Malignant melanoma is the deadliest form of skin cancer and highly chemoresistant. Melittin, an amphiphilic peptide containing 26 amino acid residues, is the major active ingredient from bee venom (BV). Although melittin is known to have several biological activities such as anti-inflammatory, antibacterial and anticancer effects, its antimelanoma effect and underlying molecular mechanism have not been fully elucidated. In the current study, we investigated the inhibitory effect and action mechanism of BV and melittin against various melanoma cells including B16F10, A375SM and SK-MEL-28. BV and melittin potently suppressed the growth, clonogenic survival, migration and invasion of melanoma cells. They also reduced the melanin formation in α-melanocyte-stimulating hormone (MSH)-stimulated melanoma cells. Furthermore, BV and melittin induced the apoptosis of melanoma cells by enhancing the activities of caspase-3 and -9. In addition, we demonstrated that the antimelanoma effect of BV and melittin is associated with the downregulation of PI3K/AKT/mTOR and MAPK signaling pathways. We also found that the combination of melittin with the chemotherapeutic agent temozolomide (TMZ) significantly increases the inhibition of growth as well as invasion in melanoma cells compared to melittin or TMZ alone. Taken together, these results suggest that melittin could be potentially applied for the prevention and treatment of malignant melanoma.

OBJECTIVES: To conduct a comprehensive analysis of prospectively measured circulating high sensitivity C reactive protein (hsCRP) concentration and risk of lung cancer overall, by smoking status (never, former, and current smokers), and histological sub-type.
DESIGN: Nested case-control study.
SETTING: 20 population based cohort studies in Asia, Europe, Australia, and the United States.
PARTICIPANTS: 5299 patients with incident lung cancer, with individually incidence density matched controls.
EXPOSURE: Circulating hsCRP concentrations in prediagnostic serum or plasma samples.
MAIN OUTCOME MEASURE: Incident lung cancer diagnosis.
RESULTS: A positive association between circulating hsCRP concentration and the risk of lung cancer for current (odds ratio associated with a doubling in hsCRP concentration 1.09, 95% confidence interval 1.05 to 1.13) and former smokers (1.09, 1.04 to 1.14) was observed, but not for never smokers (P<0.01 for interaction). This association was strong and consistent across all histological subtypes, except for adenocarcinoma, which was not strongly associated with hsCRP concentration regardless of smoking status (odds ratio for adenocarcinoma overall 0.97, 95% confidence interval 0.94 to 1.01). The association between circulating hsCRP concentration and the risk of lung cancer was strongest in the first two years of follow-up for former and current smokers. Including hsCRP concentration in a risk model, in addition to smoking based variables, did not improve risk discrimination overall, but slightly improved discrimination for cancers diagnosed in the first two years of follow-up.
CONCLUSIONS: Former and current smokers with higher circulating hsCRP concentrations had a higher risk of lung cancer overall. Circulating hsCRP concentration was not associated with the risk of lung adenocarcinoma. Circulating hsCRP concentration could be a prediagnostic marker of lung cancer rather than a causal risk factor.

BACKGROUND: Melanoma aggressiveness determines its growth and metastatic potential. This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.
METHODS: Ten metastatic melanoma cell lines were characterized by their proliferation, migration and invasion capabilities. The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients. Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment.
RESULTS: An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS. SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively. Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology. The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types. The key role of these pathways was then confirmed by functional validation. TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody. Upon such treatment melanoma cells aggressiveness was strongly reduced. Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18).
CONCLUSIONS: Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.

We used single cell Q-PCR on a micro-fluidic platform (Fluidigm) to analyse clonal, genetic architecture and phylogeny in acute myeloid leukaemia (AML) using selected mutations. Ten cases of NPM1c mutant AML were screened for 111 mutations that are recurrent in AML and cancer. Clonal architectures were relatively simple with one to six sub-clones and were branching in some, but not all, patients. NPM1 mutations were secondary or sub-clonal to other driver mutations (DNM3TA, TET2, WT1 and IDH2) in all cases. In three of the ten cases, single cell analysis of enriched CD34

BACKGROUND: Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or "Warburg effect"), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma.
METHODS: To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay.
RESULTS: In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption.
CONCLUSIONS: SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

BACKGROUND/AIMS: Melanoma is one of the most aggressive malignant tumors, with increasing incidence, poor prognosis, and lack of any effective targeted therapies. Abnormal expression of miR-29a has been found in several types of cancers, including melanoma. In this study, experiments were performed to investigate the role of miR-29a in melanoma, and the molecular mechanism by which miR-29a represses melanoma.
METHODS: miR-29 and Bmi1 expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability, apoptosis, migration and invasion were respectively determined by Cell Counting Kit-8 assay, Propidium iodide (PI) fluorescein isothiocynate (FITC)-Annexin V staining assay, wound healing assay and transwell assay. Luciferase reporter assay was performed to determine a target gene of miR-29a. Western blot was used to analyze protein expression of apoptosis-related proteins, Bmi1, Wnt/β-catenin and Nuclear factor-κB (NF-κB) pathway target genes.
RESULTS: miR-29a was down-regulated in all tested melanoma cell lines. Up-regulation of miR-29a effectively inhibited cell viability, migration, and invasion, but promoted apoptosis in A375 cells. Bmi1 was a direct target gene of miR-29a. Transfection with miR-29a mimic decreased cell migration and invasion and Bmi1 expression in Malme-3M cells, SK-MEL-2, SK-MEL-5, and M14 cell lines. Moreover, miR-29a might suppress growth, migration and invasion of A375 cells by negatively regulating Bmi1. In addition, our results demonstrated that transfection with miR-29a mimic effectively blocked Wnt/β-catenin and NF-κB pathways via down-regulating Bmi1.
CONCLUSION: miR-29a could be functioned as a potential tumor suppressor through direct regulation of Bmi1 in melanoma cells.

The present study aimed to assess the pharmacological anticancer profile of three natural and five synthetic sesquiterpenes developed by total chemical synthesis. To this end, their properties at the cellular and molecular level were evaluated in a panel of normal and cancer cell lines. The results obtained by performing cytotoxicity assays and gene expression analysis by reverse transcription-quantitative polymerase chain reaction showed that: i) Among the sesquiterpene derivatives analyzed, VDS58 exhibited a notable anticancer profile within attached (U-87 MG and MCF-7) and suspension (K562 and MEL-745) cancer cell cultures; however, U-87 MG cells were able to recover their proliferation capacity rapidly after 48 h of exposure; ii) gene expression profiling of U-87 MG cells, in contrast to K562 cells, showed a transient induction of cyclin-dependent kinase inhibitor 1A (CDKN1) expression; iii) the expression levels of transforming growth factor β1 (TGFB1) increased after 12 h of exposure of U-87 MG cells to VDS58 and were maintained at this level throughout the treatment period; iv) in K562 cells exposed to VDS58, TGFB1 expression levels were upregulated for 48 h and decrease afterwards; and v) the re-addition of VDS58 in U-87 MG cultures pretreated with VDS58 resulted in a notable increase in the expression of caspases (CASP3 and CASP9), BCL2‑associated agonist of cell death (BAD), cyclin D1, CDK6, CDKN1, MYC proto-oncogene bHLH transcription factor (MYC), TGFB1 and tumor suppressor protein p53. This upregulation persisted only for 24 h for the majority of genes, as afterwards, only the expression of TGFB1 and MYC was maintained at high levels. Through bioinformatic pathway analysis of RNA-Seq data of parental U-87 MG and K562 cells, substantial variation was reported in the expression profiles of the genes involved in the regulation of the cell cycle. This was associated with the differential pharmacological profiles observed in the same cells exposed to VDS58. Overall, the data presented in this study provide novel insights into the molecular mechanisms of action of sesquiterpene derivatives by dysregulating the expression levels of genes associated with the cell cycle of cancer cells.

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

BACKGROUND: Malignant melanoma cells can rapidly acquire phenotypic properties making them resistant to radiation and mainline chemotherapies such as decarbonize or kinase inhibitors that target RAS-proto-oncogene independent auto-activated mitogen-activated protein kinases (MAPK)/through dual specificity mitogen-activated protein kinase (MEK). Both drug resistance and inherent transition from melanocytic nevi to malignant melanoma involve the overexpression of histone deacetylases (HDACs) and a B-Raf proto-oncogene (BRAF) mutation.
MATERIALS AND METHODS: In this work, the effects of an HDAC class I and II inhibitor trichostatin A (TSA) on the whole transcriptome of SK-MEL-3 cells carrying a BRAF mutation was examined.
RESULTS: The data obtained show that TSA was an extremely potent HDAC inhibitor within SK-MEL-3 nuclear lysates, where TSA was then optimized for appropriate sub-lethal concentrations for in vitro testing. The whole-transcriptome profile shows a basic phenotype dominance in the SK-MEL-3 cell line for i) synthesis of melanin, ii) phagosome acidification, iii) ATP hydrolysis-coupled proton pumps and iv) iron transport systems. While TSA did not affect the aforementioned major systems, it evoked a dramatic change to the transcriptome: reflected by a down-regulation of 810 transcripts and up-regulation of 833, with fold-change from -15.27 to +31.1 FC (p<0.00001). Largest differentials were found for the following transcripts: Up-regulated: Tetraspanin 13 (TSPAN13), serpin family i member 1 (SERPINI1), ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), nicotinamide nucleotide adenylyl transferase 2 (NMNAT2), platelet-derived growth factor receptor-like (PDGFRL), cytochrome P450 family 1 subfamily A member 1 (CYP1A1), prostate androgen-regulated mucin-like protein 1 (PARM1), secretogranin II (SCG2), SYT11 (synaptotagmin 11), rhophilin associated tail protein 1 like (ROPN1L); down-regulated: polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), carbonic anhydrase 14 (CAXIV), BCL2-related protein A1 (BCL2A1), protein kinase C delta (PRKCD), transient receptor potential cation channel subfamily M member 1 (TRPM1), ubiquitin associated protein 1 like (UBAP1L), glutathione peroxidase 8 (GPX8), interleukin 16 (IL16), tumor protein p53 (TP53), and serpin family H member 1 (SERPINH1). There was no change to any of the HDAC transcripts (class I, II and IV), the sirtuin HDAC family (1-6) or the BRAF proto-oncogene v 599 transcripts. However, the data showed that TSA down-regulated influential transcripts that drive the BRAF-extracellular signal-regulated kinase (ERK)1/2 oncogenic pathway (namely PRKCD and MYC proto-oncogene which negatively affected the cell-cycle distribution. Mitotic inhibition was corroborated by functional pathway analysis and flow cytometry confirming halt at the G
CONCLUSION: TSA does not alter HDAC transcripts nor BRAF itself, but down-regulates critical components of the MAPK/MEK/BRAF oncogenic pathway, initiating a mitotic arrest.

Inhibition of BRAF improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to BRAF inhibitor is inevitable, and the drug resistance mechanisms still remain to be elucidated. Here, BRAF mutant cells A375 and SK-MEL-28 were chosen and treated with BRAF inhibitor vemurafenib, and the results showed that the ERK signaling pathway was blocked in these cells. Then, vemurafenib-resistant cells were constructed, and we found that drug resistance-related gene P-gp was overexpressed in the two cell lines. In addition, the histone acetylation was significantly increased on the P-gp promoter region, which suggested that the epigenetic modification participated in the P-gp overexpression. Furthermore, JQ1, a bromodomain inhibitor, was added to the vemurafenib-resistant cells and sensitizes the vemurafenib-induced melanoma cell apoptosis. In C57BL/6 mice intravenously injected with vemurafenib-resistant melanoma cells, cotreatment of vemurafenib and JQ1 also severely suppressed melanoma lung metastasis. Taken together, our findings may have important implications for the combined use of vemurafenib and JQ1 in the therapy for melanoma treatment.

Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.

DNA alterations have been extensively reported in multiple myeloma (MM); however, they cannot yet fully explain all the biological and molecular abnormalities in MM, which remains to this day an incurable disease with eventual emergence of refractory disease. Recent years have seen abnormalities at the RNA levels being reported to possess potential biological relevance in cancers. ADAR1-mediated A-to-I editing is an important posttranscriptional mechanism in human physiology, and the biological implication of its abnormality, especially at the global level, is underexplored in MM. In this study, we define the biological implications of A-to-I editing and how it contributes to MM pathogenesis. Here, we identified that the MM transcriptome is aberrantly hyperedited because of the overexpression of ADAR1. These events were associated with patients' survival independent of 1q21 amplifications and could affect patients' responsiveness to different treatment regimes. Our functional assays established ADAR1 to be oncogenic, driving cellular growth and proliferation in an editing-dependent manner. In addition, we identified NEIL1 (base-excision repair gene) as an essential and a ubiquitously edited ADAR1 target in MM. The recoded NEIL1 protein showed defective oxidative damage repair capacity and loss-of-function properties. Collectively, our data demonstrated that ADAR1-mediated A-to-I editing is both clinically and biologically relevant in MM. These data unraveled novel insights into MM molecular pathogenesis at the global RNA level.

Parkin, a critical gene of Parkinson's disease, is involved in the development of numerous cancers. However, the effect of parkin deficiency on melanoma growth and metastasis has not been reported. We showed that the tumor size and number of surface lung metastases, and expression of tumor growth and metastasis marker proteins were significantly lower in parkin-KO mice than those observed in non-transgenic controls. In an in vitro study, we also showed that parkin siRNA inhibited cell growth and migration of B16F10 and SK-Mel-28 cells. Parkin-specific ubiquitination of mitofusin-2 (MFN2) was decreased in tumors and metastasized lung tissues of parkin-KO mice. Moreover, we showed that parkin directly binds and ubiquitinates MFN2. Knockdown of MFN2 decreased the expression of Bax and apoptotic cell death, but increased that of Bcl2 and apoptotic cancer cell death. However, these effects were reversed by knockdown of parkin. Conversely, inhibitory effects on melanoma growth and migration of parkin siRNA were reversed by MFN2 siRNA. These data indicate that melanoma development was inhibited in parkin-KO mice through maintaining of MFN2 level by inhibition of ubiquitinating ability of parkin.

Extranodal NK/T-cell lymphoma, nasal type (ENKTL), is an aggressive malignancy with a poor prognosis. While the introduction of L-asparaginase in the treatment of this disease has significantly improved the prognosis, the outcome of patients relapsing after asparaginase-based chemotherapy, which occurs in up to 50% of patients with disseminated disease, remains dismal. There is hence an urgent need for effective targeted therapy especially in the relapsed/refractory setting. Gene expression profiling studies have provided new perspectives on the molecular biology, ontogeny and classification of ENKTL and further identified dysregulated signaling pathways such as Janus associated kinase (/Signal Transducer and activation of transcription (JAK/STAT), Platelet derived growth factor (PDGF), Aurora Kinase and NF-κB, which are under evaluation as therapeutic targets. Copy number analyses have highlighted potential tumor suppressor genes such as PR Domain Zinc Finger Protein 1 (PRDM1) and protein tyrosine phosphatase kappa (PTPRK) while next generation sequencing studies have identified recurrently mutated genes in pro-survival and anti-apoptotic pathways. The discovery of epigenetic dysregulation and aberrant microRNA activity has broadened our understanding of the biology of ENKTL. Importantly, immunotherapy via Programmed Cell Death -1 (PD-1) and Programmed Cell Death Ligand1 (PD-L1) checkpoint signaling inhibition is emerging as an attractive therapeutic strategy in ENKTL. Herein, we present an overview of the molecular biology and genomic landscape of ENKTL with a focus on the most promising translational opportunities.

Multiple myeloma is a heterogeneous disease with different characteristics, and genetic aberrations play important roles in this heterogeneity. Studies have shown that these genetic aberrations are crucial in prognostication and response assessment; recent efforts have focused on their possible therapeutic implications. Despite many emerging studies being published, the best way to incorporate these results into clinical practice remains unclear. In this review paper we describe the different genomic techniques available, including the latest advancements, and discuss the potential clinical application of genomics in multiple myeloma.

Matrine, one of the main alkaloid components extracted from the traditional Chinese herb, Sophora flavescens Ait, has various pharmacological effects, and has been reported to exert antitumor activity in melanoma. In the current study, the molecular mechanisms underlying the inhibitory effects of matrine were investigated in melanoma cell line. It was initially confirmed that matrine inhibited proliferation, invasion and induced apoptosis in human A375 and SK-MEL-2 melanoma cell lines in vitro. Subsequently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of microRNA (miR)-19b-3p was significantly increased in melanoma cells and was downregulated by treatment with matrine. Furthermore, downregulated miR-19b-3p exerted effects similar to 500 µg/ml matrine on cell proliferation, invasion and apoptosis. Phosphatase and tensin homolog (PTEN) mRNA was identified as a direct target of miR-19b-3p through bioinformatics analysis and a dual-luciferase reporter assay. Additionally, western blotting and RT-qPCR analysis demonstrated that the expression of PTEN protein and mRNA were increased by the treatment with matrine. Furthermore, silencing of PTEN expression reversed the effects of matrine and miR-19b-3p downregulation in A375 and SK-MEL-2 cells. Taken together, the results indicated that matrine may suppress cell proliferation and invasion and induce cell apoptosis partially via miR-19b-3p targeting of PTEN.

Chemotherapy resistance represents a major obstacle in the treatment of patients with hepatocellular carcinoma (HCC). The purpose of this study was to investigate the anti-cancer effect of MEL-pep, a novel analog of the natural antibacterial peptide melittin (MEL), on human 5-fluorouracil-resistant HCC cells (BEL-7402/5-FU) and to clarify the molecular mechanisms involved in these effects. We found that MEL-pep inhibited the proliferation of BEL-7402/5-FU cells and reversed 5-FU resistance in vitro. MEL-pep directly bound to BEL-7402/5-FU cells and disrupted the cell membrane. P-glycoprotein (P-gp) plays an important role in the development of resistance to anticancer drugs. We found that MEL-pep inhibited P-gp expression and increased the intracellular accumulation of the P-gp substrate rhodamine-123 in BEL-7402/5-FU cells. Additionally, the phosphorylation of Akt and NF-κB/p65 nuclear translocation was all inhibited by MEL-pep. Insulin - like growth factor I, a phosphatidylinositol 3 kinase(PI3K) /protein kinase B(AKT) agonist, reversed MEL-pep induced P-gp suppression. Therefore, MEL-pep inhibited P-gp expression by deactivating the PI3K/Akt signaling pathway. Finally, in a BEL-7402/5-FU cell-derived xenograft tumor model in mice, we found that the intratumoral administration of MEL-pep inhibited tumor growth in a dose-dependent manner. Thus, MEL-pep could be a promising candidate in the treatment of chemotherapy resistant HCC.

In this Review, I present evidence supporting a multifactorial causation of childhood acute lymphoblastic leukaemia (ALL), a major subtype of paediatric cancer. ALL evolves in two discrete steps. First, in utero initiation by fusion gene formation or hyperdiploidy generates a covert, pre-leukaemic clone. Second, in a small fraction of these cases, the postnatal acquisition of secondary genetic changes (primarily V(D)J recombination-activating protein (RAG) and activation-induced cytidine deaminase (AID)-driven copy number alterations in the case of ETS translocation variant 6 (ETV6)-runt-related transcription factor 1 (RUNX1)

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that responds to a diverse set of environmental cues, including amino acids

SOX4 (SRY Box 4) has attracted attention due to its important effects on cell growth, proliferation and apoptosis, among other cellular processes. However, the role of SOX4 in melanoma cell apoptosis remains unclear. In the present study, we investigated whether inhibition of SOX4 induces melanoma cell apoptosis, and explored the possible mechanism involving the NF-κB signaling pathway. SOX4 was knocked down using a lentivirus in melanoma A2058 and SK-MEL-5 cell lines. Cell proliferation was measured by MTT assay. Apoptosis was determined by flow cytometry. Western blotting was performed to detect the protein levels of SOX4, p65 and apoptosis-related proteins, such as PARP, Bcl-2, Bax and survivin. Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of SOX4 and p65. To determine whether SOX4 is able to bind to the promoter of p65, a CHIP-PCR assay was performed. Our data demonstrated that SOX4 knockdown significantly induced apoptosis in melanoma cells, which was accompanied by increases in cleaved PARP and Bax, and decreases in Bcl-2 and survivin. The expression of p65 was also decreased in SOX4-knockdown melanoma cells. The CHIP-PCR assay indicated that SOX4 was able to bind to the promoter region of p65. We also observed that apoptosis in SOX4-knockdown and p65-overexpressing A2058 cells was much lower than that in SOX4-knockdown alone cells. This revealed that the overexpression of p65 partially reversed SOX4 downregulation-induced apoptosis. In conclusion, our results demonstrated that inhibition of SOX4 markedly induced melanoma cell apoptosis via downregulation of the NF-κB signaling pathway, which thus may be a novel approach for the treatment of melanoma.

BACKGROUND: Melanoma is notoriously resistant to all current modalities of cancer therapies including chemotherapy. In recent years, microRNAs (miRNAs) have emerged as molecular regulators in the development and progression of melanoma. However, the relationship between microRNA and chemo-resistance of melanoma is little known. In present study, we aimed to investigate the miRNAs related to cisplatin-resistance in melanoma cells.
METHODS: After cisplatin (DDP) resistant melanoma cells (M8/DDP and SK-Mel-19/DDP) were established in-vitro, high-throughput screening of differentially expressed miRNAs between resistant cells and parental cells were performed.
RESULTS: It was found that a cancer-related miRNA, miR-30a-5p, was highly over-expressed in resistant cells. Transfection of miR-30a-5p mimic or inhibitor could alter the sensitivity of melanoma cells to cisplatin. Next, we showed that Insulin Like Growth Factor 1 Receptor (IGF1R) gene turned out to be a direct target of miR-30a-5p. Knockdown of IGF1R in melanoma cells could not only reduce the sensitivity to cisplatin but also lead to cell cycle arrest by regulating phosphorylation of Serine-Threonine Protein Kinase (P-AKT (Ser473)) and Tumor Protein P53 (P53).
CONCLUSION: Taken together, our study demonstrated that miR-30a-5p could influence chemo-resistance by targeting IGF1R gene in melanoma cells, which might provide a potential target for the therapy of chemo-resistant melanoma cells.

BACKGROUND: Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.
METHODS: The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential.
RESULTS: Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors.
CONCLUSIONS: The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.

Aims: MicroRNA-186 (miR-186) has been shown to be involved in various types of cancer. The purpose of this study was to investigate the expression level and functional role of miR-186 in human cutaneous malignant melanoma cells.
Subjects and Methods: Expression of miR-186 was analyzed in human cutaneous malignant melanoma (CMM) cell lines SK-MEL-1, G-361, A375 and A875, and human normal epidermal melanocytes cell line HEMn-LP by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Additionally, the functional role of miR-186 on melanoma cells was investigated by transfection of miR-186 mimic followed by analyses of cell proliferation, apoptosis, and metastasis.
Results: We found that the expression levels of miR-186 were decreased in CMM cell lines compared with normal epidermal melanocytes cell line. Moreover, overexpression of miR-186 inhibited cells proliferation through abrogating the G
Conclusions: Our findings suggested that miR-186 exhibit an inhibitory effect on CMM cell proliferation, migration, and invasion; thus, may serve as a potential therapeutic target for human CMM intervention.

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Lidocaine is a well-documented local anesthetic that has been reported to sensitize the cytotoxicity of cisplatin in cancer cells. However, little information is available concerning whether lidocaine sensitizes the cytotoxicity of 5-fluorouracil (5-FU) in melanoma cells. The study was aimed to explore the effects and mechanisms of lidocaine on the sensitivity to 5-FU in the melanoma cell line SK-MEL-2. Cell viability and apoptosis were analyzed after administration of different concentrations of lidocaine, 5-FU, or the combinations. Expression of microRNA (miR)-493 was assessed following lidocaine administration. The target genes of miR-493 were verified by luciferase reporter assay, PCR, and Western blot. The effects of abnormal expression of miR-493 and/or SRY-Box 4 (SOX4) on cell viability, apoptosis, and key proteins in phosphatidylinositol-3-kinase (PI3K)/AKT and the Smad pathways were detected. The effects of (0-100 uM) lidocaine on cell viability and apoptosis was not obvious; however, lidocaine could significantly increase the cell viability and inhibit apoptosis in 5-FU-treated cells. In addition, lidocaine induced upregulation of miR-493 in a dose-dependent manner, and we confirmed that the effects of miR-493 on the sensitivity were by upregulating miR-493. Moreover, we verified that Sox4 was a target of miR-493, and Sox4 overexpression decreased the sensitivity to 5-FU. Besides, Sox4 overexpression increased the levels of p-PI3K, p-AKT, p-Smad2 and p-Smad3, and Sox4 suppression showed contrary results. Our results suggest that lidocaine sensitizes the cytotoxicity of 5-FU in melanoma cells via upregulation of miR-493, which might be involved in SOX4-mediated PI3K/AKT and Smad pathways.

Polycomb group (PcG) proteins are repressive chromatin modifiers required for proliferation and development. PcG proteins form two large repressive complexes, namely, Polycomb Repressive Complex 1 and 2. These proteins have been shown to drive tumorigenesis by repressing cell-type specific sets of target genes. Using immunohistochemistry, we investigated the expression patterns of five human PcG proteins, including Bmi-1, Ring1b, Mel-18, Ezh2, and Suz12, in various cellular components of odontogenic keratocysts (OKCs), ameloblastomas and, pericoronal follicles (PFs). In OKCs, expression of PcG proteins were found in the majority of cases while the expression pattern was relatively different for each PcG proteins. All PcG proteins were strongly expressed in the basal cells while some proteins showed variable expression in the parabasal and luminal cell layer of OKCs. In ameloblastomas, almost all PcG proteins showed a similar expression pattern of moderate to strong staining in the peripheral ameloblast-like cells and metaplastic squamous cells. Some of the central stellate reticulum-like cells also showed positive reaction to most PcG proteins. In PFs, most PcG proteins were intensely expressed in odontogenic epithelium lining the follicles, except Mel-18 and Suz12. The present study provides the initial evidence regarding epigenetic involvement by PcG proteins in these odontogenic lesions. Although these proteins are known to be in the same repressive group proteins, differential expression patterns of these proteins in OKCs and ameloblastomas indicates that these proteins may play different roles in pathogenesis of these odontogenic lesions.

microRNA (miR)-612 shows anticancer activity in several types of cancers, yet its function in melanoma is still unclear. This study was undertaken to investigate the expression of miR-612 and its biological relevance in melanoma cell growth, invasion, and tumorigenesis. The expression and prognostic significance of miR-612 in melanoma were examined. The effects of miR-612 overexpression on cell proliferation, colony formation, tumorigenesis, and invasion were determined. Rescue experiments were conducted to identify the functional target gene(s) of miR-612. miR-612 was significantly downregulated in melanoma tissues compared to adjacent normal tissues. Low miR-612 expression was significantly associated with melanoma thickness, lymph node metastasis, and shorter overall, and disease-free survival of patients. Overexpression of miR-612 significantly decreased cell proliferation, colony formation, and invasion of SK-MEL-28 and A375 melanoma cells.

BACKGROUND: Histone deacetylase (HDAC) is an enzyme that regulates gene expression, cell cycle arrest, apoptosis and modulation of various pathways. HDAC inhibitors (HDACis) can modulate these pathways by hyper-acetylating target proteins, thereby acting as cancer chemotherapeutic agents.
OBJECTIVE: One of HDACis, suberoylanilide hydroxamic acid (SAHA), has been found to regulate the Smad signaling pathway, by an as yet unclear mechanism. This study therefore investigated the mechanism by which SAHA regulates Smad signaling in the melanoma cell line SK-Mel-5.
METHODS: Cell proliferation was assessed by MTT assays and fluorescence activated cell sorter (FACS) analysis. The activation of Smad signaling pathway was assessed by western blots analysis. The transcriptions of target genes were checked by RT-PCR and dual luciferase assay.
RESULTS: Treatment with SAHA inhibited the proliferation of SK-Mel-5 cells, enhanced the phosphorylation of R-Smad, and up-regulated p21 protein. Surprisingly, R-Smad was also activated by conditioned medium from SAHA-treated SK-Mel-5 cells. An analysis of the conditioned medium showed that activin A was responsible for the activation of R-Smad. SAHA treatment enhanced the level of activin A mRNA, increasing the level of activin A in the secretome. The activity of the SAHA-treated secretome could be eliminated by pre-incubation with antibody to activin A. In addition, activin A supplemented medium could mimic the effect of the SAHA-treated secretome.
CONCLUSION: These findings indicate that the anti-cancer function of SAHA is mediated, at least in part, by the upregulation of activin A.

BACKGROUND & AIMS: Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice.
METHODS: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRas
RESULTS: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax
CONCLUSIONS: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

BACKGROUND: The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells.
METHODS: Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study.
RESULTS: Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK
CONCLUSIONS: We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells.
GENERAL SIGNIFICANCE: The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.