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 Met high subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Met high 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. Ó2012 AACR.

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) – surprisingly – autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.

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.

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.

On the basis of our previous findings that 5'-O-tritylinosine (KIN59) behaves as an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase (TPase), we have undertaken the synthesis and enzymatic evaluation of a novel series of nucleoside analogues modified at positions 1, 2, or 6 of the purine ring and at the 5'-position of the ribose moiety of the lead compound KIN59. SAR studies indicate that quite large structural variations can be performed on KIN59 without compromising TPase inhibition. Thus, incorporation of a cyclopropylmethyl or a cyclohexylmethyl group at position N(1) of 5'-O-tritylinosine increases the inhibitory activity against TPase 10-fold compared to KIN59. Moreover, the trityl group at the 5'-position of the ribose seems to be crucial for TPase inhibition. The here reported results further substantiate that 5'-O-trityl nucleosides represent a new class of TPase inhibitors that should be further explored in those biological systems where TPase plays an instrumental role (i.e. angiogenesis).

Highlights d Total of 9% of human GBMs feature ERBB3 overexpression and nuclear accumulation d ERBB3 overexpression is sustained by miR-205 inactivation d Overexpressed ERBB3 is activated by FGFR and upregulates cell metabolism d ERBB3 targeting by a specific antibody inhibits growth of ERBB3-overexpressing GBMs

Purified bile salt hydrolase from bile-adapted Xanthomonas maltophilia displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60°C for up to 60 min, in agreement with calorimetric data.Recently, we examined the metabolism of bile salts by the bile-adapted strain Xanthomonas maltophilia CBS 827.97, which hydrolyzes bile salt conjugates, rearranging the steroid nucleus (1, 5). Bile salts have multiple functions in digestion of lipids, solubilization, and excretion of cholesterol (2,19,23). Ursodeoxycholic acid is particularly interesting in this regard, solubilizing gallbladder cholesterol stones after oral administration (20). Ursodeoxycholic acid is currently prepared by alkaline hydrolysis of bile conjugates, inversion of the hydroxyl configuration at carbon 7, and resolution of the racemic mixture (reviewed in reference 1). Procedures for enzymatic synthesis would be useful for controlling stereochemistry, increasing yields, and avoiding toxic or aggressive chemicals. In this paper we describe a solution to the hydrolysis step, in which purified bile salt hydrolase (cholylglycine hydrolase [CGH], EC 3.5.1.24) from our bacterial strain is used.Procedures for purification of CGH and of other enzymes of bile salt metabolism are covered by an Italian patent (1). For purification of CGH, the enzyme was isolated from 10 g of bacteria grown as described previously (5) and lysed by overnight stirring in 10 volumes of 20 mM sodium phosphate-1 mM EDTA-2 mM mercaptoethanol (pH 7.5) (NaP buffer) containing 0.3 mg of egg lysozyme per ml and 1 mM phenylmethylsulfonyl fluoride. Cell debris was removed by centrifugation (200,000 ϫ g, 20 min), and nucleic acids were removed by precipitation with protamine sulfate (2 mg/ml). The supernatant, adjusted to pH 8.3, was chromatographed on a DEAESepharose column (30 ml) and eluted with a linear 0 to 0.25 M NaCl gradient in NaP buffer (pH 8.3). The second protein peak, displaying hydrolase activity, was fractionated with ammonium sulfate in order to collect proteins insoluble between 45 and 75% saturation. After dialysis against NaP buffer, the phosphate concentration of the solution was adjusted to 200 mM at pH 7.5 by adding solid salts, and the solution was heated to 55°C with continuous swirling in a water bath at 70°C. After the solution was cooled on ice, denatured aggregated proteins were removed by high-speed centrifugation, and the supernatant, diluted with 1 volume of water, was loaded onto a phenyl-Sepharose column (2 ml) equilibrated in 100 mM phosphate buffer (pH 7.5). Hydrolase activity was eluted with NaP buffer. The CGH activity assay was performed by measuring glycine release from 5 mM cholylglycine in NaP buffer by a cadmium-ninhydrin procedure (6). For the cholyltaurine hydrolysis assay, we employed a different ninhydrin formulation (17). The protein concentration was determined as described previously (18).The yield was 0...

The Saccharomyces cerevisiae atypical protein kinase Bud32p is a member of the nuclear endopeptidase‐like, kinase, chromatin‐associated/kinase, endopeptidase‐like and other protein of small size (EKC/KEOPS) complex, known to be involved in the control of transcription and telomere homeostasis. Complex subunits (Pcc1p, Pcc2p, Cgi121p, Kae1p) represent, however, a small subset of the proteins able to interact with Bud32p, suggesting that this protein may be endowed with additional roles unrelated to its participation in the EKC/KEOPS complex. In this context, we investigated the relationships between Bud32p and the nuclear glutaredoxin Grx4p, showing that it is actually a physiological substrate of the kinase and that Bud32p contributes to the full functionality of Grx4p in vivo. We also show that this regulatory system is influenced by the phosphorylation of Bud32p at Ser258, which is specifically mediated by the Sch9p kinase [yeast homolog of mammalian protein kinase B (Akt/PKB)]. Notably, Ser258 phosphorylation of Bud32p does not alter the catalytic activity of the protein kinase per se, but positively regulates its ability to interact with Grx4p and thus to phosphorylate it. Interestingly, this novel signaling pathway represents a function of Bud32p that is independent from its role in the EKC/KEOPS complex, as the known functions of the complex in the regulation of transcription and telomere homeostasis are unaffected when the cascade is impaired. A similar relationship has already been observed in humans between Akt/PKB and p53‐related protein kinase (Bud32p homolog), and could indicate that this pathway is conserved throughout evolution.