Combining onartuzumab with erlotinib inhibits growth of non-small cell lung cancer with activating EGFR mutations and HGF overexpression.

Erlotinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR-TKI), benefits survival of patients with non-small cell lung cancer (NSCLC) who harbor activating EGFR mutations. However, elevated expression of hepatocyte growth factor (HGF), a ligand of the MET receptor tyrosine kinase, causes erlotinib resistance. Because onartuzumab, a monovalent antibody to MET, blocks HGF-induced MET activation, the addition of onartuzumab to erlotinib may improve therapeutic efficacy. We engineered the human NSCLC cell line PC-9 (MET-positive cells harboring an exon 19 deletion of EGFR) to overexpress hHGF and evaluated the effects of an onartuzumab and erlotinib combination in vitro and in vivo in xenograft models. A stable clone of PC-9/hHGF was less sensitive to erlotinib than the parental PC-9, and the addition of onartuzumab to erlotinib suppressed the proliferation of these cells in vitro. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone minimally inhibited tumor growth; however, combining onartuzumab and erlotinib markedly suppressed tumor growth. The total MET protein level was decreased in PC-9/hHGF cells, because MET is constitutively phosphorylated by autocrine HGF, leading to its ubiquitination and degradation. Onartuzumab reduced phospho-MET levels, inhibited MET ubiquitination, and consequently restored MET protein levels. Moreover, in NSCLC clinical specimens harboring activating EGFR mutations, more than 30% of patients expressed high levels of HGF. Our findings raised the possibility that patients with NSCLC with EGFR mutations who express high levels of HGF may benefit from onartuzumab and erlotinib combination therapy, and that HGF can be a novel biomarker for selecting such patients.

Integration of proteomic analyses to monitor the activity status of phosphorylation signaling.

We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling. We also performed whole cell proteomics using an off-gel fractionator, and found 68.9% of the proteins identified by phosphoproteomics. Thus, the expression levels of phosphoproteins were roughly estimated. In addition to many uncharacterized phosphorylation sites, the dataset includes 136 sites that were experimentally verified elsewhere to be phosphorylated by a total of 83 kinases and kinase groups out of the 256 registered in the Phospho.ELM database. With the integration of various proteomic analyses and information from database, the responsible kinases of the identified phosphorylation sites and possibly their activity status were predicted by phosphorylation status and expression levels of their substrates, and thus our method may be able to monitor the activity status of phosphorylation signaling.