Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy.

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT-mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

The two faces of the Integrated Stress Response in cancer progression and therapeutic strategies.

In recent years considerable progress has been made in identifying the impact of mRNA translation in tumour progression. Cancer cells hijack the pre-existing translation machinery to thrive under the adverse conditions originating from intrinsic oncogenic programs, that increase their energetic demand, and from the hostile microenvironment. A key translation program frequently dysregulated in cancer is the Integrated Stress Response, that reprograms translation by attenuating global protein synthesis to decrease metabolic demand while increasing translation of specific mRNAs that support survival, migration, immune escape. In this review we provide an overview of the Integrated Stress Response, emphasise its dual role during tumorigenesis and cancer progression, and highlight the therapeutic strategies available to target it.

BRN2 is a non-canonical melanoma tumor-suppressor.

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.

MITF induces escape from innate immunity in melanoma.

BACKGROUND: The application of immune-based therapies has revolutionized cancer treatment. Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood. In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair. Whether MITF also controls the immune response is unknown. METHODS: By using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response. ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene. Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing. RESULTS: Here we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells. By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system. Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance. CONCLUSION: Our results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.

PRMT1-dependent methylation of BRCA1 contributes to the epigenetic defense of breast cancer cells against ionizing radiation.

The therapeutic effect of irradiation is thought to come from DNA damage that affects rapidly proliferating cancer cells; however, resistant cells rapidly initiate mechanisms to repair such damage. While DNA repair mechanisms responsible for cancer cell survival following DNA damage are understood, less is known about the epigenetic mechanisms resulting in resistance to radiotherapy. Although changes in DNA methylation are related to mechanisms of long-term resistance, it is more likely that the methylation state of a series of proteins could be responsible for the first-line of defense of cancer cells against irradiation. In this study, we observed that irradiation of breast cancer cells was accompanied by an overproduction in S-adenosylmethionine, which increases the activity of cellular methylases. We found that by activating PRMT1, irradiation triggers a BRCA1-dependent program that results in efficient DNA repair and inhibition of apoptosis. Depletion of PRMT1 in irradiated cells resulted in a switch of BRCA1 functions from repair and survival in the nucleus to activation of cell death signals in the cytoplasm. We conclude that by modulating the cellular localization of BRCA1, PRMT1 is an important regulator of the oncogenic functions of BRCA1, contributing to the epigenetic defense of breast cancer cells against ionizing radiation.

Acriflavine, a Potent Inhibitor of HIF-1α, Disturbs Glucose Metabolism and Suppresses ATF4-Protective Pathways in Melanoma under Non-Hypoxic Conditions.

Hypoxia-inducible factor (HIF)-1α is constitutively expressed in melanoma cells under normoxic conditions and its elevated expression correlates with the aggressiveness of melanoma tumors. Here, we used acriflavine, a potent inhibitor of HIF-1α dimerization, as a tool to investigate whether HIF-1α-regulated pathways contribute to the growth of melanoma cells under normoxia. We observed that acriflavine differentially modulated HIF-1α-regulated targets in melanoma under normoxic conditions, although acriflavine treatment resulted in over-expression of vascular endothelial growth factor (VEGF), its action clearly downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1), a well-known target of HIF-1α. Consequently, downregulation of PDK1 by acrifavine resulted in reduced glucose availability and suppression of the Warburg effect in melanoma cells. In addition, by inhibiting the AKT and RSK2 phosphorylation, acriflavine also avoided protective pathways necessary for survival under conditions of oxidative stress. Interestingly, we show that acriflavine targets activating transcription factor 4 (ATF4) for proteasomal degradation while suppressing the expression of microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and a melanoma oncogene. Since acriflavine treatment results in the consistent death of melanoma cells, our results suggest that inhibition of HIF-1α function in melanoma could open new avenues for the treatment of this deadly disease regardless of the hypoxic condition of the tumor.

Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells.

Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.

MITF is a critical regulator of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in malignant melanoma.

The multifunctional Ig-like carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is neo-expressed in the majority of malignant melanoma lesions. CEACAM1 acts as a driver of tumor cell invasion, and its expression correlates with poor patient prognosis. Despite its importance in melanoma progression, how CEACAM1 expression is regulated is largely unknown. Here, we show that CEACAM1 expression in melanoma cell lines and melanoma tissue strongly correlates with that of the microphthalmia-associated transcription factor (MITF), a key regulator of melanoma proliferation and invasiveness. MITF is revealed as a direct and positive regulator for CEACAM1 expression via binding to an M-box motif located in the CEACAM1 promoter. Taken together, our study provides novel insights into the regulation of CEACAM1 expression and suggests an MITF-CEACAM1 axis as a potential determinant of melanoma progression.

Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development.

The mechanisms by which arterial fate is established and maintained are not clearly understood. Although a number of signaling pathways and transcriptional regulators have been implicated in arterio-venous differentiation, none are essential for arterial formation, and the manner in which widely expressed factors may achieve arterial-specific gene regulation is unclear. Using both mouse and zebrafish models, we demonstrate here that arterial specification is regulated combinatorially by Notch signaling and SoxF transcription factors, via direct transcriptional gene activation. Through the identification and characterization of two arterial endothelial cell-specific gene enhancers for the Notch ligand Delta-like ligand 4 (Dll4), we show that arterial Dll4 expression requires the direct binding of both the RBPJ/Notch intracellular domain and SOXF transcription factors. Specific combinatorial, but not individual, loss of SOXF and RBPJ DNA binding ablates all Dll4 enhancer-transgene expression despite the presence of multiple functional ETS binding sites, as does knockdown of sox7;sox18 in combination with loss of Notch signaling. Furthermore, triple knockdown of sox7, sox18 and rbpj also results in ablation of endogenous dll4 expression. Fascinatingly, this combinatorial ablation leads to a loss of arterial markers and the absence of a detectable dorsal aorta, demonstrating the essential roles of SoxF and Notch, together, in the acquisition of arterial identity.

Suppression of antifolate resistance by targeting the myosin Va trafficking pathway in melanoma.

Human melanoma is a significant clinical problem. As most melanoma patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is one of the highest priorities to improve melanoma therapy. Melanosomal sequestration and the cellular exportation of cytotoxic drugs have been proposed to be important melanoma-specific mechanisms that contribute to multidrug resistance in melanoma. Concretely, we found that treatment of melanoma with methotrexate (MTX) altered melanogenesis and accelerated the exportation of melanosomes; however, the cellular and molecular processes by which MTX is trapped into melanosomes and exported out of cells have not been elucidated. In this study, we identified myosin Va (MyoVa) as a possible mediator of these cellular processes. The results demonstrated that melanoma treatment with MTX leads to Akt2-dependent MyoVa phosphorylation, which enhances its ability to interact with melanosomes and accelerates their exportation. To understand the mechanism(s) by which MTX activates Akt2, we examined the effects of this drug on the activity of protein phosphatase 2A, an Akt inhibitor activated by the methylation of its catalytic subunit. Taken together, this study identified a novel trafficking pathway in melanoma that promotes tumor resistance through Akt2/MyoVa activation. Because of these findings, we explored several MTX combination therapies to increase the susceptibility of melanoma to this drug. By avoiding MTX exportation, we observed that the E2F1 apoptotic pathway is functional in melanoma, and its induction activates p73 and apoptosis protease-activating factor 1 following a p53-autonomous proapoptotic signaling event.

Directed phenotype switching as an effective antimelanoma strategy.

Therapeutic resistance in melanoma and other cancers arises via irreversible genetic, and dynamic phenotypic, heterogeneity. Here, we use directed phenotype switching in melanoma to sensitize melanoma cells to lineage-specific therapy. We show that methotrexate (MTX) induces microphthalmia-associated transcription factor (MITF) expression to inhibit invasiveness and promote differentiation-associated expression of the melanocyte-specific Tyrosinase gene. Consequently, MTX sensitizes melanomas to a tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), that inhibits the essential enzyme DHFR with high affinity. The combination of MTX and TMECG leads to depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in culture and in vivo. Importantly, this drug combination delivers an effective and tissue-restricted antimelanoma therapy in vitro and in vivo irrespective of BRAF, MEK, or p53 status.

Melanoma coordinates general and cell-specific mechanisms to promote methotrexate resistance.

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current modalities of cancer therapy, including to the drug methotrexate. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mechanisms that contribute to the resistance of melanoma to methotrexate. In addition, other mechanisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This report elucidates how melanoma orchestrates these mechanisms to become extremely resistant to methotrexate, where both E2F1 and checkpoint kinase 1 (Chk1), two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of dihydrofolate in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate expression of dihydrofolate reductase and thymidylate synthase, two E2F1-target genes involved in folate metabolism and required for G(1) progression, favored dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1-mediated apoptosis in melanoma cells.

Comparison of a pair of synthetic tea-catechin-derived epimers: synthesis, antifolate activity, and tyrosinase-mediated activation in melanoma.

Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin-derived compounds, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG) and 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro-apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non-melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.

Mechanism of dihydrofolate reductase downregulation in melanoma by 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin.

In our search to improve the stability and cellular absorption of tea polyphenols, we synthesized 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), which showed high antiproliferative activity against melanoma. TMECG downregulates dihydrofolate reductase (DHFR) expression in melanoma cells and we detail the sequential mechanisms that result from this even. TMECG is specifically activated in melanoma cells to form a stable quinone methide (TMECG-QM). TMECG-QM has a dual action on these cells. First, it acts as a potent antifolate compound, disrupting folate metabolism and increasing intracellular oxidized folate coenzymes, such as dihydrofolate, which is a non-competitive inhibitor of dihydropterine reductase, an enzyme essential for tetrahydrobiopterin (H(4)B) recycling. Such inhibition results in H(4)B deficiency, endothelial nitric oxide synthase (eNOS) uncoupling and superoxide production. Second, TMECG-QM acts as an efficient superoxide scavenger and promotes intra-cellular H(2)O(2) accumulation. Here, we present evidence that TMECG markedly reduces melanoma H(4)B and NO bioavailability and that TMECG action is abolished by the eNOS inhibitor N(omega)-nitro-L-arginine methyl ester or the H(2)O(2) scavenger catalase, which strongly suggests H(2)O(2)-dependent DHFR downregulation. In addition, the data presented here indicate that the simultaneous targeting of important pathways for melanoma survival, such as the folate cycle, H(4)B recycling, and the eNOS reaction, could represent an attractive strategy for fighting this malignant skin pathology.

Purification and kinetic properties of human recombinant dihydrofolate reductase produced in Bombyx mori chrysalides.

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.

The critical role of alpha-folate receptor in the resistance of melanoma to methotrexate.

Although methotrexate (MTX) is an effective drug for several types of cancer, it is not active against melanoma. Experiments following methotrexate treatment indicated a reduced accumulation of the drug in the cytosolic compartment in melanoma cells, suggesting that the mechanisms that control the transport and retention of this drug could be altered in melanoma. For this reason, we analyzed the presence and function of folate receptor-alpha (FRalpha) in melanoma cells. In this study, we have identified the presence of FRalpha in normal and pathological melanocytes and demonstrated that MTX is preferentially transported through this receptor in melanoma cells. FRalpha-induced endocytic transport of MTX, together with drug melanosomal sequestration and cellular exportation, ensures reduced accumulation of this cytotoxic compound in intracellular compartments. The critical role of FRalpha in this mechanism of resistance and the therapeutic consequences of these findings are also discussed.

Melanoma activation of 3-o-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin to a potent irreversible inhibitor of dihydrofolate reductase.

Human melanoma is a significant clinical problem because it is resistant to treatment by most chemotherapeutic agents, including antifolates. It is therefore a desirable goal to develop a second generation of low-toxicity antifolate drugs to overcome acquired resistance to the prevention and treatment of this skin pathology. In our efforts to improve the stability and bioavailability of green tea polyphenols for cancer therapy, we synthesized a trimethoxy derivative of epicatechin-3-gallate, which showed high antiproliferative and proapoptotic activity against melanoma. This derivative, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), is a prodrug that is selectively activated by the specific melanocyte enzyme tyrosinase. Upon activation, TMECG generates a stable quinone methide product that strongly inhibits dihydrofolate reductase in an irreversible manner. The treatment of melanoma cells with TMECG also affected cellular folate transport and the gene expression of DHFR, which supported the antifolate nature of this compound. In addition, its pharmacological efficacy has been confirmed in a mouse melanoma model, in which tumor growth and metastasis were inhibited, significantly enhancing the mean survival of the treated groups. TMECG, therefore, shows a potential for clinical use in melanoma therapy.

Binding of natural and synthetic polyphenols to human dihydrofolate reductase.

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent inhibitors of DHFR with dissociation constants (K(D))of 0.9 and 1.8 microM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin; TMECG), which effectively binds to DHFR (K(D) = 2.1 microM). In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a K(D) of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a K(D) of 0.6 microM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.