H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Alcohol as a Non-UV Social-Environmental Risk Factor for Melanoma.

Although cancer mortality has declined among the general population, the incidence of melanoma continues to rise. While identifying high-risk cohorts with genetic risk factors improves public health initiatives and clinical care management, recognizing modifiable risk factors such as social-environmental risk factors would also affect the methods of patient outreach and education. One major modifiable social-environmental risk factor associated with melanoma is ultraviolet (UV) radiation. However, not all forms of melanoma are correlated with sun exposure or occur in sun-exposed areas. Additionally, UV exposure is rarely associated with tumor progression. Another social-environmental factor, pregnancy, does not explain the sharply increased incidence of melanoma. Recent studies have demonstrated that alcohol consumption is positively linked with an increased risk of cancers, including melanoma. This perspective review paper summarizes epidemiological data correlating melanoma incidence with alcohol consumption, describes the biochemical mechanisms of ethanol metabolism, and discusses how ethanol and ethanol metabolites contribute to human cancer, including melanoma.

EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses.

Over the last decade, therapies targeting immune checkpoints, such as programmed death-1 (PD-1), have revolutionized the field of cancer immunotherapy. However, low response rates and immune-related adverse events remain a major concern. Here, we report that epigallocatechin gallate (EGCG), the most abundant catechin in green tea, inhibits melanoma growth by modulating an immune response against tumors. In vitro experiments revealed that EGCG treatment inhibited interferon-gamma (IFN-γ)-induced PD-L1 and PD-L2 expression and JAK-STAT signaling. We confirmed that this effect was driven by inhibiting STAT1 gene expression and STAT1 phosphorylation, thereby downregulating the PD-L1/PD-L2 transcriptional regulator IRF1 in both human and mouse melanoma cells. Animal studies revealed that the in vivo tumor-inhibitory effect of EGCG was through CD8+ T cells and that the inhibitory effect of EGCG was comparable to anti-PD-1 therapy. However, their mechanisms of action were different. Dissimilar to anti-PD-1 treatment that blocks PD-1/PD-L1 interaction, EGCG inhibited JAK/STAT signaling and PD-L1 expression in tumor cells, leading to the re-activation of T cells. In summary, we demonstrate that EGCG enhances anti-tumor immune responses by inhibiting JAK-STAT signaling in melanoma. EGCG could be used as an alternative treatment strategy to target the PD-L1/PD-L2-PD-1 axis in cancers.

Computational Modeling of NLRP3 Identifies Enhanced ATP Binding and Multimerization in Cryopyrin-Associated Periodic Syndromes.

Cyropyrin-associated periodic syndromes (CAPS) are clinically distinct syndromes that encompass a phenotypic spectrum yet are caused by alterations in the same gene, NLRP3. Many CAPS cases and other NLRP3-autoinflammatory diseases (NLRP3-AIDs) are directly attributed to protein-coding alterations in NLRP3 and the subsequent dysregulation of the NLRP3 inflammasome leading to IL-1ß-mediated inflammatory states. Here, we used bioinformatics tools, computational modeling, and computational assessments to explore the proteomic consequences of NLRP3 mutations, which potentially drive NLRP3 inflammasome dysregulation. We analyzed 177 mutations derived from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and the non-hereditary chronic infantile neurologic cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease (CINCA/NOMID), as well as other NLRP3-AIDs. We found an inverse relationship between clinical severity and the severity of predicted structure changes resulting from mutations in NLRP3. Bioinformatics tools and computational modeling revealed that NLRP3 mutations that are predicted to be structurally severely-disruptive localize around the ATP binding pocket and that specific proteo-structural changes to the ATP binding pocket lead to enhanced ATP binding affinity by altering hydrogen-bond and charge interactions. Furthermore, we demonstrated that NLRP3 mutations that are predicted to be structurally mildly- or moderately-disruptive affect protein-protein interactions, such as NLRP3-ASC binding and NLRP3-NLRP3 multimerization, enhancing inflammasome formation and complex stability. Taken together, we provide evidence that proteo-structural mechanisms can explain multiple mechanisms of inflammasome activation in NLRP3-AID.

Inflammasome Sensor NLRP1 Confers Acquired Drug Resistance to Temozolomide in Human Melanoma.

Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1ß maturation and NF-κB activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1ß secretion, and NF-κB activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.

Epigenetics and metabolism at the crossroads of stress-induced plasticity, stemness and therapeutic resistance in cancer.

Despite the recent advances in the treatment of cancers, acquired drug resistance remains a major challenge in cancer management. While earlier studies suggest Darwinian factors driving acquired drug resistance, recent studies point to a more dynamic process involving phenotypic plasticity and tumor heterogeneity in the evolution of acquired drug resistance. Chronic stress after drug treatment induces intrinsic cellular reprogramming and cancer stemness through a slow-cycling persister state, which subsequently drives cancer progression. Both epigenetic and metabolic mechanisms play an important role in this dynamic process. In this review, we discuss how epigenetic and metabolic reprogramming leads to stress-induced phenotypic plasticity and acquired drug resistance, and how the two reprogramming mechanisms crosstalk with each other.

Clinical implications of ALDH1A1 and ALDH1A3 mRNA expression in melanoma subtypes.

Aldehyde dehydrogenase (ALDH) activity is not only a valuable marker for cancer cells with stem-like features, but also plays a vital role in drug resistance and disease progression in many tumors including melanoma. However, the precise role of ALDH activity in patient prognosis remains unclear. In this study, using the Cancer Genome Atlas (TCGA) RNA-sequencing expression data, we analyzed gene expression of ALDH isozymes in melanoma tumors to define the expression patterns and the prognostic and predictive values of these enzymes. We found that ALDH1A1 and ALDH1A3 had both higher and broader expression ranges in melanoma patients, and that ALDH1A3 expression correlated with better overall survival in metastatic melanoma. Further, stratification of the TCGA cohorts by the mutational subtypes of melanoma specifically revealed that expression of ALDH1A3 correlated with better prognosis in metastatic BRAF-mutant melanoma while expression of ALDH1A1 correlated with better prognosis in BRAF wild-type melanoma. Gene set enrichment analysis (GSEA) of these cohorts identified upregulation in oxidative phosphorylation, adipogenesis, and fatty acid metabolism signaling in ALDH1Alo patients, suggesting BRAF/MEK inhibitor resistance in that subset of patients. On the other hand, GSEA of ALDH1A3hi cohorts revealed upregulation in glycolysis, hypoxia and angiogenesis, suggesting BRAF/MEK inhibitor sensitivity in that subset of patients. Gene expression analysis using pre-treatment tumor samples supports high ALDH1A3 expression before BRAF/MEK inhibitor treatment as predictive of better treatment response in BRAF-mutant melanoma patients. Our study provides evidence that high ALDH1A3 mRNA expression is not only a prognostic marker but also a predictive marker for BRAF/MEK inhibitor treatment response in BRAF-mutant metastatic melanoma patients.

CDK1 Interacts with Sox2 and Promotes Tumor Initiation in Human Melanoma.

: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.

Acetylsalicylic Acid Governs the Effect of Sorafenib in RAS-Mutant Cancers.

Purpose: Identify and characterize novel combinations of sorafenib with anti-inflammatory painkillers to target difficult-to-treat RAS-mutant cancer.Experimental Design: The cytotoxicity of acetylsalicylic acid (aspirin) in combination with the multikinase inhibitor sorafenib (Nexavar) was assessed in RAS-mutant cell lines in vitro The underlying mechanism for the increased cytotoxicity was investigated using selective inhibitors and shRNA-mediated gene knockdown. In vitro results were confirmed in RAS-mutant xenograft mouse models in vivoResults: The addition of aspirin but not isobutylphenylpropanoic acid (ibruprofen) or celecoxib (Celebrex) significantly increased the in vitro cytotoxicity of sorafenib. Mechanistically, combined exposure resulted in increased BRAF/CRAF dimerization and the simultaneous hyperactivation of the AMPK and ERK pathways. Combining sorafenib with other AMPK activators, such as metformin or A769662, was not sufficient to decrease cell viability due to sole activation of the AMPK pathway. The cytotoxicity of sorafenib and aspirin was blocked by inhibition of the AMPK or ERK pathways through shRNA or via pharmacologic inhibitors of RAF (LY3009120), MEK (trametinib), or AMPK (compound C). The combination was found to be specific for RAS/RAF-mutant cells and had no significant effect in RAS/RAF-wild-type keratinocytes or melanoma cells. In vivo treatment of human xenografts in NSG mice with sorafenib and aspirin significantly reduced tumor volume compared with each single-agent treatment.Conclusions: Combination sorafenib and aspirin exerts cytotoxicity against RAS/RAF-mutant cells by simultaneously affecting two independent pathways and represents a promising novel strategy for the treatment of RAS-mutant cancers. Clin Cancer Res; 24(5); 1090-102. ©2017 AACR.

Notch4 Signaling Induces a Mesenchymal-Epithelial-like Transition in Melanoma Cells to Suppress Malignant Behaviors.

The effects of Notch signaling are context-dependent and both oncogenic and tumor-suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the reexpression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting. Cancer Res; 76(7); 1690-7. ©2016 AACR.