Vacuolar Proton-Translocating ATPase May Take Part in the Drug Resistance Phenotype of Glioma Stem Cells.

The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.

The Role of NMNAT2/SARM1 in Neuropathy Development.

Chemotherapy-induced peripheral neuropathy (CIPN) commonly arises as a side effect of diverse cancer chemotherapy treatments. This condition presents symptoms such as numbness, tingling, and altered sensation in patients, often accompanied by neuropathic pain. Pathologically, CIPN is characterized by an intensive "dying-back" axonopathy, starting at the intra-epidermal sensory innervations and advancing retrogradely. The lack of comprehensive understanding regarding its underlying mechanisms explains the absence of effective treatments for CIPN. Recent investigations into axon degeneration mechanisms have pinpointed nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and TIR motif-containing 1 protein (SARM1) as pivotal mediators of injury-induced axonal degeneration. In this review, we aim to explore various studies shedding light on the interplay between NMNAT2 and SARM1 proteins and their roles in the progression of CIPN.

Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy.

BACKGROUND: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. METHODS: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. RESULTS: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. CONCLUSIONS: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

AMPK and Diseases: State of the Art Regulation by AMPK-Targeting Molecules.

5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that regulates cellular energy homeostasis, glucose, fatty acid uptake, and oxidation at low cellular ATP levels. AMPK plays an important role in several molecular mechanisms and physiological conditions. It has been shown that AMPK can be dysregulated in different chronic diseases, such as inflammation, diabetes, obesity, and cancer. Due to its fundamental role in physiological and pathological cellular processes, AMPK is considered one of the most important targets for treating different diseases. Over decades, different AMPK targeting compounds have been discovered, starting from those that activate AMPK indirectly by altering intracellular AMP:ATP ratio to compounds that activate AMPK directly by binding to its activation sites. However, indirect altering of intracellular AMP:ATP ratio influences different cellular processes and induces side effects. Direct AMPK activators showed more promising results in eliminating side effects as well as the possibility to engineer drugs for specific AMPK isoforms activation. In this review, we discuss AMPK targeting drugs, especially concentrating on those compounds that activate AMPK by mimicking AMP. These compounds are poorly described in the literature and still, a lot of questions remain unanswered about the exact mechanism of AMP regulation. Future investigation of the mechanism of AMP binding will make it possible to develop new compounds that, in combination with others, can activate AMPK in a synergistic manner.

Sodium-Calcium Exchanger 2: A Pivotal Role in Oxaliplatin Induced Peripheral Neurotoxicity and Axonal Damage?

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines.

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Breast radiotherapy with kilovoltage photons and gold nanoparticles as radiosensitizer: An in vitro study.

PURPOSE: We investigated the dose enhancement and internalization of gold nanoparticles (AuNPs) used as a radiosensitizer agent for rotational radiotherapy of breast cancer using a kilovoltage (kV) X-ray beam. METHODS: Human breast cancer cells MDA-MB-231 were incubated with or without 100 µg/mL (4.87 nM) or 200 µg/mL (9.74 nM) 15 nm AuNPs and irradiated with 100 kV, 190 kV, or 6 MV X-rays. To assess the toxicity of the AuNPs, we performed a Sulforhodamine B assay. Using atomic absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, and time-lapse optical microscopy (rate of 2 frames per minute), we carried out a quantitative assessment of the amount of gold internalized by MDA-MB-231 cells and a characterization of the static and dynamical aspects of this internalization process. RESULTS: No effect of AuNPs alone was shown on cell viability. Time-lapse optical microscopy showed for the first time AuNPs cellular uptake and the dynamics of AuNPs internalization. Electron microscopy demonstrated AuNPs localization in endosomal vesicles, preferentially in the perinuclear region. After irradiation at doses up to 2 Gy, cell survival fraction curves showed increased mortality with AuNPs, with respect to irradiation without AuNPs. The highest effect of radioenhancement by AuNPs (at 9.74 nM AuNPs concentration) was observed at 190 kV showing a dose enhancement factor of 1.33 ± 0.06 (1.34 ± 0.02 at 100 kV), while at 6 MV it was 1.14 ± 0.06. CONCLUSIONS: The observed radio-sensitization effect is promising for future radio-enhanced kV radiotherapy of breast cancer and quantitatively in the order of previous observations for 15 nm AuNPs. These results of a significant dose enhancement were obtained at 15 nm AuNPs concentration as low as several nanomolar units, at dose levels typical of a single dose fraction in a radiotherapy session. Dynamical behavior of the 3D spatial distribution of 15 nm AuNPs outside the nucleus of single breast cancer cell was observed, with possible implications for future models of AuNPs sensitization.

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin.

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

On-cell saturation transfer difference NMR study of Bombesin binding to GRP receptor.

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.

3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line.

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.

Anti-Multiple Myeloma Potential of Secondary Metabolites from Hibiscus sabdariffa.

Multiple myeloma (MM) belongs to hematological cancers and its incidence is increasing worldwide. Despite recent advances in its therapy, MM still causes many deaths every year. In fact, current therapies sometimes fail and are associated with severe adverse effects, including neurotoxicity. As a part of our ongoing efforts to discover new potential therapies against MM, we prepared Hibiscus sabdariffa extracts obtained by a microwave-assisted solvent extraction and investigate their activity by in vitro assays on the RPMI-8226 cell line. The bioguided fractionation of the crude ethanolic extract allowed the identification of HsFC as the most effective extract. We assessed cell viability (MTT and Tripan blue test), cell migration (Boyden chamber assay), and neurotoxicity (DRG neurotoxicity assay). The promising results prompted us to further fractionate HsFC and we obtained two molecules effective against RPMI-8226 cells without neurotoxic effects at their active concentrations. Moreover, both compounds are able to significantly reduce cell migration.

Chemotherapy-Induced Peripheral Neuropathy and Changes in Cytoskeleton.

Despite the different antineoplastic mechanisms of action, peripheral neurotoxicity induced by all chemotherapy drugs (anti-tubulin agents, platinum compounds, proteasome inhibitors, thalidomide) is associated with neuron morphological changes ascribable to cytoskeleton modifications. The "dying back" degeneration of distal terminals (sensory nerves) of dorsal root ganglia sensory neurons, observed in animal models, in in vitro cultures and biopsies of patients is the most evident hallmark of the perturbation of the cytoskeleton. On the other hand, in highly polarized cells like neurons, the cytoskeleton carries out its role not only in axons but also has a fundamental role in dendrite plasticity and in the organization of soma. In the literature, there are many studies focused on the antineoplastic-induced alteration of microtubule organization (and consequently, fast axonal transport defects) while very few studies have investigated the effect of the different classes of drugs on microfilaments, intermediate filaments and associated proteins. Therefore, in this review, we will focus on: (1) Highlighting the fundamental role of the crosstalk among the three filamentous subsystems and (2) investigating pivotal cytoskeleton-associated proteins.

An actionable axis linking NFATc2 to EZH2 controls the EMT-like program of melanoma cells.

Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+ EZH2+ melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

Facial emotion recognition in schizophrenia: An exploratory study on the role of comorbid alcohol and substance use disorders and COMT Val158Met.

OBJECTIVES: To explore whether facial emotion recognition (FER), impaired in both schizophrenia and alcohol and substance use disorders (AUDs/SUDs), is additionally compromised among comorbid subjects, also considering the role of catechol-O-methyltransferase (COMT) Val158Met. METHODS: We conducted a cross-sectional study, randomly recruiting 67 subjects with a DSM-IV-TR diagnosis of schizophrenia, and rigorously assessing AUDs/SUDs and COMT Val158Met polymorphism. FER was assessed using the Ekman 60 Faces Test- EK-60F. RESULTS: As a whole, the sample scored significantly lower than normative data on EK-60F. However, subjects with comorbid AUDs/SUDs did not perform worse on EK-60F than those without, who had a better performance on EK-60F if they carried the COMT Val/Met variant. CONCLUSIONS: This study is the first to date examining the impact of AUDs/SUDs and COMT variants on FER in an epidemiologically representative sample of subjects with schizophrenia. Our findings do not suggest an additional impairment from comorbid AUDs/SUDs on FER among subjects with schizophrenia, whilst COMT Val158Met, though based on a limited sample, might have a role just among those without AUDs/SUDs. Based on our results, additional research is needed also exploring differential roles of various substances.

Executive control in schizophrenia: a preliminary study on the moderating role of COMT Val158Met for comorbid alcohol and substance use disorders.

BACKGROUND: A functional polymorphism in the catechol-O-methyltransferase (COMT) gene (Val158Met) appears to influence cognition in people with alcohol/substance use disorders (AUD/SUD) and in those with psychosis. METHODS: To explore the potential moderating effect of these factors, a cross-sectional study was conducted, randomly recruiting subjects with DSM-IV diagnosis of schizophrenia. AUD/SUD was rigorously assessed, as well as COMT Val158Met polymorphism. Executive control functioning was measured using the Intra-Extra Dimensional Set Shift (IED). The effect of a possible interaction between comorbid AUD/SUD and COMT Val158Met polymorphism on IED scores was explored. RESULTS: Subjects with schizophrenia, comorbid AUD/SUD, and MetMet carriers for SNP rs4680 of the COMT gene showed worse performance on IED completed stages scores, as compared with individuals with ValVal genotype. However, among subjects without AUD/SUD, those with the MetMet variant performed better than people carrying ValVal genotype. CONCLUSIONS: This study is the first to date examining the impact of COMT on cognition in a highly representative sample of people with schizophrenia and comorbid AUD/SUD. Differential moderating effects of COMT Val/Met genotype variations may similarly influence executive functions in people with schizophrenia and comorbid AUD/SUD.

Histologic and metabolic assessment in a cohort of patients with genital prolapse: preoperative stage and recurrence investigations.

BACKGROUND: Biological basis of prolapse development and recurrence are still unclear. Aim of this observational and prospective study is to correlate clinical stage of anterior vaginal wall prolapse and anatomical recurrence to histological and metabolic characteristics of vaginal tissue. METHODS: Patients undergoing surgery were divided into two groups according to anterior stage ≤II (group A) and ≥III (group B). Full-thickness excisional biopsies of the anterior vaginal wall were obtained after hysterectomy. Hystological characteristics and metalloproteinases activity (MMP-2) were analyzed. RESULTS: Sixty-nine patients (35 group A; 34 group B) completed evaluation. Mean follow-up was 35 months. Collagen amount and organization were significantly higher in group B both in lamina propria and fascia specimens, but MMP-2 activity was significantly lower in this group. Recurrence rate of anterior compartment was 10.1%. Collagen cellularity of fascia was higher in recurrence groups. On the contrary MMP-2 activity showed a close to significant correlation to surgical success (P=0.07). CONCLUSIONS: Patients with advanced stages of prolapse have increased collagen amount associated to decreased MMP-2 activity. This suggests that connective tissue is more abundant but less metabolically active in patients with severe prolapse. A similar trend can be found in recurrences.

Antitumoral Effect of Hibiscus sabdariffa on Human Squamous Cell Carcinoma and Multiple Myeloma Cells.

Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 µM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

A melanoma subtype with intrinsic resistance to BRAF inhibition identified by receptor tyrosine kinases gene-driven classification.

Dysregulation of receptor tyrosine kinases (RTKs) contributes to several aspects of oncogenesis including drug resistance. In melanoma, distinct RTKs have been involved in BRAF inhibitors (BRAFi) resistance, yet the utility of RTKs expression pattern to identify intrinsically resistant tumors has not been assessed. Transcriptional profiling of RTKs and integration with a previous classification, reveals three robust subtypes in two independent datasets of melanoma cell lines and one cohort of melanoma samples. This classification was validated by Western blot in a panel of patient-derived melanoma cell lines. One of the subtypes identified here for the first time displayed the highest and lowest expression of EGFR and ERBB3, respectively, and included BRAF-mutant tumors all intrinsically resistant to BRAFi PLX4720, as assessed by analysis of the Cancer Cell Line Encyclopedia pharmacogenomic study and by in vitro growth inhibition assays. High levels of EGFR were detected, even before therapy, in tumor cells of one of three melanoma patients unresponsive to BRAFi. Use of different pharmacological inhibitors highlighted the relevance of PI3K/mTOR signaling for growth of this PLX4720-resistant subtype. Our results identify a specific molecular profile of melanomas intrinsically resistant to BRAFi and suggest the PI3K/mTOR pathway as a potential therapeutic target for these tumors.

Axonal Transport Impairment in Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a dose-limiting side effect of several antineoplastic drugs which significantly reduces patients' quality of life. Although different molecular mechanisms have been investigated, CIPN pathobiology has not been clarified yet. It has largely been recognized that Dorsal Root Ganglia are the main targets of chemotherapy and that the longest nerves are the most damaged, together with fast axonal transport. Indeed, this bidirectional cargo-specific transport has a pivotal role in neuronal function and its impairment is involved in several neurodegenerative and neurodevelopmental diseases. Literature data demonstrate that, despite different mechanisms of action, all antineoplastic agents impair the axonal trafficking to some extent and the severity of the neuropathy correlates with the degree of damage on this bidirectional transport. In this paper, we will examine the effect of the main old and new chemotherapeutic drug categories on axonal transport, with the aim of clarifying their potential mechanisms of action, and, if possible, of identifying neuroprotective strategies, based on the knowledge of the alterations induced by each drugs.