Impact of Physical Exercise on Melanoma Hallmarks: Current Status of Preclinical and Clinical Research.

In recent years, accumulating evidence from preclinical and clinical studies consistently indicated that physical activity/exercise plays a crucial role in reducing the incidence and recurrence of various malignancies, by exerting a beneficial modulation of cancer hallmarks. Moreover, physical activity is suggested to attenuate certain adverse effects of anticancer therapy, including the reduction of cardiovascular toxicity and symptoms related to depression and anxiety, among others, while preserving muscular strength. In the case of melanoma, the relationship with physical activity has been critically debated. Historically, several cohort studies and meta-analyses reported a positive association between physical activity/exercise and melanoma risk. This association was primarily attributed to outdoor activities that may expose the skin to UV radiation, a well-known risk factor for melanocyte transformation. However, more recent evidence does not support such association and recognizes physical activity/exercise role in both melanoma prevention and progression. Nevertheless, sun protection is recommended during outdoor training to minimize UV radiation exposure. This narrative review summarizes preclinical and clinical data about physical activity effects on melanoma hallmarks. Specifically, experimental evidence is reported concerning (i) invasion and metastasis, (ii) reprogramming of energy metabolism, (iii) angiogenesis, (iv) resistance to cell death, (v) evasion from immune destruction, and (vi) tumor-promoting inflammation.

Targeting of PDGF-C/NRP-1 autocrine loop as a new strategy for counteracting the invasiveness of melanoma resistant to braf inhibitors.

Melanoma resistance to BRAF inhibitors (BRAFi) is often accompanied by a switch from a proliferative to an invasive phenotype. Therefore, the identification of signaling molecules involved in the development of metastatic properties by resistant melanoma cells is of primary importance. We have previously demonstrated that activation of neuropilin-1 (NRP-1) by platelet-derived growth factor (PDGF)-C confers melanoma cells with an invasive behavior similar to that of BRAFi resistant tumors. Aims of the present study were to evaluate the role of PDGF-C/NRP-1 autocrine loop in the acquisition of an invasive and BRAFi-resistant phenotype by melanoma cells and the effect of its inhibition on drug resistance and extracellular matrix (ECM) invasion. Furthermore, we investigated whether PDGF-C serum levels were differentially modulated by drug treatment in metastatic melanoma patients responsive or refractory to BRAFi as single agents or in combination with MEK inhibitors (MEKi). The results indicated that human melanoma cells resistant to BRAFi express higher levels of PDGF-C and NRP-1 as compared to their susceptible counterparts. Overexpression occurs early during development of drug resistance and contributes to the invasive properties of resistant cells. Accordingly, silencing of NRP-1 or PDGF-C reduces tumor cell invasiveness. Analysis of PDGF-C in the serum collected from patients treated with BRAFi or BRAFi+MEKi, showed that in responders PDGF-C levels decrease after treatment and raise again at tumor progression. Conversely, in non-responders treatment does not affect PDGF-C serum levels. Thus, blockade of NRP-1 activation by PDGF-C might represent a new therapeutic approach to counteract the invasiveness of BRAFi-resistant melanoma.

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5ß1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Targeting Tumor-Associated Macrophages to Increase the Efficacy of Immune Checkpoint Inhibitors: A Glimpse into Novel Therapeutic Approaches for Metastatic Melanoma.

Immune checkpoint inhibitors (ICIs) represent a promising therapeutic intervention for a variety of advanced/metastatic solid tumors, including melanoma, but in a large number of cases, patients fail to establish a sustained anti-tumor immunity and to achieve a long-lasting clinical benefit. Cells of the tumor micro-environment such as tumor-associated M2 macrophages (M2-TAMs) have been reported to limit the efficacy of immunotherapy, promoting tumor immune evasion and progression. Thus, strategies targeting M2-TAMs have been suggested to synergize with immune checkpoint blockade. This review recapitulates the molecular mechanisms by which M2-TAMs promote cancer immune evasion, with focus on the potential cross-talk between pharmacological interventions targeting M2-TAMs and ICIs for melanoma treatment.

Targeting the vascular endothelial growth factor receptor-1 by the monoclonal antibody D16F7 to increase the activity of immune checkpoint inhibitors against cutaneous melanoma.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a membrane receptor for VEGF-A, placenta growth factor (PlGF) and VEGF-B that plays a crucial role in melanoma invasiveness, vasculogenic mimicry and tumor-associated angiogenesis. Furthermore, activation of VEGFR-1 is involved in the mobilization of myeloid progenitors from the bone marrow that infiltrate the tumor. Myeloid-derived suppressor cells and tumor-associated macrophages have been involved in tumor progression and resistance to cancer treatment with immune checkpoint inhibitors (ICIs). We have recently demonstrated that the anti-VEGFR-1 monoclonal antibody (mAb) D16F7 developed in our laboratories is able to inhibit melanoma growth in preclinical in vivo models and to reduce monocyte/macrophage progenitor mobilization and tumor infiltration by myeloid cells. Aim of the study was to investigate whether the anti-VEGFR-1 mAb D16F7 affects the activity of protumoral M2 macrophages in vitro in response to PlGF and inhibits the recruitment of these cells to the melanoma site in vivo. Finally, we tested whether, through its multi-targeted action, D16F7 mAb might increase the efficacy of ICIs against melanoma. The results indicated that VEGFR-1 expression is up-regulated in human activated M2 macrophages compared to activated M1 cells and exposure to the D16F7 mAb decreases in vitro chemotaxis of activated M2 macrophages. In vivo treatment with the anti-VEGFR-1 mAb D16F7 of B6D2F1 mice injected with syngeneic B16F10 melanoma cells resulted in tumor growth inhibition associated with the modification of tumor microenvironment that involves a decrease of melanoma infiltration by M2 macrophages and PD-1+ and FoxP3+ cells. These alterations result in increased M1/M2 and CD8+/FoxP3+ ratios, which favor an antitumor and immunostimulating milieu. Accordingly, D16F7 mAb increased the antitumor activity of the ICIs anti-CTLA-4 and anti-PD-1 mAbs. Overall, these data reinforce the role of VEGFR-1-mediated-signalling as a valid target for reducing tumor infiltration by protumoral macrophages and for improving the efficacy of immunotherapy with ICIs.

Role of VEGFs/VEGFR-1 Signaling and its Inhibition in Modulating Tumor Invasion: Experimental Evidence in Different Metastatic Cancer Models.

The vascular endothelial growth factor (VEGF) family members, VEGF-A, placenta growth factor (PlGF), and to a lesser extent VEGF-B, play an essential role in tumor-associated angiogenesis, tissue infiltration, and metastasis formation. Although VEGF-A can activate both VEGFR-1 and VEGFR-2 membrane receptors, PlGF and VEGF-B exclusively interact with VEGFR-1. Differently from VEGFR-2, which is involved both in physiological and pathological angiogenesis, in the adult VEGFR-1 is required only for pathological angiogenesis. Besides this role in tumor endothelium, ligand-mediated stimulation of VEGFR-1 expressed in tumor cells may directly induce cell chemotaxis and extracellular matrix invasion. Furthermore, VEGFR-1 activation in myeloid progenitors and tumor-associated macrophages favors cancer immune escape through the release of immunosuppressive cytokines. These properties have prompted a number of preclinical and clinical studies to analyze VEGFR-1 involvement in the metastatic process. The aim of the present review is to highlight the contribution of VEGFs/VEGFR-1 signaling in the progression of different tumor types and to provide an overview of the therapeutic approaches targeting VEGFR-1 currently under investigation.

Role of VEGFR-1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF-A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR-1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour-associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro-angiogenic pathways, we have investigated VEGFR-1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF-A and express higher VEGFR-1 levels compared with their BRAFi-sensitive counterparts. Transient VEGFR-1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR-1 expression, by stable gene transfection in receptor-negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR-1 are more invasive than VEGFR-1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF-A and PlGF. These data suggest that VEGFR-1 up-regulation might contribute to melanoma progression and spreading after acquisition of a drug-resistant phenotype. Thus, VEGFR-1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR-1 positive tumours with acquired resistance to vemurafenib.

Poly(ADP-ribose) polymerase inhibitor olaparib hampers placental growth factor-driven activation of myelomonocytic cells.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor activated by the angiogenic factors VEGF­A and placental growth factor (PlGF). While VEGF­A binds to both VEGFR­1 and VEGFR­2, PlGF interacts exclusively with VEGFR­1 triggering a signaling pathway involved in: i) tumor­associated angiogenesis; ii) chemotaxis and invasion of the extracellular matrix (ECM) by cancer cells; and iii) mobilization of bone marrow­derived myeloid cells that generate tumor­associated macrophages. By using a novel anti­VEGFR­1 monoclonal antibody (D16F7 mAb), which hampers receptor activation without avoiding ligand binding, we recently demonstrated that VEGFR­1 blockade reduced myeloid progenitor mobilization and monocyte/macrophage cell infiltration of tumor grafts in vivo. Since poly(ADP­ribose) polymerase (PARP)­1 exerts a pro­inflammatory role favoring monocyte activation, in the present study we investigated whether the PARP inhibitor (PARPi) olaparib hampers PlGF­induced activation of human myelomonocytic cells. HL­60 cells induced to differentiate towards the monocytic/macrophage lineage were tested and the results were confirmed in freshly isolated monocytes obtained from healthy donors. Cells were treated with olaparib, at clinically achievable concentrations, before exposure to PlGF and were analyzed for migration and ECM invasion in response to PlGF. Olaparib effects were compared to those obtained with D16F7 mAb used as single agent or in combination with the PARPi. The results indicate that differentiated HL­60 cells and monocytes expressed VEGFR­1 and migrated in response to PlGF. Moreover, olaparib and D16F7 inhibited PlGF­induced chemotaxis and ECM invasion in a dose­dependent manner and with similar efficacy. However, in combination studies the PARPi and D16F7 did not exert synergistic effects. Olaparib also hampered PlGF­induced monocyte adhesion to fibronectin, while it did not affect NF­κB activation in response to the angiogenic factor. These data suggest that olaparib likely interferes with the same pathway affected by the anti­VEGFR­1 mAb and that inhibition of PlGF-induced monocyte activation may contribute to PARPi antitumor activity.

The Anti-Vascular Endothelial Growth Factor Receptor-1 Monoclonal Antibody D16F7 Inhibits Glioma Growth and Angiogenesis In Vivo.

The vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1) is a tyrosine kinase receptor that does not play a relevant role in physiologic angiogenesis in adults, whereas it is important in tumor angiogenesis. In high-grade glioma VEGFR-1 expression by tumor endothelium and neoplastic cells contributes to the aggressive phenotype. We recently generated an anti-VEGFR-1 monoclonal antibody (D16F7 mAb) characterized by a novel mechanism of action, since it hampers receptor activation without interfering with ligand binding. The mAb is able to inhibit chemotaxis and extracellular matrix invasion of glioma cells in vitro stimulated by VEGF-A and by the VEGFR-1-selective ligand placental growth factor (PlGF). In this study, we have investigated the influence of D16F7 on glioma growth and angiogenesis in vivo using C6 glioma cells transfected with the human VEGFR-1. D16F7 was able to inhibit receptor activation and migration and extracellular matrix invasion of C6 cells overexpressing the receptor in response to PlGF and VEGF-A. In nude mice, treatment with 10 and 20 mg/kg D16F7 as a single agent was well tolerated and significantly inhibited glioma growth (P < 0.001). Strikingly, in an intracranial orthotopic model, mice dosed with 20 mg/kg D16F7 demonstrated a 65% increase in median survival time compared with vehicle-treated controls (P < 0.001) with a high percentage of long-term survivors (46%). These effects were associated with glioma cell apoptosis and decreased tumor-associated vessel formation. Overall, these results highlight the therapeutic potential of D16F7 in glioma treatment, deserving further investigation after a humanization process as single agent or in combination therapies.

Platelet-derived growth factor-C promotes human melanoma aggressiveness through activation of neuropilin-1.

Despite recent progress in advanced melanoma therapy, identification of signalling pathways involved in melanoma switch from proliferative to invasive states is still crucial to uncover new therapeutic targets for improving the outcome of metastatic disease. Neuropilin-1 (NRP-1), a co-receptor for vascular endothelial growth factor-A (VEGF-A) tyrosine kinase receptors (VEGFRs), has been suggested to play a relevant role in melanoma progression. NRP-1 can be activated by VEGF-A also in the absence of VEGFRs, triggering specific signal transduction pathways (e.g. p130Cas phosphorylation). Since melanoma cells co-expressing high levels of NRP-1 and platelet derived growth factor-C (PDGF-C) show a highly invasive behaviour and PDGF-C shares homology with VEGF-A, in this study we have investigated whether PDGF-C directly interacts with NRP-1 and promotes melanoma aggressiveness. Results demonstrate that PDGF-C specifically binds in vitro to NRP-1. In melanoma cells expressing NRP-1 but lacking PDGFRα, PDGF-C stimulates extra-cellular matrix (ECM) invasion and induces p130Cas phosphorylation. Blockade of PDGF-C function by neutralizing antibodies or reduction of its secretion by specific siRNA inhibit ECM invasion and vasculogenic mimicry. Moreover, PDGF-C silencing significantly down-modulates the expression of Snail, a transcription factor involved in tumour invasiveness that is highly expressed in NRP-1 positive melanoma cells. In conclusion, our results demonstrate for the first time a direct activation of NRP-1 by PDGF-C and strongly suggest that autocrine and/or paracrine stimulation of NRP-1 by PDGF-C might contribute to the acquisition of a metastatic phenotype by melanoma cells.

The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells.

BACKGROUND: Glioblastoma (GBM) is a highly migratory, invasive, and angiogenic brain tumor. Like vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF) promotes GBM angiogenesis. VEGF-A is a ligand for both VEGF receptor-1 (VEGFR-1) and VEGFR-2, while PlGF interacts exclusively with VEGFR-1. We recently generated the novel anti-VEGFR-1 monoclonal antibody (mAb) D16F7 that diminishes VEGFR-1 homodimerization/activation without affecting VEGF-A and PlGF binding. METHODS: In the present study, we evaluated the expression of VEGFR-1 in human GBM tissue samples (n = 42) by immunohistochemistry, in cell lines (n = 6) and GBM stem cells (GSCs) (n = 18) by qRT-PCR and/or western blot analysis. In VEGFR-1 positive GBM or GSCs we also analyzed the ability of D16F7 to inhibit GBM invasiveness in response to VEGF-A and PlGF. RESULTS: Most of GBM specimens stained positively for VEGFR-1 and all but one GBM cell lines expressed VEGFR-1. On the other hand, in GSCs the expression of the receptor was heterogeneous. D16F7 reduced migration and invasion of VEGFR-1 positive GBM cell lines and patient-derived GSCs in response to VEGF-A and PlGF. Interestingly, this effect was also observed in VEGFR-1 positive GSCs transfected to over-express wild-type EGFR (EGFRwt+) or mutant EGFR (ligand binding domain-deficient EGFRvIII+). Furthermore, D16F7 suppressed intracellular signal transduction in VEGFR-1 over-expressing GBM cells by reducing receptor auto-phosphorylation at tyrosine 1213 and downstream Erk1/2 activation induced by receptor ligands. CONCLUSION: The results from this study suggest that VEGFR-1 is a relevant target for GBM therapy and that D16F7-derived humanized mAbs warrant further investigation.

Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth.

Ellagic acid (EA) is a polyphenolic compound that can be found as a naturally occurring hydrolysis product of ellagitannins in pomegranates, berries, grapes, green tea and nuts. Previous studies have reported the antitumor properties of EA mainly using in vitro models. No data are available about EA influence on bladder cancer cell invasion of the extracellular matrix triggered by vascular endothelial growth factor-A (VEGF-A), an angiogenic factor associated with disease progression and recurrence, and tumor growth in vivo. In this study, we have investigated EA activity against four different human bladder cancer cell lines (i.e., T24, UM-UC-3, 5637 and HT-1376) by in vitro proliferation tests (measuring metabolic and foci forming activity), invasion and chemotactic assays in response to VEGF-A and in vivo preclinical models in nude mice. Results indicate that EA exerts anti-proliferative effects as a single agent and enhances the antitumor activity of mitomycin C, which is commonly used for the treatment of bladder cancer. EA also inhibits tumor invasion and chemotaxis, specifically induced by VEGF-A, and reduces VEGFR-2 expression. Moreover, EA down-regulates the expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint involved in immune escape. EA in vitro activity was confirmed by the results of in vivo studies showing a significant reduction of the growth rate, infiltrative behavior and tumor-associated angiogenesis of human bladder cancer xenografts. In conclusion, these results suggest that EA may have a potential role as an adjunct therapy for bladder cancer.

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.

BACKGROUND: Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. METHODS: AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. RESULTS: TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects. CONCLUSION: NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.