Purpose: MET, the tyrosine kinase receptor for hepatocyte growth factor, is frequently overexpressed in colon cancers with high metastatic tendency. We aimed to evaluate the role of its negative regulators, miR-1 and miR-199a*, and its transcriptional activator, the metastasis-associated in colon cancer 1 (MACC1), in controlling MET expression in human colon cancer samples. Experimental Design: The expression of MET, miR-1, miR-199a*, and MACC1 was evaluated by real-time PCR in 52 matched pairs of colorectal cancers and nontumoral surrounding tissues. The biological role of miR-1 in controlling MET expression and biological activity was assessed in colon cancer cells either by its forced expression or by AntagomiR-mediated inhibition. Results: MiR-1 was downregulated in 84.6% of the tumors and its decrease significantly correlated with MET overexpression, particularly in metastatic tumors. We found that concurrent MACC1 upregulation and miR-1 downregulation are required to elicit the highest increase of MET expression. Consistent with a suppressive role of miR-1, its forced in vitro expression in colon cancer cells reduced MET levels and impaired MET-induced invasive growth. Finally, we identified a feedback loop between miR-1 and MET, resulting in their mutual regulation. Conclusions: This study identifies an oncosuppressive role of miR-1 in colorectal cancer in which it acts by controlling MET expression through a feedback loop. Concomitant downregulation of miR-1 and increase of MACC1 can thus contribute to MET overexpression and to the metastatic behavior of colon cancer cells. Clin Cancer Res; 18(3); 737–47. ©2011 AACR.

Background Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. Methods PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. Results PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. Conclusions These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP⁺ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP⁺ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP⁺ MCs (1/42) compared with the eGFP⁻ counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP⁺ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

Receptor tyrosine kinases (RTKs) inhibitors’ activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.

The objectives of this narrative review are as follows: an evaluation of the bromatological composition of hazelnuts and a comparison of the nutritional properties of raw versus roasted hazelnuts, taking into account potential differences among varieties from different production territories such as Turkey, Italy, Chile, and New Zealand; an evaluation of nutrients contained in hazelnut skin; and an evaluation of nutrients contained in hazelnut oil. This review incorporates 27 scientific articles that measured and reported the concentrations of macro- and micro-nutrients in hazelnuts. These hazelnuts were subjected to different processing methods, originated from various geographical areas, or belonged to different varieties. Our results showed that the different varieties and territories where the hazelnuts were cultivated influence their bromatological composition, and we found that different processing steps can largely influence the concentration of specific nutrients. The removal of the skin, which contains a very high concentration of compounds with antioxidant action, is particularly critical. We should give greater attention to the skin, considering it not as a waste product, but as an important part of the hazelnut due to its nutritional properties of primary relevance in the Mediterranean diet. We provide a detailed assessment of the nutritional properties of the hazelnut kernel, skin, and oil, evaluating nutrient compositions and possible modifications (increases or reductions) that occur during the roasting process or that depend on the production territory and origin.

The relevant role in cancer played by the tyrosine kinase receptor encoded by the MET oncogene led to the development of specific inhibitors, some of which are now in advanced phases of clinical trials. Previous experience has shown that the main limit to the efficacy of most targeted treatments is the advent of resistance. Mechanisms underlying resistance to MET‐specific small tyrosine kinase inhibitors (TKIs) have been already described, while nothing is known about resistance to MET monoclonal antibodies, nor about bypassing resistance to chemical TKIs by antibodies or vice‐versa. EBC1 lung cancer cells are MET‐addicted as a consequence of gene amplification and thus sensitive to MET inhibitors, including the monovalent form of a MET monoclonal antibody (MV‐DN30). We generated cells resistant to this antibody and found that resistance was due to a further increase of gene copy number and a dramatic overexpression of the MET receptor. Such an excess of expression saturated the ‘shedding’ activity of MV‐DN30, and prevented both the efficient down‐regulation of the MET receptor from the surface and the inhibition of the ensuing constitutive activation. Notably, antibody‐resistant cells remained MET‐‘addicted’ and were still sensitive to MET TKIs. Moreover, antibody‐resistant cells became ‘drug‐dependent’, since the removal of MV‐DN30 led them to death due to excess of signal. In the mirror experiment, cells made resistant to MET‐specific TKIs were still sensitive to treatment with the antibody MV‐DN30. These findings suggest that a discontinuous, combined treatment by antibodies and chemical kinase inhibitors may increase the clinical response and bypass resistance to anti‐MET targeted therapies.