A preclinical algorithm of soluble surrogate biomarkers that correlate with therapeutic inhibition of the MET oncogene in gastric tumors.

The MET oncogene is amplified in a fraction of human gastric carcinoma cell lines, with consequent overexpression and constitutive activation of the corresponding protein product, the Met tyrosine kinase receptor. This genetically driven hyperactivation of Met is necessary for cancer cell growth and survival, so that Met pharmacological blockade results in cell-cycle arrest or apoptosis (oncogene addiction). MET gene amplification also occurs in vivo in a number of human gastric carcinomas, and clinical trials are now ongoing to assess the therapeutic efficacy of Met inhibitors in this type of malignancy. The aim of our study was to identify a preclinical algorithm of soluble surrogate biomarkers indicative of response to Met inhibition in gastric tumors, as a potential tool to integrate imaging criteria during patient follow-up. We started from a survey of candidate molecules based on antibody proteomics and gene expression profiling; after ELISA validation and analytical quantification, four biomarkers were identified that appeared to be strongly and consistently modulated by Met inhibition in a panel of Met-addicted gastric carcinoma cell lines, but not in Met-independent cell lines. Pharmacologic blockade of Met using specific small-molecule inhibitors led to reduced secretion of IL-8, GROα and the soluble form of uPAR and to increased production of IL-6 both in vitro (in culture supernatants) and in vivo (in the plasma of xenografted mice). If confirmed in patients, this information might prove useful to monitor clinical response to Met-targeted therapies in MET-amplified gastric carcinomas.

Genetic and expression analysis of MET, MACC1, and HGF in metastatic colorectal cancer: response to met inhibition in patient xenografts and pathologic correlations.

PURPOSE: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease. EXPERIMENTAL DESIGN: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets. RESULTS: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer. CONCLUSIONS: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation.

B-cell-activating factor in rituximab-treated patients with anti-MAG polyneuropathy.

BACKGROUND: Antimyelin-associated glycoprotein (MAG) polyneuropathy is a slowly progressive distal form of mixed motor-sensory polyneuropathy that is scarcely responsive to conventional immunosuppressive therapy. Rituximab, a B-cell depleting antibody, is a promising therapeutic choice for anti-MAG polyneuropathy, and the evaluation of factors, such as B-cell-activating factor (BAFF), that control B-cell homeostasis is important to understand how this drug works. METHODS: Using an ELISA method, the authors measured serum BAFF concentrations in 23 patients with anti-MAG polyneuropathy, before and after rituximab therapy, in 20 neurological controls and in 14 healthy subjects. The patients were followed up over a mean period of 38±12 months and categorised as responders/non-responders, and, between the responders, as relapsing/non-relapsing. RESULTS: Pretherapy serum BAFF concentrations in non-responders were higher than in responders (cut-off 1665 pg/ml; sensitivity 71.4%; specificity 93.7%; likelihood ratio 11.4), with the highest post-therapy increases in responders. In the responders who relapsed, relapses occurred when serum BAFF concentrations returned to baseline values, 1-2 years after blood B-cell reappearance. CONCLUSIONS: Before and during therapy, measurements of serum BAFF in rituximab-treated patients with anti-MAG polyneuropathy may help predict the response to the therapy. The findings in this study also provide information about rituximab-induced modifications on B-cell homeostatic regulation.

The Met oncogene and basal-like breast cancer: another culprit to watch out for?

Recent findings suggest the involvement of the MET oncogene, encoding the tyrosine kinase receptor for hepatocyte growth factor, in the onset and progression of basal-like breast carcinoma. The expression profiles of basal-like tumors - but not those of other breast cancer subtypes - are enriched for gene sets that are coordinately over-represented in transcriptional signatures regulated by Met. Consistently, tissue microarray analyses have revealed that Met immunoreactivity is much higher in basal-like cases of human breast cancer than in other tumor types. Finally, mouse models expressing mutationally activated forms of Met develop a high incidence of mammary tumors, some of which exhibit basal characteristics. The present review summarizes current knowledge on the role and activity of Met in basal-like breast cancer, with a special emphasis on the correlation between this tumor subtype and the cellular hierarchy of the normal mammary gland.

Inhibition of Src impairs the growth of met-addicted gastric tumors.

PURPOSE: We examined whether inhibition of Src tyrosine kinase, a downstream effector of the MET oncogene, can hinder the malignant properties of gastric tumors dependent on Met for growth and survival. EXPERIMENTAL DESIGN: Sensitivity to Src inhibition was determined in vitro by measuring clonogenic survival (anchorage-independent growth) and in vivo by establishing xenograft models. Four "Met-addicted" gastric carcinoma cell lines (GTL16, MKN45, HS746T, and SNU5) and three Met-independent gastric carcinoma cell lines (KATO III, AGS, and NCI-N87) were treated with the Src inhibitor saracatinib (AZD0530). In GTL16 and KATO III, Src neutralization was also achieved by dasatinib and RNA interference. The biochemical and transcriptional consequences of Src inhibition were explored using anti-phosphoprotein antibodies and oligonucleotide microarrays. RESULTS: Inhibition of Src in Met-addicted gastric carcinoma cell lines (a) decreased the phosphorylation/activation levels of signaling intermediates involved in cell proliferation and protection from apoptosis and down-modulated the expression of several cell cycle regulators; (b) reduced anchorage-independent growth; (c) enhanced impairment of cell viability produced by Met inhibition; and (d) delayed tumorigenesis in xenotransplantation models. Immunohistochemical analysis of tumor xenograft tissues following systemic treatment with saracatinib showed a reduction of tumor cell proliferation index, increased apoptosis, and diminished phospho-focal adhesion kinase and phospho-paxillin staining. Tumor stroma parameters such as angiogenesis or inflammatory infiltration were unaffected. In clonogenic survival assays, gastric carcinoma cells without addiction to Met were less sensitive than Met-addicted cells to Src inhibition. CONCLUSIONS: Src is as a key downstream transducer of Met-driven tumor growth. Targeting Src might provide therapeutic benefit in Met-addicted tumors.

Only a subset of Met-activated pathways are required to sustain oncogene addiction.

Tumor onset and progression require the accumulation of many genetic and epigenetic lesions. In some cases, however, cancer cells rely on only one of these lesions to maintain their malignant properties, and this dependence results in tumor regression upon oncogene inactivation ("oncogene addiction"). Determining which nodes of the many networks operative in the transformed phenotype specifically mediate this response to oncogene neutralization is crucial to identifying the vulnerabilities of cancer. Using the Met receptor as the major model system, we combined multiplex phosphoproteomics, genome-wide expression profiling, and functional assays in various cancer cells addicted to oncogenic receptor tyrosine kinases. We found that Met blockade affected a limited subset of Met downstream signals: Little or no effect was observed for several pathways downstream of Met; instead, only a restricted and pathway-specific signature of transducers and transcriptional effectors downstream of Ras or phosphoinositide 3-kinase (PI3K) was inactivated. An analogous signature was also generated by inhibition of epidermal growth factor receptor in a different cellular context, suggesting a stereotyped response that likely is independent of receptor type or tissue origin. Biologically, Met inhibition led to cell-cycle arrest. Inhibition of Ras-dependent signals and PI3K-dependent signals also resulted in cell-cycle arrest, whereas cells in which Met was inhibited proliferated when Ras or PI3K signaling was active. These findings uncover "dominant" and "recessive" nodes among the numerous oncogenic networks regulated by receptor tyrosine kinases and active in cancer, with the Ras and PI3K pathways as determinants of therapeutic response.

Only a subset of Met-activated pathways are required to sustain oncogene addiction.

Tumor onset and progression require the accumulation of many genetic and epigenetic lesions. In some cases, however, cancer cells rely on only one of these lesions to maintain their malignant properties, and this dependence results in tumor regression upon oncogene inactivation ("oncogene addiction"). Determining which nodes of the many networks operative in the transformed phenotype specifically mediate this response to oncogene neutralization is crucial to identifying the vulnerabilities of cancer. Using the Met receptor as the major model system, we combined multiplex phosphoproteomics, genome-wide expression profiling, and functional assays in various cancer cells addicted to oncogenic receptor tyrosine kinases. We found that Met blockade affected a limited subset of Met downstream signals: Little or no effect was observed for several pathways downstream of Met; instead, only a restricted and pathway-specific signature of transducers and transcriptional effectors downstream of Ras or phosphoinositide 3-kinase (PI3K) was inactivated. An analogous signature was also generated by inhibition of epidermal growth factor receptor in a different cellular context, suggesting a stereotyped response that likely is independent of receptor type or tissue origin. Biologically, Met inhibition led to cell-cycle arrest. Inhibition of Ras-dependent signals and PI3K-dependent signals also resulted in cell-cycle arrest, whereas cells in which Met was inhibited proliferated when Ras or PI3K signaling was active. These findings uncover "dominant" and "recessive" nodes among the numerous oncogenic networks regulated by receptor tyrosine kinases and active in cancer, with the Ras and PI3K pathways as determinants of therapeutic response.

Hemopexin prevents endothelial damage and liver congestion in a mouse model of heme overload.

Intravascular hemolysis results in the release of massive amounts of hemoglobin and heme into plasma, where they are rapidly bound by haptoglobin and hemopexin, respectively. Data from haptoglobin and hemopexin knockout mice have shown that both proteins protect from renal damage after phenylhydrazine-induced hemolysis, whereas double-mutant mice were especially prone to liver damage. However, the specific role of hemopexin remains elusive because of the difficulty in discriminating between hemoglobin and heme recovery. To study the specific role of hemopexin in intravascular hemolysis, we established a mouse model of heme overload. Under these conditions, both endothelial activation and vascular permeability were significantly higher in hemopexin-null mice compared with wild-type controls. Vascular permeability was particularly altered in the liver, where congestion in the centrolobular area was believed to be associated with oxidative stress and inflammation. Liver damage in hemopexin- null mice may be prevented by induction of heme oxygenase-1 before heme overload. Furthermore, heme-treated hemopexin-null mice exhibited hyperbilirubinemia, prolonged heme oxygenase-1 expression, excessive heme metabolism, and lack of H-ferritin induction in the liver compared with heme-treated wild-type controls. Moreover, these mutant mice metabolize an excess of heme in the kidney. These studies highlight the importance of hemopexin in heme detoxification, thus suggesting that drugs mimicking hemopexin activity might be useful to prevent endothelial damage in patients suffering from hemolytic disorders.

Decrease of serotonin transporters in blood platelets after epileptic seizures.

Serotonin transporter (SERT) was studied by [3H]-paroxetine binding in blood platelets from controls and epileptic patients with generalized convulsive seizures. The average KD and BMax were not different in the two cases. However, a significant decrease was found in the serotonin transporter density in the platelet membranes from patients having undergone an epileptic seizure less than 4 days before. This circumstance may indicate a homeostatic reaction to the epileptic attack.