoma that regulates cell migration, and therefore may be correlated with poor patient survival.

Antiproliferative Effects of 1\u03b1-OH-vitD3 in Malignant Melanoma: Potential Therapeutic implications

Early detection and surgery represent the mainstay of treatment for superficial melanoma, but for high risk lesions (Breslow’s thickness >0.75 mm) an effective adjuvant therapy is lacking. Vitamin D insufficiency plays a relevant role in cancer biology. The biological effects of 1α hydroxycholecalciferol on experimental melanoma models were investigated. 105 melanoma patients were checked for 25-hydroxycholecalciferol (circulating vitamin D) serum levels. Human derived melanoma cell lines and in vivo xenografts were used for studying 1α-hydroxycholecalciferol-mediated biological effects on cell proliferation and tumor growth. 99 out of 105 (94%) melanoma patients had insufficient 25-hydroxycholecalciferol serum levels. Interestingly among the six with vitamin D in the normal range, five had a diagnosis of in situ/microinvasive melanoma. Treatment with 1α-hydroxycholecalciferol induced antiproliferative effects on melanoma cells in vitro and in vivo, modulating the expression of cell cycle key regulatory molecules. Cell cycle arrest in G1 or G2 phase was invariably observed in vitamin D treated melanoma cells. The antiproliferative activity induced by 1α-hydroxycholecalciferol in experimental melanoma models, together with the discovery of insufficient 25-hydroxycholecalciferol serum levels in melanoma patients, provide the rationale for using vitamin D in melanoma adjuvant therapy, alone or in association with other therapeutic options.

Evaluation of anti-diabetic and anti-tumoral activities of bioactive compounds from Phoenix dactylifera L's leaf: In vitro and in vivo approach.

Among various chronic disorders, cancer and diabetes mellitus are the most common disorders. This study was designed to evaluate the effectiveness of hydroalcoholic extract of Phoenix dactylifera L. leaves (HEPdL) in animal models of type II diabetes in vitro/in vivo and in a human melanoma-derived cell line (IGR-39). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was also performed to determine the amount of phenolic and flavonoid compounds in this plant. The physicochemical results by LC-MS/MS analysis of HEPdL showed the presence of 10 phenolic compounds. The in vitro study showed that the extract exhibited a more specific and potent inhibitor of α-glucosidase than α-amylase with an IC50 value of 20±1μg/mL and 30±0.8μg/mL, respectively. More importantly, the in vivo study of the postprandial hyperglycemia activity with (20mg/kg) of HEPdL showed a decrease in plasma glucose levels after 60min in resemblance to the glucor (acarbose) (50mg/kg) effect. The oral administration of HEPdL (20mg/kg) in alloxan-induced diabetic mices for 28days showed a more significant anti-diabetic activity than that of the drug (50mg/kg). Moreover, cytotoxicity effects of HEPdL in IGR-39 cancer cell lines were tested by MTT assay. This extract was effective in inhibiting cancer cells growth (IGR-39) at dose 35 and 75μg/mL. These results confirm ethnopharmacological significance of the plant and could be taken further for the development of an effective pharmaceutical drug against diabetes and cancer.