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.

Antitumour activity of resveratrol on human melanoma cells: A possible mechanism related to its interaction with malignant cell telomerase.

BACKGROUND: trans-Resveratrol (tRES) is a polyphenolic stilbene found in plant products which has attracted great attention because of its antioxidant, anti-inflammatory and anticancer properties. METHODS: The possible correlation between tRES-induced suppression of melanoma cell growth and its influence on telomerase expression has been investigated by biological assays. Moreover, in order to gain new knowledge about possible mechanisms of action of tRES as antineoplastic agent, its interaction with biologically relevant secondary structure-forming DNA sequences, its aggregation properties and copper-binding activity have been studied by CD, UV and fluorescence spectroscopies. RESULTS: Biological assays have confirmed that growth inhibitory properties of tRES well correlate with the reduction of telomerase activity and hTERT gene transcript levels in human melanoma cells. Biophysical studies in solution have proved that tRES binds all the studied DNA model systems with low affinity, however showing high ability to discriminate G-quadruplex vs. duplex DNA. In addition, tRES has shown no propensity to form aggregates in the explored concentration range and has been found able to bind Cu2+ ions with a 2:1 stoichiometry. CONCLUSIONS: From these biological and biophysical analyses it has emerged that tRES produces cytotoxic effects on human melanoma cells and, at a molecular level, is able to bind Cu2+ and cancer-involved G-quadruplexes, suggesting that multiple mechanisms of action could be involved in its antineoplastic activity. GENERAL SIGNIFICANCE: Expanding the knowledge on the putative mechanisms of action of tRES as antitumour agent can help to develop novel, effective tRES-based anticancer drugs.

Circulating miR-1246 and miR-485-3p as Promising Biomarkers of Clinical Response and Outcome in Melanoma Patients Treated with Targeted Therapy.

Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan-Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.

Exogenous control of the expression of Group I CD1 molecules competent for presentation of microbial nonpeptide antigens to human T lymphocytes.

Group I CD1 (CD1a, CD1b, and CD1c) glycoproteins expressed on immature and mature dendritic cells present nonpeptide antigens (i.e., lipid or glycolipid molecules mainly of microbial origin) to T cells. Cytotoxic CD1-restricted T lymphocytes recognizing mycobacterial lipid antigens were found in tuberculosis patients. However, thanks to a complex interplay between mycobacteria and CD1 system, M. tuberculosis possesses a successful tactic based, at least in part, on CD1 downregulation to evade CD1-dependent immunity. On the ground of these findings, it is reasonable to hypothesize that modulation of CD1 protein expression by chemical, biological, or infectious agents could influence host's immune reactivity against M. tuberculosis-associated lipids, possibly affecting antitubercular resistance. This scenario prompted us to perform a detailed analysis of the literature concerning the effect of external agents on Group I CD1 expression in order to obtain valuable information on the possible strategies to be adopted for driving properly CD1-dependent immune functions in human pathology and in particular, in human tuberculosis.

Reduction of T Lymphoma Cells and Immunological Invigoration in a Patient Concurrently Affected by Melanoma and Sezary Syndrome Treated With Nivolumab.

Despite the recent availability of several new drugs in hemato-oncology, T-cell lymphomas are still incurable and PD-1 blockade could represent a therapeutic chance for selected patients affected by these malignancies, although further studies are required to understand the biological effects of anti-PD-1 mAbs on neoplastic T-cells and to identify biomarkers for predicting and/or monitoring patients' response to therapy. Sezary Syndrome (SS) represents a rare and aggressive variant of cutaneous T cell lymphoma (CTCL) with a life expectancy of less than 5 years, characterized by the co-presence of neoplastic lymphocytes mainly in the blood, lymph nodes and skin. In this study we analyzed longitudinal blood samples and lesional skin biopsies of a patient concurrently affected by SS and melanoma who underwent 22 nivolumab administrations. In blood, we observed a progressive reduction of SS cell number and a raise in the percentage of normal CD4+ and CD8+ T cells and NK cells over total leukocytes. Eight weeks from the start of nivolumab, these immune cell subsets showed an increase of Ki67 proliferation index that positively correlated with their PD-1 expression. Conversely, SS cells displayed a strong reduction of Ki67 positivity despite their high PD-1 expression. On skin biopsies we observed a marked reduction of SS cells which were no more detectable at the end of therapy. We also found an increase in the percentage of normal CD4+ T cells with a concomitant decrease of that of CD8+ and CD4+ CD8+ T cells, two cell subsets that, however, acquired a cytotoxic phenotype. In summary, our study demonstrated that nivolumab marked reduced SS tumor burden and invigorated immune responses in our patient. Our data also suggest, for the first time, that Ki67 expression in circulating neoplastic and immune cell subsets, as well as an enrichment in T cells with a cytotoxic phenotype in lesional skin could be valuable markers to assess early on treatment SS patients' response to PD-1 blockade, a therapeutic strategy under clinical investigation in CTCL (ClinicalTrials.gov NCT03385226, NCT04118868).

miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

BACKGROUND: Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma. Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi. METHODS: miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR. The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA. ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting. Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines. Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development. VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA. RESULTS: miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity. VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines. ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance. At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not. In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2. CONCLUSIONS: Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity. Circulating VEGF-A is a promising biomarker for predicting patients' response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.

BCG-infected adherent mononuclear cells release cytokines that regulate group 1 CD1 molecule expression.

Increasing evidence is now available showing that CD1-restricted T cell responses against non-peptide mycobacterial antigens could play a role in the immune resistance against tuberculosis. BCG, widely used in anti-tubercular vaccination, shares various constituents with Mycobacterium tuberculosis, but does not provide full protection. In the present study we have investigated the pattern of group 1 CD1 molecule expression in adherent mononuclear cells (AMNC) of human peripheral blood, infected in vitro with BCG. Shortly after exposure to BCG, both BCG-positive and BCG-negative AMNC showed a moderate CD1 expression elicited by BCG-induced release of GM-CSF presumably acting through an autocrine and a paracrine mechanism. This was demonstrated using two-color flow cytometry with green fluorescent BCG and anti-CD1 PE-labeled antibodies. However, high CD1 expression induced by exogenously added GM-CSF in AMNC was reduced if target cells were cocultivated with BCG. Monoclonal antibodies against IL-10 partially restored CD1 expression, thus showing that IL-10, released from infected AMNC, is involved, at least in part, in CD1 negative modulation. Therefore, through a complex cytokine network, including not yet identified factor(s), BCG triggers but does not allow full expression of CD1 on AMNC. It cannot be excluded that this mechanism could play a role in the limited efficiency of BCG vaccination.

Targeting the PTTG1 oncogene impairs proliferation and invasiveness of melanoma cells sensitive or with acquired resistance to the BRAF inhibitor dabrafenib.

The pituitary tumor transforming gene 1 (PTTG1) is implicated in tumor growth, metastasis and drug resistance. Here, we investigated the involvement of PTTG1 in melanoma cell proliferation, invasiveness and response to the BRAF inhibitor (BRAFi) dabrafenib. We also preliminary assessed the potential value of circulating PTTG1 protein to monitor melanoma patient response to BRAFi or to dabrafenib plus trametinib. Dabrafenib-resistant cell lines (A375R and SK-Mel28R) were more invasive than their drug-sensitive counterparts (A375 and SK-Mel28), but expressed comparable PTTG1 levels. Dabrafenib abrogated PTTG1 expression and impaired invasion of the extracellular matrix (ECM) in A375 and SK-Mel28 cells. In contrast, it affected neither PTTG1 expression in A375R and SK-Mel28R cells, nor ECM invasion in the latter cells, while further stimulated A375R cell invasiveness. Assessment of proliferation and ECM invasion in control and PTTG1-silenced A375 and SK-Mel28 cells, exposed or not to dabrafenib, demonstrated that the inhibitory effects of this drug were, at least in part, dependent on its ability to down-regulate PTTG1 expression. PTTG1-silencing also impaired proliferation and invasiveness of A375R and SK-Mel28R cells, and counteracted dabrafenib-induced stimulation of ECM invasion in A375R cells. Further experiments performed in A375R cells indicated that PTTG1-silencing impaired cell invasiveness through inhibition of MMP-9 and that PTTG1 expression and ECM invasion could be also reduced by the CDK4/6 inhibitor LEE011. PTTG1 targeting might, therefore, represent a useful strategy to impair proliferation and metastasis of melanomas resistant to BRAFi. Circulating PTTG1 also appeared to deserve further investigation as biomarker to monitor patient response to targeted therapy.

A novel telomerase-based approach to detect natural cell-mediated cytotoxic activity against tumor cells in vitro.

This study was designed to develop a novel technical approach based on tumor-associated telomerase activity to detect cytotoxic activity of effector cells of the natural immune system against neoplastic cells. Human K562, DAUDI or Raji leukemia cells were co-cultured with NK or LAK effector cells at 37 degrees C for 4 h. Target cell killing was evaluated by 51Cr-release assay (CRA) or reduction of telomerase activity (R-TRAPCTX) of the target after exposure to effector cells. NK and LAK effector cells tested against K562 target cells at effector/target ratio of 50:1 showed cytotoxicity of 65% and 78%, respectively, with CRA and 51% and 74%, respectively, with R-TRAPCTX. Incorrect results were obtained with CRA when target cells were admixed with normal fibroblasts, whereas R-TRAPCTX was not influenced by the presence of normal cells. Control experiments performed with telomerase-negative cells showed that telomerase activity of effector cells was not altered during the cytolytic reaction. Moreover, supernatants obtained from effector-target cell co-cultures did not influence telomerase activity of targets. This novel R-TRAPCTX method to assay anti-tumor natural and possibly antigen-dependent cell-mediated cytotoxicity appears to provide sensible advantages over classical CRA or gamma-interferon release by effector cells in presence of target cells (ELISPOT), since (a) it furnishes reliable data on effector cell killing against neoplastic cells, even when malignant cells are admixed with normal cells, as frequently occurs in tumor biopsies, not manageable with CRA; (b) it provides an actual measure of target cell killing, not furnished by ELISPOT technique.

Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.

Interferon-Beta combined with interleukin-2 restores human natural cytotoxicity impaired in vitro by ionizing radiations.

It is well known that ionizing radiations induce a marked downregulation of antigen-dependent and natural immunity for a prolonged period of time. This is due, at least in part, to radiation-induced apoptosis of different lymphocyte subpopulations, including natural killer (NK) cells. Aim of this study was to investigate the capability of Beta Interferon (ß-IFN) and Interleukin-2 (IL2), alone or in combination, to restore the functional activity of the natural immune system. Mononuclear cells (MNCs) obtained from intact or in vitro irradiated human peripheral blood were treated in vitro with ß-IFN immediately before or at the end of the 4-day treatment with IL2. Time-course analysis was performed on the NK activity, the total number and the apoptotic fraction of CD16+ and CD56+ cells, the 2 main NK effector cell subpopulations. The results indicate that radiation-induced impairment of natural cytotoxicity of MNC could be successfully antagonized by the ß-IFN+IL2 combination, mainly when exposure to ß-IFN preceded IL2 treatment. This radioprotective effect is paralleled by lower levels of radiation-induced apoptosis and increased expression of the antiapoptotic Bcl-2 protein. Since natural immunity can play a significant role in antitumor host's resistance, these results could provide the rational basis for a cytokine-based pharmacological strategy able to restore immune responsiveness and to afford possible therapeutic benefits in cancer patients undergoing radiotherapy.

The antiretroviral agent saquinavir enhances hTERT expression and telomerase activity in human T leukaemia cells in vitro.

BACKGROUND: Saquinavir, a protease inhibitor utilized in HIV infection, shows antitumor activity in various experimental models. In previous studies performed in our laboratory the drug was found to induce a substantial increase of telomerase activity in normal peripheral blood mononuclear cells. Aim of the present investigation was to test whether saquinavir was able to increase telomerase activity and the expression of the catalytic subunit of telomerase, hTERT, in human malignant hematopoietic cells. METHODS: Human Jurkat CD4+ T cell leukaemia cell line was used throughout the present study. The antiproliferative effect of saquinavir was tested by the MTT assay. Telomerase activity was determined according to the telomeric repeat amplification protocol. The expression of hTERT mRNA was semi-quantitative evaluated by RT-PCR amplification and quantitative Real Time PCR. The binding of the transcription factor c-Myc to its specific E-Box DNA binding-site of hTERT promoter was analyzed by Electophoretic Mobility Shift Assay (EMSA). The amount of c-Myc in cytoplasm and nucleus of leukemia cells was determined by Western Blot analysis, and c-Myc down-regulation was obtained by siRNA transfection. RESULTS: Saquinavir produced a substantial increase of telomerase activity in Jurkat cells in vitro without increasing but rather reducing target cell proliferation rate. Telomerase up-regulation appeared to be the result of enhanced expression of hTERT. Saquinavir-mediated up-regulation of hTERT gene was the result of the increased binding of proteins to the E-Box sequence of the promoter. Moreover, saquinavir amplified the expression of c-Myc especially in the nuclear cell fraction. The direct influence of saquinavir on this transcription factor was also demonstrated by the antagonistic effect of the drug on siRNA induced c-Myc suppression. Since c-Myc is the main responsible for hTERT transcription, these findings suggest that the main mechanism underlying saquinavir-induced telomerase activation is mediated by c-Myc up-regulation. CONCLUSIONS: Saquinavir augments hTERT expression while inhibiting leukemic cell growth. Experimental evidences show that this effect is mediated by saquinavir-influenced increase of c-Myc levels. This could have relevance in terms of enhanced hTERT-dependent tumor cell immunogenicity and suggests new paharmacological approaches interfering with c-Myc dependent pathways.

Circulating tumor cells in colorectal cancer patients.

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.

Detection of circulating tumor cells is improved by drug-induced antigen up-regulation: preclinical and clinical studies.

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.

Combined effects of 5-fluorouracil, folinic acid and oxaliplatin on the expression of carcinoembryonic antigen in human colon cancer cells: pharmacological basis to develop an active antitumor immunochemotherapy.

BACKGROUND: Five-fluorouracil (FU), mainly associated with leucovorin (L), plays an essential role in chemotherapy of colorectal carcinoma. Moreover, FU +/- L has been found to increase the expression of tumor-associated carcinoembryonic antigen (CEA), that may be an important target in therapeutic protocols of active specific immunotherapy. FU + L (FUL) are frequently combined with oxaliplatin (OXA) in advanced colon cancer patients. Thus, we investigated whether FUL in combination with OXA according to 2 different schedules may influence CEA expression in human colon cancer cells in vitro. METHODS: CEA protein expression was evaluated by cytofluorimetric and western blot analysis. Relative quantification of CEA mRNA was assessed by real time RT-PCR analysis. RESULTS: Levels of CEA protein and transcript were found to be higher in FUL-treated cells than in controls. However, when target cells were exposed to OXA before but not after FUL treatment, the up-regulation of CEA was partially inhibited. CONCLUSION: These results suggest that target cells must be exposed to OXA after but not before treatment with the fluoropyrimidine in order to exploit drug-induced up-regulation of CEA. This finding appears to provide useful information to design chemo-immunotherapy protocols based on FUL + OXA, combined with host's immunity against CEA directed cancer vaccines.