Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519–561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis1

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis

Role and Therapeutic Targeting of the HGF/MET Pathway in Glioblastoma

Glioblastoma (GBM) is a lethal brain tumor with dismal prognosis. Current therapeutic options, consisting of surgery, chemotherapy and radiation, have only served to marginally increase patient survival. Receptor tyrosine kinases (RTKs) are dysregulated in approximately 90% of GBM; attributed to this, research has focused on inhibiting RTKs as a novel and effective therapy for GBM. Overexpression of RTK mesenchymal epithelial transition (MET), and its ligand, hepatocyte growth factor (HGF), in GBM highlights a promising new therapeutic target. This review will discuss the role of MET in cell cycle regulation, cell proliferation, evasion of apoptosis, cell migration and invasion, angiogenesis and therapeutic resistance in GBM. It will also discuss the modes of deregulation of HGF/MET and their regulation by microRNAs. As the HGF/MET pathway is a vital regulator of multiple pro-survival pathways, efforts and strategies for its exploitation for GBM therapy are also described

MicroRNA-608 and MicroRNA-34a Regulate Chordoma Malignancy by Targeting EGFR, Bcl-xL and MET

<div><p>Chordomas are rare malignant tumors that originate from the notochord remnants and occur in the skull base, spine and sacrum. Due to a very limited understanding of the molecular pathogenesis of chordoma, there are no adjuvant and molecular therapies besides surgical resection and radiation therapy. microRNAs (miRNAs) are small noncoding regulatory RNA molecules with critical roles in cancer. The role of miRNAs in chordomas is mostly unknown. We uncover microRNA-608 (miR-608) and microRNA-34a (miR-34a) as novel tumor suppressive microRNAs that regulate malignancy in chordoma. We find that miR-608 and miR-34a expressions are downregulated in human chordoma cell lines and primary cells at least partially via alteration of their genes’ copy numbers. We identify the commonly deregulated oncogenes EGFR and Bcl-xL as direct targets of miR-608 and the receptor tyrosine kinase MET as direct target of miR-34a. We show that EGFR and MET activations promote chordoma cell proliferation and invasion and that pharmacological inhibition of EGFR and MET inhibits chordoma cell proliferation and survival. We demonstrate that restoration of miR-608 and miR-34a inhibits cell proliferation and invasion and induces apoptosis in chordoma cells. We find that miR-34a inversely correlates with MET expression and miR-608 inversely correlates with EGFR expression in chordoma cells. These findings demonstrate for the first time that miR-608 and miR-34a regulate chordoma malignancy by regulating EGFR, MET and Bcl-xL.</p></div

microRNAs are differentially expressed in chordoma cells.

<p>Small RNAs were extracted from chordoma UCH1 and UCH2 cells and control fibroblasts. miRNA levels were measured using qRT-PCR relative to control U6B snRNA.</p

miR-608 and miR-34a are downregulated via gene copy number alteration, and miR-608 and miR-34a expressions inversely correlate with EGFR and MET in chordoma cells.

<p>A) miR-608 and miR-34a levels in chordoma cell lines and primary cells were measured by qRT-PCR and compared with those in fibroblasts, B) lystaes from chordoma cells were immunoblotted for EGFR and normalized to β-actin (left panel), and the level of miR-608 was correlated with EGFR protein level (right panel) (R² = 0.8, P<0.05). C) MET protein levels in chordoma cells were determined by immunoblotting (left panel) and miR-34a levels correlated with MET protein (right panel) (R² = 0.0.61, P<0.05). D) miR-608 gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-608 expression levels were correlated with gene copy number (R² = 0.77, P<0.05) (right panel). E) miR-34a gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-34a expression levels are correlated with gene copy number (R² = 0.51, P<0.05).</p