EGFR targeted monoclonal antibodies are effective in a subset of metastatic colorectal tumors (mCRC). Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in patients who do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies demonstrate that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived CRC xenografts, MET amplification correlated with resistance to EGFR blockade which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in CRC and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.

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.

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Semaphorins, a large family of membranebound 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. IntroductionSemaphorins belong to a large family of transmembrane, glycosylphosphatidylinositol (GPI)-anchored, and secreted proteins that have been divided into subclasses according to their structural features. 1 Semaphorins act by binding their cognate receptors, the plexins, a family of transmembrane molecules sharing structural homology with semaphorins themselves. 2 All plexins have a large cytoplasmic domain, highly conserved during evolution, that is endowed with R-Ras GAP activity 3 and is able to interact, directly or indirectly, with small G proteins, thus exerting control over cytoskeletal structures. [4][5][6][7] Semaphorins were originally characterized in the nervous system, where they are involved in the establishment of a correct neuronal network, steering axon growth cones and dendrites to their final targets. 8 According to the widespread expression of semaphorins and plexins, an increasing amount of data involve these 2 protein families in the development of different tissues and organs beyond the nervous system. In particular, a significant role for these molecules has been established in cardiac and skeletal development, 9 immune response, 10,11 epithelial morphogenesis, 12 and tumor growth and metastasis. 13,14 Although secreted class 3 semaphorins have been involved in the control of endothelial cell (EC) adhesion, motility, and vascular development, the role of semaphorins belonging to other classes remains largely unknown. Indeed, it has been shown that the prototypic secreted class 3 semaphorin, Sema3A, inhibits EC migration and in vitro angiogenesis. 15 Furthermore, during vascular development and experimental angiogenesis, ECs generate autocrine chemorepulsive signals, driven by class 3 semaphorins, that inhibit integrin activity, thus endowing the vascular system with the plasticity required for reshaping. 16 We focused our interest on Sema4D, a semaphorin originally identified for its activity in the immune system, where it promotes B ...

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.

The establishment of the role of MET in human cancer has led to the development of small-molecule inhibitors, many of which are currently in clinical trials. Thus far, nothing is known about their therapeutic efficacy and the possible emergence of resistance to treatment, a problem that has been often observed with other receptor tyrosine kinase (RTK) inhibitors. To predict mechanisms of acquired resistance, we generated resistant cells by treating MET-addicted cells with increasing concentrations of the MET small-molecule inhibitors PHA-665752 or JNJ38877605. Resistant cells displayed MET gene amplification, leading to increased expression and constitutive phosphorylation of MET, followed by subsequent amplification and overexpression of wild-type (wt) KRAS. Cells harboring KRAS amplification progressively lost their MET dependence and acquired KRAS dependence. Our results suggest that MET and KRAS amplification is a general mechanism of resistance to specific MET inhibitors given that similar results were observed with two small inhibitors and in different cell lines of different histotypes. To our knowledge, this is the first report showing that overexpression of wt KRAS can overcome the inhibitory effect of a RTK inhibitor. In view of the fact that cellular models of resistance to inhibitors targeting other tyrosine kinases have predicted and corroborated clinical findings, our results provide insights into strategies for preventing and/or overcoming drug resistance.

The microenvironment influences cancer drug response and sustains resistance to therapies targeting receptor-tyrosine kinases. However, if and how the tumor microenvironment can be altered during treatment, contributing to resistance onset, is not known. We show that, under prolonged treatment with tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a metabolic shift toward increased glycolysis and lactate production. We identified secreted lactate as the key molecule instructing cancer-associated fibroblasts to produce hepatocyte growth factor (HGF) in a nuclear factor κB-dependent manner. Increased HGF, activating MET-dependent signaling in cancer cells, sustained resistance to TKIs. Functional or pharmacological targeting of molecules involved in the lactate axis abrogated in vivo resistance, demonstrating the crucial role of this metabolite in the adaptive process. This adaptive resistance mechanism was observed in lung cancer patients progressed on EGFR TKIs, demonstrating the clinical relevance of our findings and opening novel scenarios in the challenge to drug resistance.

MicroRNAs (miRNA) are a recently identified class of noncoding, endogenous, small RNAs that regulate gene expression, mainly at the translational level. These molecules play critical roles in several biological processes, such as cell proliferation and differentiation, development, and aging. It is also known that miRNAs play a role in human cancers where they can act either as oncogenes, down-regulating tumor suppressor genes, or as onco-suppressors, targeting molecules critically involved in promotion of tumor growth. One of such molecules is the tyrosine kinase receptor for hepatocyte growth factor, encoded by the MET oncogene. The MET receptor promotes a complex biological program named ''invasive growth'' that results from stimulation of cell motility, invasion, and protection from apoptosis. This oncogene is deregulated in many human tumors, where its most frequent alteration is overexpression. In this work, we have identified three miRNAs (miR-34b, miR-34c, and miR199a*) that negatively regulate MET expression. Inhibition of these endogenous miRNAs, by use of antagomiRs, resulted in increased expression of MET protein, whereas their exogenous expression in cancer cells blocked MET-induced signal transduction and the execution of the invasive growth program, both in cells expressing normal levels of MET and in cancer cells overexpressing a constitutively active MET. Moreover, we show that these same miRNAs play a role in regulating the MET-induced migratory ability of melanomaderived primary cells. In conclusion, we have identified miRNAs that behave as oncosuppressors by negatively targeting MET and might thus provide an additional option to inhibit this oncogene in tumors displaying its deregulation.