Simultaneous Targeting of Two Distinct Epitopes on MET Effectively Inhibits MET- and HGF-Driven Tumor Growth by Multiple Mechanisms.

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro The importance of these effector functions was confirmed in vivo using an Fc-effector function-attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780-91. ©2017 AACR.

SOX11 and HIG-2 are cross-regulated and affect growth in mantle cell lymphoma.

The transcriptional factor SOX11 is a disease-defining antigen in mantle cell lymphoma (MCL) and absent in most non-malignant tissues. To explore the role of SOX11-related cell signaling, and potentially take benefit from these for targeted therapy, associated networks and proteins need to be defined. In this study, we used an inducible SOX11 knock-down system followed by gene expression analysis to identify co-regulated genes and associated signaling pathways. A limited number (n = 27) of significantly co-regulated genes were identified, including SETMAR, HIG-2, and CD24. Further analysis confirmed co-regulation of SOX11 with HIG-2 and CD24 at the protein level. Of major interest, knock-down of HIG-2 reduced SOX11 levels and increased proliferation, the proteins are thus cross-regulated. HIG-2 was localized at the plasma cell membrane in both cell lines and primary MCL cells, and could potentially be of interest for targeted therapy.

Knock-down of SOX11 induces autotaxin-dependent increase in proliferation in vitro and more aggressive tumors in vivo.

The transcription factor SOX11 is a novel diagnostic marker for mantle cell lymphoma (MCL), distinguishing this aggressive tumor from potential simulators. Recent data also show that the level of SOX11 correlates to in vitro growth properties in MCL, as well as the clinical progression. We have previously shown that MCL-associated pathways, such as Rb-E2F, are dysregulated leading to decreased proliferation upon overexpression of SOX11, emphasizing the impact of SOX11 on MCL-specific gene expression and growth control. However, it remains to be determined which growth regulatory pathways that are induced upon SOX11 knock-down, leading to an increased cellular growth. Consequently, we established a model cell line with constitutive down-regulation of SOX11. The highly proliferative features of this cell line were investigated by gene expression analysis, proliferation assay, cell cycle distribution and potential to induce tumors in NOD-SCID mice. Our in vitro studies demonstrated a SOX11-dependent regulation of MCL-specific gene expression. In addition, we identified autotaxin (ATX) to be regulated by SOX11. Our results clearly showed a correlation between SOX11 level and cellular growth rate, which was dependent on ATX, as well as a direct relation between the level of SOX11 in tumorigenic cells and the growth rate of these tumors in NOD-SCID mice.

Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.

Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages.

Increased evidence suggests that cancer-associated inflammation supports tumor growth and progression. We have previously shown that semaphorin 4D (Sema4D), a ligand produced by different cell types, is a proangiogenic molecule that acts by binding to its receptor, plexin B1, expressed on endothelial cells (Conrotto, P., D. Valdembri, S. Corso, G. Serini, L. Tamagnone, P.M. Comoglio, F. Bussolino, and S. Giordano. 2005. Blood. 105:4321-4329). The present work highlights the role of Sema4D produced by the tumor microenvironment on neoplastic angiogenesis. We show that in an environment lacking Sema4D, the ability of cancer cells to generate tumor masses and metastases is severely impaired. This condition can be explained by a defective vascularization inside the tumor. We demonstrate that tumor-associated macrophages (TAMs) are the main cells producing Sema4D within the tumor stroma and that their ability to produce Sema4D is critical for tumor angiogenesis and vessel maturation. This study helps to explain the protumoral role of inflammatory cells of the tumor stroma and leads to the identification of an angiogenic molecule that might be a novel therapeutic target.

Lys Tag: an easy and robust chemical modification for improved de novo sequencing with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer.

Mass spectrometry using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) instrument is a widespread technique for various types of proteomic analysis. Along with an expanding interest in proteomics, there is a strong requirement for the identification of proteins with high confidence from biological samples. Peptide modification by a wide variety of post-translational modifications (PTMs), the existence of different protein isoforms and the presence of uncharacterized genomes of many species, make protein identification through peptide mass fingerprinting (PMF) often unachievable. Peptide de novo sequencing has been proven to be a useful approach to overcome these variables, and efficient derivatization processes are important tools to achieve this goal. In the present work we describe the methodology and experimental applications of a fast, efficient and cheap lysine derivatization. This chemical modification improves the signals from lysine-terminated peptides and can be efficiently used as a lysine-blocking agent in combination with other derivatization techniques. Most importantly, upon peptide fragmentation it generates a neat series of predominantly y-ions, allowing the determination of unambiguous amino acid sequences. Moreover, this chemical compound was used with target-eluted samples, enabling a second, alternative analysis of the same sample in the MALDI mass spectrometer.

p53 targets identified by protein expression profiling.

p53 triggers cell cycle arrest and apoptosis through transcriptional regulation of specific target genes. We have investigated the effect of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells carrying wild-type p53. Approximately 5,800 protein spots were separated in overlapping narrow-pH-range gel strips, and 115 protein spots showed significant expression changes upon p53 activation. The identity of 55 protein spots was obtained by mass spectrometry. The majority of the identified proteins have no previous connection to p53. The proteins fall into different functional categories, such as mRNA processing, translation, redox regulation, and apoptosis, consistent with the idea that p53 regulates multiple cellular pathways. p53-dependent regulation of five of the up-regulated proteins, eIF5A, hnRNP C1/C2, hnRNP K, lamin A/C, and Nm23-H1, and two of the down-regulated proteins, Prx II and TrpRS, was examined in further detail. Analysis of mRNA expression levels demonstrated both transcription-dependent and transcription-independent regulation among the identified targets. Thus, this study reveals protein targets of p53 and highlights the role of transcription-independent effects for the p53-induced biological response.

Interactome of transforming growth factor-beta type I receptor (TbetaRI): inhibition of TGFbeta signaling by Epac1.

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGFbeta receptor (TbetaRI) is the key receptor for initiation of intracellular signaling by TGFbeta. Here we report proteomics-based identification of proteins that form a complex with TbetaRI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused TbetaRI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), beta-spectrin, PIASy, and beta-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of TbetaRI but was not affected by deletion of cAMP-binding domain of Epac1. TGFbeta1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGFbeta1/TbetaRI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGFbeta/TbetaRI-dependent decrease of cell adhesion and TGFbeta/TbetaRI-induced stimulation of cell migration. Thus, we have reported novel TRI-interacting proteins and have shown that Epac1 inhibited TGFbeta-dependent regulation of cell migration and adhesion.

Sema4D induces angiogenesis through Met recruitment by Plexin B1.

Semaphorins, a large family of membrane-bound and secreted proteins, signal through their transmembrane receptors, the plexins. Semaphorins and plexins share structural homologies with scatter factor receptors, a family of tyrosine kinase receptors for which Met is the prototype. Semaphorins have been studied primarily in the developing nervous system, where they act as repelling cues in axon guidance. However, they are widely expressed in several tissues, and their role in epithelial morphogenesis has been recently established. Not much is known about their role in angiogenesis, a key step during embryonic development and adulthood. Here we demonstrate that a semaphorin, Sema4D, is angiogenic in vitro and in vivo and that this effect is mediated by its high-affinity receptor, Plexin B1. Moreover, we prove that biologic effects elicited by Plexin B1 require coupling and activation of the Met tyrosine kinase. In sum, we identify a proangiogenic semaphorin and provide insight about the signaling machinery exploited by Plexin B1 to control angiogenesis.

Sulfonation chemistry as a powerful tool for MALDI TOF/TOF de novo sequencing and post-translational modification analysis.

Mass spectrometry using matrix-assisted laser desorption/ionization (MALDI) is a widespread technique for various types of proteomic analysis. In the identification of proteins using peptide mass fingerprinting, samples are enzymatically digested and resolved into a number of peptides, whose masses are determined and matched with a sequence data-base. However, the presence inside the cell of several splicing variants, protein isoforms, or fusion proteins gives rise to a complex picture, demanding more complete analysis. Moreover, the study of species with yet uncharacterized genomes or the investigation of post-translational modifications are not possible with classical mass fingerprinting, and require specific and accurate de novo sequencing. In the last several years, much effort has been made to improve the performance of peptide sequencing with MALDI. Here we present applications using a fast and robust chemical modification of peptides for improved de novo sequencing. Post-source decay of derivatized peptides generates at the same time peaks with high intensity and simple spectra, leading to a very easy and clear sequence determination.

Plexin-B3 is a functional receptor for semaphorin 5A.

Semaphorins are a large family of molecular cues implicated in neural development and in a variety of functions outside the nervous system. Semaphorin 5A (Sema5A) is a transmembrane semaphorin, containing seven thrombospondin type-1 repeats, which was recently found to control axon guidance. Here we show that plexin-B3 is a high-affinity receptor specific for Sema5A. We further demonstrate that plexin-B3 activation by Sema5A mediates functional responses in plexin-B3-expressing cells (either fibroblasts, epithelial and primary endothelial cells). In addition, Sema5A can trigger the intracellular signalling of the hepatocyte growth factor/scatter factor receptor, Met, associated in a complex with plexin-B3. We thus conclude that Sema5A is able to elicit multiple functional responses through its receptor plexin-B3.

Interplay between scatter factor receptors and B plexins controls invasive growth.

Met and Ron tyrosine kinases are members of the Scatter Factor Receptor family. Met is the receptor for hepatocyte growth factor while Ron is that for macrophage stimulating protein. On ligand stimulation, activation of these receptors induces 'invasive growth', a complex biological response involved in tissue morphogenesis and, when deregulated, in tumor progression and metastasis. Scatter Factor Receptors share structural homology with Plexins, transmembrane receptors for Semaphorins, a family of ligands originally identified as axon guidance molecules. A physical and functional association between Met and Plexin B1, the prototype of class B Plexin subfamily, has been previously demonstrated. Here, we show that both Met and Ron receptors can interact with each of the three members of class B Plexins, even in the absence of their ligands and that Plexin B1 ligand, Sema 4D, can induce activation of Met and Ron receptors, promoting an invasive response. Furthermore, in some human neoplastic cell lines Plexin B1 is overexpressed, constitutively tyrosine phosphorylated, and associated with Scatter Factor Receptors. These data extend the crosstalk previously described between Met and Plexin B1 to the entire families of Scatter Factor Receptors and class B Plexins and show that interaction with multiple upstream activators can finely tune the invasive growth process both in physiological conditions and in tumor growth and metastatization.

The semaphorin 4D receptor controls invasive growth by coupling with Met.

Semaphorins are cell surface and soluble signals that control axonal guidance. Recently, semaphorin receptors (plexins) have been discovered and shown to be widely expressed. Their biological activities outside the nervous system and the signal transduction mechanism(s) they utilize are largely unknown. Here, we show that in epithelial cells, Semaphorin 4D (Sema 4D) triggers invasive growth, a complex programme that includes cell#150;cell dissociation, anchorage-independent growth and branching morphogenesis. Interestingly, the same response is also controlled by scatter factors through their tyrosine kinase receptors, which share striking structural homology with plexins in their extracellular domain. We found that in cells expressing the endogenous proteins, Plexin B1 (the Sema 4D Receptor) and Met (the Scatter Factor 1/ Hepatocyte Growth Factor Receptor) associate in a complex. In addition, binding of Sema 4D to Plexin B1 stimulates the tyrosine kinase activity of Met, resulting in tyrosine phosphorylation of both receptors. Finally, cells lacking Met expression do not respond to Sema 4D unless exogenous Met is expressed. This work identifies a novel biological function of semaphorins and suggests the involvement of an unexpected signalling mechanism, namely, the coupling of a plexin to a tyrosine kinase receptor.