Melanoma Lines Research Articles

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

BackgroundMicroenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described.MethodsWe studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis.Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression.ResultsWe found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive.We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis.A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases.ConclusionsA crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

Effect of Differences in Metabolic Activity of Melanoma Models on Response to Lonidamine plus Doxorubicin

Lonidamine (LND), a metabolic modulator, sensitizes DB-1 human melanoma to doxorubicin (DOX) chemotherapy by acidifying and de-energizing the tumor. This report compares the effects of LND on two human melanoma lines, DB-1 and WM983B, which exhibit different metabolic properties. Using liquid chromatography mass spectrometry and Seahorse analysis, we show that DB-1 was more glycolytic than WM983B in vitro. 31P magnetic resonance spectroscopy (MRS) indicates that LND (100 mg/kg, i.p.) induces similar selective acidification and de-energization of WM983B xenografts in immunosuppressed mice. Over three hours, intracellular pH (pHi) of WM983B decreased from 6.91 ± 0.03 to 6.59 ± 0.10 (p = 0.03), whereas extracellular pH (pHe) of this tumor changed from 7.03 ± 0.05 to 6.89 ± 0.06 (p = 0.19). A decline in bioenergetics (β-NTP/Pi) of 55 ± 5.0% (p = 0.03) accompanied the decline in pHi of WM983B. Using 1H MRS with a selective multiquantum pulse sequence and Hadamard localization, we show that LND induced a significant increase in tumor lactate levels (p < 0.01). LND pre-treatment followed by DOX (10 mg/kg, i.v.) produced a growth delay of 13.7 days in WM983B (p < 0.01 versus control), a growth delay significantly smaller than the 25.4 days that occurred with DB-1 (p = 0.03 versus WM983B). Differences in relative levels of glycolysis may produce differential therapeutic responses of DB-1 and WM983B melanomas.

Biomimetic quantum dot-labeled B16F10 murine melanoma cells as a tool to monitor early steps of lung metastasis by in vivo imaging.

BackgroundNumerous studies have proposed the use of fluorescent semiconductor nanoparticles or quantum dots (QDs) as novel tools to label cells and tumors. However, QD applications are limited by their toxicity in biological systems and little is known about whether QDs affect the capacity of cancer cells to metastasize. Previously, we described the “biomimetic” synthesis of CdTe-QDs (QDs-glutathione [GSH]) with increased biocompatibility and the potential utility in labeling cells.PurposeIn order to determine the feasibility of using QDs-GSH as a tool for tracking tumor cells during early metastasis, we characterized here for the first time, the in vitro and in vivo effects of the incorporation of green or red biomimetic QDs-GSH into B16F10 cells, a syngeneic mouse melanoma line for metastasis assays in C57BL/6 mice.MethodsB16F10 cells were labeled with green or red biomimetic QDs-GSH in the presence or absence of n-acetylcysteine. Then, migration, invasion and proliferation of labeled B16F10 were evaluated in vitro. Finally, the B16F10 cells labeled with red QDs-GSH were used to monitor in vivo lung metastasis at early time points (5 minutes to 24 hours) or after 21 days in C57BL/6 mice.ResultsWe developed a methodology that allows obtaining QDs-GSH-labeled B16F10 cells (nearly 100% viable labeled cells), which remained viable for at least 5 days and migrated similarly to control cells. However, proliferation, invasion, and the capacity to form metastatic nodules in the lungs were severely attenuated. Fluorescence imaging revealed that distribution/accumulation of QDs-GSH-labeled B16F10 cells could be tracked following injection into C57BL/6 mice (syngeneic preclinical metastasis model) and that these cells preferentially accumulated in the perialveolar area in lungs as early as 5 minutes post-injection.ConclusionThe methodology described here represents a useful alternative for monitoring initial events during tumor cell metastasis.

Antiproliferative Activity of Non-Calcemic Vitamin D Analogs on Human Melanoma Lines in Relation to VDR and PDIA3 Receptors.

Vitamin D is a precursor for secosteroidal hormones, which demonstrate pleiotropic biological activities, including the regulation of growth and the differentiation of normal and malignant cells. Our previous studies have indicated that the inhibition of melanoma proliferation by a short side-chain, low calcemic analog of vitamin D—21(OH)pD is not fully dependent on the expression of vitamin D receptor (VDR). We have examined the effects of classic vitamin D metabolites, 1,25(OH)2D3 and 25(OH)D3, and two low calcemic vitamin D analogs, (21(OH)pD and calcipotriol), on proliferation, mRNA expression and vitamin D receptor (VDR) translocation in three human melanoma cell lines: WM98, A375 and SK-MEL-188b (subline b of SK-MEL-188, which lost responsiveness to 1,25(OH)2D3 and became VDR−/−CYP27B1−/−). All tested compounds efficiently inhibited the proliferation of WM98 and A375 melanoma cells except SK-MEL-188b, in which only the short side-chain vitamin D analog—21(OH)pD was effective. Overall, 21(OH)pD was the most potent compound in all three melanoma cell lines in the study. The lack of responsiveness of SK-MEL-188b to 1,25(OH)2D3, 25(OH)D3 and calcipotriol is explained by a lack of characteristic transcripts for the VDR, its splicing variants as well as for vitamin D-activating enzyme CYP27B1. On the other hand, the expression of VDR and its splicing variants and other vitamin D related genes (RXR, PDIA3, CYP3A4, CYP2R1, CYP27B1, CYP24A1 and CYP11A1) was detected in WM98 and A375 melanomas with the transcript levels being modulated by vitamin D analogs. The expression of VDR isoforms in WM98 cells was stimulated strongly by calcipotriol. The antiproliferative activities of 21(OH)pD appear not to require VDR translocation to the nucleus, which explains the high efficacy of this noncalcemic pregnacalciferol analog in SK-MEL-188b melanoma, that is, VDR−/−. Therefore, we propose that 21(OH)pD is a good candidate for melanoma therapy, although the mechanism of its action remains to be defined.

Melanoma LAMP-2C Modulates Tumor Growth and Autophagy.

Autophagy plays critical but diverse roles in cellular quality control and homeostasis potentially checking tumor development by removing mutated or damaged macromolecules, while conversely fostering tumor survival by supplying essential nutrients during cancer progression. This report documents a novel inhibitory role for a lysosome-associated membrane protein, LAMP-2C in modulating autophagy and melanoma cell growth in vitro and in vivo. Solid tumors such as melanomas encounter a variety of stresses in vivo including inflammatory cytokines produced by infiltrating lymphocytes directed at limiting tumor growth and spread. Here, we report that in response to the anti-tumor, pro-inflammatory cytokine interferon-gamma, melanoma cell expression of LAMP2C mRNA significantly increased. These results prompted an investigation of whether increased melanoma cell expression of LAMP-2C might represent a mechanism to control or limit human melanoma growth and survival. In this study, enhanced expression of human LAMP-2C in melanoma cells perturbed macroautophagy and chaperone-mediated autophagy in several human melanoma lines. In vitro analysis showed increasing LAMP-2C expression in a melanoma cell line, triggered reduced cellular LAMP-2A and LAMP-2B protein expression. Melanoma cells with enhanced LAMP-2C expression displayed increased cell cycle arrest, increased expression of the cell cycle regulators Chk1 and p21, and greater apoptosis and necrosis in several cell lines tested. The increased abundance of Chk1 protein in melanoma cells with increased LAMP-2C expression was not due to higher CHEK1 mRNA levels, but rather an increase in Chk1 protein abundance including Chk1 molecules phosphorylated at Ser345. Human melanoma cell xenografts with increased LAMP-2C expression, displayed reduced growth in immune compromised murine hosts. Melanomas with high LAMP-2C expression showed increased necrosis and reduced cell density upon histological analysis. These results reveal a novel role for LAMP-2C in negatively regulating melanoma growth and survival.

Abstract 5518: Bi-allelic loss of CDKN2A initiates melanoma invasion and metastasis via E2F1-BRN2 axis

Abstract CDKN2A acts as a critical tumor suppressor in melanoma, as evidenced by frequent loss of function mutations and deletion. Loss of CDKN2A is believed to permit escape from senescent pre-neoplastic cell populations through relieve of a cell cycle block mediated by its two gene products. We performed a comprehensive analysis of CDKN2A gene status, mRNA and protein expression levels of p16 and p14 in a cohort of melanomas and their adjacent pre-neoplastic lesions and observed that bi-allelic CDKN2A loss coincides with the progression stage when primary melanomas become invasive. In melanoma lines, p16INK4A, one of the protein products of the CDKN2A locus, is a potent barrier to metastasis, independent of its known role inhibiting cell proliferation. We genetically engineered primary human melanocytes to harbor CDKN2A deletions and/or BRAF V600E mutation at their endogenous BRAF locus. Using this physiologic model for the early phases of neoplastic transformation, we found no evidence for BRAF-induced senescence, rather observing that p16INK4A loss activates a master regulator of melanoma invasion, BRN2, through Rb-E2F1 pathway. These results demonstrate that one of the most frequently altered genes across human cancers, CDKN2A, has an unexpected novel role in inhibiting cellular invasion through lineage specific transcription factors and acts as an essential gatekeeper of early metastatic dissemination. Citation Format: Hanlin Zeng, Aparna Jorapur, A. Hunter Shain, Ursula E. Lang, Rodrigo Torres, Yuntian Zhang, Thomas Botton, Jue Lin, Andrew S. Mcneal, Matthew Donne, Ingmar N. Bastian, Jeffrey North, Laura Pincus, Richard Yu, Beth S. Ruben, Nancy Joseph, Iwei Ye, Boris C. Bastian, Robert L. Judson. Bi-allelic loss of CDKN2A initiates melanoma invasion and metastasis via E2F1-BRN2 axis [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 5518.

Abstract 3541: Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma

Abstract Purpose: Abnormal activation of the NF-κB pathway promotes a more aggressive phenotype of cutaneous malignant melanoma. Understanding the mechanisms regulating the NF-κB pathway in melanoma is of critical importance. KPC1 (RNF123) is an E3 ubiquitin ligase that leads to proteasomal processing of precursor NF-κB1 p105 into mature p50, one of the most important steps in the NF-κB pathway regulation. We demonstrated novel epigenetic mechanisms affecting KPC1 expression that lead to an abnormal activation of the NF-κB pathway, which was significant during tumor progression in melanoma patients. Experimental Design: Initially using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including microRNA targeted interaction and DNA methylation. The clinical impact of KPC1 expression and these epigenetic regulations were further assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and melanoma TCGA database cohort (n=370). High-throughput RNA sequencing, reverse-phase protein array and human methylation 450k platform were utilized for comprehensive analyses. Results: Initially using metastatic melanoma cell lines, we verified that KPC1 promotes processing of NF-κB1 p105 into p50, thereby modulates NF-κB-target gene expression and suppresses melanoma cell proliferation. Concordantly in melanoma tissue, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (JWCI cohort stage I/II p=0.013, stage III p=0.004), whereby low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (tissue micro-arrays, n=137, Hazard Ratio 1.810, p=0.006). Furthermore, epigenetic mechanisms regulating KPC1, particularly miR-155-5p and DNA methylation level at its promoter region, was shown to be significant in melanoma lines. This regulatory mechanism was validated in clinical melanoma tissues. High miR-155-5p expression, which is negatively regulated by its promoter DNA methylation level (melanoma TCGA database cohort; Pearson's r -0.455, p&amp;lt;0.001), is significantly associated with KPC1 down-regulation (JWCI cohort; p=0.028, melanoma TCGA database cohort; p=0.003). Conclusions: We identified miR-155-5p, which is epigenetically controlled by its promoter methylation, has a regulatory role on KPC1 expression. These interactions promote to down-regulation of KPC1 and abnormal NF-κB pathway activation, leading to highly proliferative melanoma cells and poor clinical outcomes. These findings suggest utility of KPC1 expression level for stratification of stage IV melanoma patients, and the importance of the miR-155-5p-KPC1-NF-κB-axis in controlling melanoma proliferation. Citation Format: Yuuki Iida, Aaron Ciechanover, Diego M. Marzese, Keisuke Hata, Matias Bustos, Shigeshi Ono, Jinhua Wang, Matthew P. Salomon, Kevin Tran, Yelena Kravtsova-Ivantsiv, Gordon B. Mills, Michael A. Davies, Dave S.B. Hoon. Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic 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 3541.

10 Extracellular vesicles that carry signalling networks drive phenocopying of migratory behaviour between cancer cells in vivo

IntroductionTumours consist of heterogeneous populations of cancer cells with various abilities to metastasize. Recent data shows that cancer cells from different subtypes exchange biomolecules through extracellular vesicles (EVs), influencing each other’s metastatic behaviour. How EVs can mediate this effect is still largely unknown, especially in tumours from the same subtype in which molecular differences are small but instrumental drivers of metastasis.Material and methodsHere, we study EVs shed by two B16 melanoma lines with different metastatic potential. Using label-free tandem mass spectrometry and RNA sequencing we profile the tumour microenvironmental EVs shed by these clones in vivo. We use the Cre-LoxP system to monitor EV transfer in vivo in combination with intravital microscopy to study phenotypic changes induced by EV exchange.Results and discussionsWe show that melanoma cells with different metastatic capacities functionally exchange EVs in vivo. EVs shed into the tumour microenvironment by cancer cells contain interconnected protein and RNA signalling networks involved in a variety of processes, including cell migration. Transfer of EVs from highly metastatic and migratory cells copies their phenotype to other cancer cells.ConclusionWe show that cancer cells influence each other’s behaviour in the tumour microenvironment through EVs. Profiling of tumor-derived EVs suggests that this is mediated by a diverse range of EV-molecules that together amplify numerous nodes of signalling networks in recipient cells.

Bi-allelic Loss of CDKN2A Initiates Melanoma Invasion via BRN2 Activation

Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage-restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16INK4A permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.

Generation of murine tumor cell lines to assess in vivo anti-tumor immune responses to melanoma.

Abstract Mouse models of cancer remain the preferred means to assess the efficacy of anti-cancer therapies, and several models have been used to address the effect of cancer drugs or in the discovery of biomarkers. The Yale University Mutant melanoma lines, YUMM1.5 and YUMMER1.7 murine tumor models were developed from the BrafV600E/Pten/−/−Cdkn2a−/− engineered mouse model to more closely reflect poorly immunogenic and highly mutated melanomas, respectively, that are driven by human disease-relevant mutations; however, the dominant T cell-recognized epitopes specific for these tumors remains unknown. We therefore sought to engineer OVA-expressing versions of the YUMM1.5 and YUMMER1.7 tumor lines, hereafter referred to as YUMM1.5-OVA and YUMMER1.7-OVA. Using stable transfection to insert a plasmid expressing full-length secretory ovalbumin, we have generated polyclonal and monoclonal cell lines that generate an in vivo OVA-specific response as validated by MHC class I tetramer staining and cytokine production following OVA peptide stimulation. Together, these two new models for melanoma tumors will allow us to assess intrinsic and extrinsic mechanisms regulating the generation of effective and recovery of exhausted T cell responses to melanoma in vivo.

Mutated MITF-E87R in Melanoma Enhances Tumor Progression via S100A4.

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.

Characterization of Drug Resistance Protein Expression Levels in Human Melanoma Cells and Subsequent Treatment with Antifungal Agents

The multidrug resistance (MDR) phenotype allows cancer cells to display decreased sensitivity to chemotherapy. A well‐characterized drug resistance gene in cancer is MDR1, which encodes for the p‐glycoprotein/ATP‐binding cassette (ABC) transporter. Lesser known are the roles of the multidrug resistance‐associated protein 1 (MRP1) and lung resistance‐related protein (LRP) in melanoma. Because of the previously demonstrated presence of MRP1 and LRP in human melanoma cells and the continued difficulty of overcoming MDR in melanoma treatment, here we characterized the expression levels of drug resistance genes in melanoma cell lines derived from human patients and determined their susceptibility to novel antifungal treatment. Expression levels were determined by real‐time PCR (qPCR) and immunoblot analyses. Susceptibility of cell lines to antifungal agents was analyzed by flow cytometry, cell proliferation assays, and immunoblotting after drug treatments.Several lines of human melanoma and normal skin cells were analyzed. MDR1 expression was observed in multiple melanoma lines, while increased levels of MRP1 were observed in normal skin cells and one melanoma line. Interestingly, LRP expression was highest in normal skin cells and several melanoma lines. Novel treatment with clotrimazole, an agent previously shown to eradicate melanoma cells, caused significant decreases in proliferation and increases in cell death in all MDR melanoma lines. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cells. These results indicated that clotrimazole selectively induces cytotoxicity in drug‐resistant melanoma lines. However, decreased efficacy was observed in melanoma lines that overexpressed LRP, as compared to all other melanoma lines that overexpressed MDR1 and/or MRP1. These results indicated that LRP, mostly known for facilitating drug resistance in lung and breast cancer, may also have a significant role in the ability of melanoma cells to resist the effects of conventional melanoma treatments.In conclusion, this study demonstrated the existence and differential expression levels of MDR1, MRP1, and LRP in melanoma and normal skin cell lines. Although all melanoma lines were susceptible to treatment with clotrimazole, decreased efficacy was observed in melanoma lines that overexpressed LRP. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug‐resistant human melanoma cells. However, these preliminary studies also indicated that LRP is potentially a significant facilitator of drug resistance in human melanoma.Support or Funding InformationThis research was funded by the Ohio Northern University Summer Research Stipend.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis.

The incidence of melanoma is increasing, particularly in young women, and the disease remains incurable for many because of its aggressive, metastatic nature and its high rate of resistance to conventional, targeted, and immunological agents. Cathepsins are proteases that are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave or degrade proteins that restrict cancer progression, whereas extracellular cathepsins directly cleave the extracellular matrix and activate proinvasive proteases in the tumor microenvironment. Cathepsin secretion is markedly increased in cancer cells. We investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells. We found that the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) promoted the secretion of cathepsin B and cathepsin L by activating transcription factors (namely, Ets1, Sp1, and NF-κB/p65) that have key roles in the epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance. In some melanoma cell lines, Abl/Arg promoted the Ets1/p65-induced secretion of cathepsin B and cathepsin L in a kinase-independent manner, whereas in other melanoma lines, Abl/Arg promoted the kinase-dependent, Sp1/Ets1/p65-mediated induction of cathepsin L secretion and the Sp1/p65-mediated induction of cathepsin B secretion. As an indication of clinical relevance, the abundance of mRNAs encoding Abl/Arg, Sp1, Ets1, and cathepsins was positively correlated in primary melanomas, and Abl/Arg-driven invasion in culture and metastasis in vivo required cathepsin secretion. These data suggest that drugs targeting Abl kinases, many of which are FDA-approved, might inhibit cathepsin secretion in some melanomas and potentially other aggressive cancers harboring activated Abl kinases.

Linker Variation and Structure-Activity Relationship Analyses of Carboxylic Acid-based Small Molecule STAT3 Inhibitors.

The molecular determinants for the activities of the reported benzoic acid (SH4-54), salicylic acid (BP-1-102), and benzohydroxamic acid (SH5-07)-based STAT3 inhibitors were investigated to design optimized analogues. All three leads are based on an N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities and the CH2 group of the glycinamide scaffold were separately modified. The replacement of the pentafluorobenzene or cyclohexylbenzene, or replacing the benzene ring of the aromatic carboxylic or hydroxamic acid motif with heterocyclic components (containing nitrogen and oxygen elements) all decreased potency. Notably, the Ala-linker analogues, 1a and 2v, and the Pro-based derivative 5d, all with (R)-configuration at the chiral center, had improved inhibitory activity and selectivity against STAT3 DNA-binding activity in vitro, with IC50 of 3.0 ± 0.9, 1.80 ± 0.94, and 2.4 ± 0.2 μM, respectively. Compounds 1a, 2v, 5d, and other analogues inhibited constitutive STAT3 phosphorylation and activation in human breast cancer and melanoma lines, and blocked tumor cell viability, growth, colony formation, and migration in vitro. Pro-based analogue, 5h, with a relatively polar tetrahydropyranyl (THP) ring, instead of the cyclohexyl, showed improved permeability. In general, the (R)-configuration Pro-based analogs showed the overall best profile, including physicochemical properties (e.g., microsomal metabolic stability, Caco-2 permeability), and in particular, 5d showed improved tumor-cell specificity.

Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin’s favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Abstract A06: MicroRNA-195 acts as a tumor suppressor miRNA in human melanoma cells by targeting Prohibitin 1

Abstract Melanoma is the most lethal type of skin cancer. Since chemo-resistance is a significant barrier, identification of regulators affecting chemo-sensitivity is necessary in order to create new forms of intervention. Here, we investigate the role of miR-195, its impact on Prohibitin 1 (PHB1) expression, and on chemo-sensitivity in melanoma cells. TCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by qRT-PCR and Western blot, luciferase reporter, and rescue experiments. Cell proliferation, hypodiploid, and caspase 3/7 assays were performed to investigate the potential action of miR-195 as chemo-sensitizer in melanoma cells treated with cisplatin and temozolomide. Analysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, qRT-PCR data showed that miR-195 is downregulated while PHB1 is upregulated in a collection of melanoma lines. We demonstrated that miR-195 regulates PHB1 directly by qRT-PCR and Western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect of miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as a tumor suppressor and potential chemo-sensitivity agent. Citation Format: Priscila D. R. Cirilo, Bruna R. S. Côrrea, Mei Qiao, Luciana N. S. Andrade, Roger Chammas, Luiz O. F. Penalva. MicroRNA-195 acts as a tumor suppressor miRNA in human melanoma cells by targeting Prohibitin 1 [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A06.

Abstract A150: Complex 3D cultures for combination compound screens reveal new insights

Abstract 3D tumor cell cultures more closely mimic the tumor environment in drug screening assays. The tumor environment includes endothelial cells, infiltrating cells, and stromal cells and the extracellular matrix excreted by these cells. It is likely that the tumor microenvironment affects the response of tumor cells to therapeutic agents. We have used a complex spheroid model consisting of tumor cells, endothelial cells (HUVEC), and mesenchymal stem cells (MSCs) to explore response to selected therapeutic agents. Using 9-10 tumor lines of each of NSCLC, melanoma, and pancreatic cancer, we tested combinations of FDA-approved agents and investigational agents on viability. All agents were studied at multiple concentrations at their reported clinical Cmax values and below in a 6 by 5 concentration matrix. Bliss independence calculations identified drug combinations that were additive, synergistic, or antagonistic. Gemcitabine plus the Chk1/2 inhibitor AZD 7762 showed strong synergy in PANC-1 cells and weaker synergy in MiaPAca-2 complex spheroids. Under the same conditions, gemcitabine and AZD7762 were additive to antagonistic in cell killing and strong antagonism was observed in CFPAC-1 cells. A combination of gemcitabine with the BET inhibitor (GSK525762) was extremely cytotoxic even at low concentrations (Gem, 0.2uM; GSK525762, 10uM)–demonstrating synergy between the Bromodomain (BET) inhibitor GSK525762 and gemcitabine in PANC-1 cells. In one NSCLC cell line (NCI-H23) the combination of GSK525762 (concentrations &amp;gt;1uM) was synergistic with carboplatin; however, in 5 NSCLC PDX lines, the combination had no synergy. In the LOX-IMVI melanoma line, GSK525762 was synergistic with cobimetinib. Cobimetinib and rabusertib were synergistic in the MDA-MB-435 line with &amp;gt;99% cell killing at concentrations &amp;lt; clinical Cmax. The results demonstrate that complex multicellular spheroids can be utilized in prolonged compound exposure screens (7 days) with robust and statistically relevant results using cell viability as an initial measure of cytotoxicity and can inform selection of combinations for progression. The observed additivity, synergy, or antagonism was cell line and tumor type dependent. Citation Format: David M. Evans, Selby H. Michael, Lori Bowles, Rene M. Delosh, Laudeman Julie, Chad C. Ogle, Reinhart Russell, Silvers Thomas, Ralph Parchment, James Doroshow, Beverly Teicher. Complex 3D cultures for combination compound screens reveal new insights [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A150.

ANTICANCER ACTIVITY OF DOXORUBICIN CONJUGATES WITH DENDRITIC POLYMERS OF SECOND GENERATION AND VECTOR PROTEIN IN MODEL SYSTEMS IN VITRO AND IN VIVO

Anticancer activity of doxorubicin (Dox), its conjugates with the second generation dendritic polymer (G2-Dox) and vector protein (recombinant third domain of alpha-fetoprotein - 3D-G2-Dox were studied in vitro and in vivo within the framework of the development of selective transport system of anticancer drugs to the target cells. The objects of the study were MCF-7 and MCF-7/MDR1 breast cancer cells, differing in chemosensitivity as well as mouse melanoma line B16. Anticancer activity of G2-Dox and 3D-G2-Dox was demonstrated in vivo (for B16 melanoma) and in vitro (for chemoresistant MCF-7/MDR1 cells, expressing P-glycoprotein). The effectiveness of anticancer action of G2-Dox and 3D-G2-Dox did not differ from that for free Dox. However for chemosensitive line MCF-7 free Dox proved to be more effective drug. It is important that the conjugates, and particularly 3D-G2-Dox, accumulated much weaker than the free Dox in normal cells isolated from bone marrow, spleen and liver of mice. The findings suggest that the main benefit from the possible use of such conjugates can be associated with reduced toxic effect of Dox on normal tissues and organs.

MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

BackgroundMelanoma is the most lethal type of skin cancer. Since chemoresistance is a significant barrier, identification of regulators affecting chemosensitivity is necessary in order to create new forms of intervention. Prohibitin 1 (PHB1) can act as anti-apoptotic or tumor suppressor molecule, depending on its subcellular localization. Our recent data shown that accumulation of PHB1 protects melanoma cells from chemotherapy-induced cell death. Lacking of post-transcriptional regulation of PHB1 could explain this accumulation. Interestingly, most of melanoma patients have down-regulation of microRNA-195. Here, we investigate the role of miR-195, its impact on PHB1 expression, and on chemosensitivity in melanoma cells.MethodsTCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma cell lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by RT-qPCR and western blot, luciferase reporter and genetic rescue experiments. Cell proliferation, cell-cycle analysis and caspase 3/7 assay were performed to investigate the potential action of miR-195 as chemosensitizer in melanoma cells treated with cisplatin and temozolomide.ResultsAnalysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, RT-qPCR data showed that miR-195 is down-regulated while PHB1 is up-regulated in a collection of melanoma cells. We demonstrated that miR-195 regulates PHB1 directly by RT-qPCR and western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as potential anti-proliferative agent, with potential impact in sensitization of melanoma cell death.ConclusionsThis study support the role of miR-195 as anti-proliferative miRNA via targeting of PHB1 in melanoma cells.

Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells

Flubendazole (FLU) is a widely used anthelmintic drug belonging to benzimidazole group. Recently, several studies have been published demonstrating its potential to inhibit growth of various tumor cells including those derived from colorectal cancer, breast cancer or leukemia via several mechanisms. In the present study we have investigated cytotoxic effects of FLU on malignant melanoma using A-375, BOWES and RPMI-7951 cell lines representing diverse melanoma molecular types. In all three cell lines, FLU inhibited cell growth and proliferation and disrupted microtubule structure and function which was accompanied by dramatic changes in cellular morphology. In addition, FLU-treated cells accumulated at the G2/M phase of cell cycle and displayed the features of mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and subsequent apoptotic demise. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.