The existence of treatment-resistant cancer stem cells contributes to the aggressive phenotype of glioblastoma. However, the molecular alterations that drive stem cell proliferation in these tumors remain unknown. In this study, we found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma. Met expression was almost absent from neurospheres expressing the signature of the classical subtype and was mutually exclusive with amplification and expression of the EGF receptor (EGFR) gene. Met-positive and Met-negative neurospheres displayed distinct growth factor requirements, differentiated along divergent pathways, and generated tumors with distinctive features. The Methigh subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Methigh cells, the Met ligand HGF further sustained proliferation, clonogenicity, expression of self-renewal markers, migration, and invasion in vitro. Together, our findings suggest that Met is a functional marker of glioblastoma stem cells and a candidate target for identification and therapy of a subset of glioblastomas. Cancer Res; 72(17); 4537–50. ©2012 AACR.

Glioblastoma (GBM) contains stem‐like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti‐apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC‐positive selection, induced by radiotherapy, into GSC eradication. Synopsis GBM radioresistance relies on the inherent properties of its stem‐like cell population (GSCs). MET, which encodes for the HGF receptor, is shown to be a functional marker of GSC radioresistance and a therapeutic target for GSC radiosensitization. MET expression identifies radioresistant GSCs derived from a subset of patients and propagated as neurospheres. In GBMs recurring after radiotherapy, MET expression is more frequent, as compared with matched primary tumors (85% versus 45%). MET drives positive selection of irradiated GSCs, by promoting the DNA damage response via AKT, which sustains ATM activation, and p21 phosphorylation and cytoplasmic retention. When combined with radiotherapy, MET inhibition impairs the GSC DNA damage response and converts GSC selection from positive to negative.

The inflammatory cytokine Tumor Necrosis Factor Alpha (TNF‐α) is known to trigger invasive growth, a physiological property for tissue healing, turning into a hallmark of progression in cancer. However, the invasive response to TNF‐α relies on poorly understood molecular mechanisms. We thus investigated whether it involves the MET oncogene, which regulates the invasive growth program by encoding the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF). Here we show that the TNF‐α pro‐invasive activity requires MET function, as it is fully inhibited by MET‐specific inhibitors (small‐molecules, antibodies, and siRNAs). Mechanistically, we show that TNF‐α induces MET transcription via NF‐κB, and exploits MET to sustain MEK/ERK activation and Snail accumulation, leading to E‐cadherin downregulation. We then show that TNF‐α not only induces MET expression in cancer cells, but also HGF secretion by fibroblasts. Consistently, we found that, in human colorectal cancer tissues, high levels of TNF‐α correlates with increased expression of both MET and HGF. These findings suggest that TNF‐α fosters a HGF/MET pro‐invasive paracrine loop in tumors. Targeting this ligand/receptor pair would contribute to prevent cancer progression associated with inflammation.

In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.

Doxorubicin anti‐cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its administration is hampered by low biodistribution. In this study we investigated whether the activation of the HGF receptor ‐ encoded by the Met gene ‐ by an agonist monoclonal antibody (mAb) protects from doxorubicin‐induced cardiotoxicity. MAb (5 mg/kg) was injected in vivo into C57BL/6J mice prior to doxorubicin (three doses of 7 mg/kg). The cardiac functions were evaluated through magnetic resonance imaging (MRI) after treatment termination. Heart histological staining and mRNA levels of genes associated with heart failure (Acta1, Nppa), inflammation (IL‐6) and fibrosis (Ctgf, Col1a2, Timp1, and Mmp9) were assessed. MAb (100 nM) was administered in vitro to H9c2 cardiomyoblasts before addition of doxorubicin (25 μM). DDR and apoptosis markers were evaluated by quantitative western blotting, flow cytometry and immunofluorescence. Stattic was used for pharmacological inactivation of signal transducer and activation of transcription 3 (Stat3). In vivo, administration of mAb alleviated doxorubicin‐induced cardiac dysfunction and fibrosis. In vitro, mAb mimicked the response to HGF by (i) inhibiting histone H2AX phosphorylation at S139, (ii) quenching the expression of the DNA repair enzyme poly (ADP‐ribose) polymerase 1, and (iii) reducing the proteolytic activation of caspase 3. The Met‐driven cardioprotection involved, at least in vitro, the phosphorylation of Stat3. This paper shows that Met agonist mAb provides a new tool for cardioprotection against anthracycline cardiotoxicity.

Melanomas are characterised by accelerated cell proliferation and metabolic reprogramming resulting from the contemporary dysregulation of the MAPK pathway, glycolysis and the tricarboxylic acid (TCA) cycle. Here, we suggest that the oncogenic transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and function, controls melanoma tumour growth through a transcriptional programme targeting ERK1/2 activity and glucose, glutamine and cholesterol metabolism. Mechanistically, TFEB binds and negatively regulates the promoter of DUSP-1, which dephosphorylates ERK1/2. In melanoma cells, TFEB silencing correlates with ERK1/2 dephosphorylation at the activation-related p-Thr185 and p-Tyr187 residues. The decreased ERK1/2 activity synergises with TFEB control of CDK4 expression, resulting in cell proliferation blockade. Simultaneously, TFEB rewires metabolism, influencing glycolysis, glucose and glutamine uptake, and cholesterol synthesis. In TFEB-silenced melanoma cells, cholesterol synthesis is impaired, and the uptake of glucose and glutamine is inhibited, leading to a reduction in glycolysis, glutaminolysis and oxidative phosphorylation. Moreover, the reduction in TFEB level induces reverses TCA cycle, leading to fatty acid production. A syngeneic BRAFV600E melanoma model recapitulated the in vitro study results, showing that TFEB silencing sustains the reduction in tumour growth, increase in DUSP-1 level and inhibition of ERK1/2 action, suggesting a pivotal role for TFEB in maintaining proliferative melanoma cell behaviour and the operational metabolic pathways necessary for meeting the high energy demands of melanoma cells.