Purpose We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling. Experimental Design Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice. Results c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa. Conclusions These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.

The epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription (STATs) are commonly expressed and activated in many malignancies. EGFR is an upstream activator of several pathways involved in tumor progression, and STATs activate selected genes involved in oncogenesis. There are several different mechanisms by which STAT proteins can mediate intracellular EGFR signaling, including direct activation of STATs by EGFR binding and indirect activation of STATs through Src-mediated EGFR signaling. EGFR likely activates STAT in a manner distinctive from other mechanisms of STAT activation; STAT5 can be phosphorylated in an EGF-dependent manner at unique sites, conferring novel functions. Cumulative evidence suggests that targeting EGFR signaling pathways at several levels may demonstrate synergistic therapeutic effects compared with targeting the upstream receptor alone. Thus, methods to inhibit EGFR in conjunction with oncogenic STATs may represent a novel therapeutic strategy for cancers characterized by upregulation of EGFR signaling.

PURPOSE Strategies to inhibit the epidermal growth factor receptor (EGFR) using the tyrosine kinase inhibitor (TKI) erlotinib have been associated with limited clinical efficacy in head and neck squamous cell carcinoma (HNSCC). Co-activation of alternative kinases may contribute to erlotinib resistance. EXPERIMENTAL DESIGN We generated HNSCC cells expressing dominant-active c-Src (DA-Src) to determine the contribution of c-Src activation to erlotinib response. RESULTS Expression of DA-Src conferred resistance to erlotinib in vitro and in vivo compared with vector-transfected control cells (VC). Phospho-Met was strongly upregulated by DA-Src, and DA-Src cells did not produce hepatocyte growth factor (HGF). Knockdown of c-Met enhanced sensitivity to erlotinib in DA-Src cells in vitro, as did combining a c-Met or c-Src inhibitor with erlotinib. Inhibiting EGFR resulted in minimal reduction of phospho-Met in DA-Src cells, whereas complete phospho-Met inhibition was achieved by inhibiting c-Src. A c-Met inhibitor significantly sensitized DA-Src tumors to erlotinib in vivo, resulting in reduced Ki67 labeling and increased apoptosis. In parental cells, knockdown of endogenous c-Src enhanced sensitivity to erlotinib, while treatment with HGF to directly induce phospho-Met resulted in erlotinib resistance. The level of endogenous phospho-c-Src in HNSCC cell lines was also significantly correlated with erlotinib resistance. CONCLUSIONS Ligand-independent activation of c-Met contributes specifically to erlotinib resistance, not cetuximab resistance, in HNSCC with activated c-Src, where c-Met activation is more dependent on c-Src than on EGFR, providing an alternate survival pathway. Addition of a c-Met or c-Src inhibitor to erlotinib may increase efficacy of EGFR inhibition in patients with activated c-Src.

Genome-wide aCGH can be used to detect and map CNAs in OSCC tissue specimens with high resolution. These data implicate several candidate genes and gene pathways in the tumorigenesis of sporadic OSCC.

Ischemia/reperfusion (IR) injury occurs when oxygen is rapidly reintroduced into ischemic tissue, resulting in cell death and necrotic tissue damage. This is a major concern during liver transplantation procedures since there is an inevitable interruption and subsequent restoration of circulation. IR injury in liver tissue is initiated through reactive oxygen species (ROS), which are generated by hepatocytes during IR insult. Although these ROS are thought to play a protective roll since they are known to activate several pathways involved in the hypoxic response, they also trigger a localized sterile immune response that results in the recruitment of Kupffer cells and neutrophils to the site of IR insult. These immune cells generate larger quantities of ROS that trigger apoptosis and oncotic necrosis in liver tissue. In this review, we will summarize what is currently known about the response of liver tissue to IR insult at the molecular level.

These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.

Purpose Acquired resistance to cetuximab, a chimeric EGFR-targeting monoclonal antibody, is a widespread problem in the treatment of solid tumors. The paucity of preclinical models has limited investigations to determine the mechanism of acquired therapeutic resistance thereby limiting the development of effective treatments. The purpose of this study was to generate cetuximab-resistant tumors in vivo to characterize mechanisms of acquired resistance. Experimental Design We generated cetuximab-resistant clones from a cetuximab-sensitive bladder cancer cell line in vivo by exposing cetuximab-sensitive xenografts to increasing concentrations of cetuximab followed by validation of the resistant phenotype in vivo and in vitro using invasion assays. A candidate-based approach was used to examine the role of HER2 on mediating cetuximab resistance both in vitro and in vivo. Results We generated a novel model of cetuximab resistance and, for the first time in the context of EGFR-inhibitor resistance, we identified increased phosphorylation of a c-terminal fragment of HER2 (611-CTF) in cetuximab-resistant cells. Afatinib (BIBW-2992), an irreversible kinase inhibitor targeting EGFR and HER2, successfully inhibited growth of the cetuximab-resistant cells in vitro. When afatinib was combined with cetuximab in vivo, we observed an additive growth inhibitory effect in cetuximab-resistant xenografts. Conclusions These data suggest that the use of dual EGFR-HER2 kinase inhibitors can enhance responses to cetuximab, perhaps in part due to downregulation of 611-CTF. This study in a novel in vivo model provides a mechanistic rationale for ongoing phase I clinical trials using this combination treatment modality.

The Cub and Sushi Multiple Domains-1 (CSMD1) is a tumor suppressor gene on 8p23.2, where allelic loss is both frequent and associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). To understand the extent of CSMD1 aberrations in vivo, we characterized 184 primary tumors from the head and neck, lung, breast and skin for gene copy number and analyzed expression in our HNSCCs and lung squamous cell carcinomas (SCCs).We detected loss of CSMD1 in a large proportion of HNSCCs (50%), lung (46%) and breast cancers (55%), and to a lesser extent in cutaneous SCCs (29%) and basal cell carcinomas (BCCs, 17%) using array-based comparative genomic hybridization (aCGH). Studying the region more closely with quantitative real-time PCR (qPCR), the loss of CSMD1 increased to 80% in HNSCCs and 93% in lung SCCs. CSMD1 expression was decreased in tumors compared to adjacent benign tissue (65%, 13/20) and was likely due to gene loss in 45% of cases (9/20). We also identified truncated transcripts lacking exons due to DNA copy number loss (30%, 5/17) or aberrant splicing (24%, 4/17).We show loss of CSMD1 in primary HNSCC tissues, and document for the first time that CSMD1 is lost in breast, lung and cutaneous SCCs. We also show that deletions of CSMD1 and aberrant splicing contribute to altered CSMD1 function in vivo.