Kelly Quesnelle

STAT‐mediated EGFR signaling in cancer

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. J. Cell. Biochem. 102: 311–319, 2007.

HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer

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.

Dual Kinase Inhibition of EGFR and HER2 Overcomes Resistance to Cetuximab in a Novel In Vivo Model of Acquired Cetuximab Resistance

Purpose: Acquired resistance to cetuximab, a chimeric epidermal growth factor receptor (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 conducted in a novel in vivo model provides a mechanistic rationale for ongoing phase I clinical trials using this combination treatment modality. Clin Cancer Res; 17(18); 5935–44. ©2011 AACR.

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion

AIMS Nitrite (NO2(-)), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. METHODS AND RESULTS Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. CONCLUSIONS 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.

c-Src Activation Mediates Erlotinib Resistance in Head and Neck Cancer by Stimulating c-Met

Purpose: Strategies to inhibit the EGF receptor (EGFR) using the tyrosine kinase inhibitor 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. 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, whereas 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. Clin Cancer Res; 19(2); 380–92. ©2012 AACR.

Quantitative Proteomics Analysis Reveals Molecular Networks Regulated by Epidermal Growth Factor Receptor Level in Head and Neck Cancer

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck cancer (HNC), where increased expression levels of EGFR correlate with poor prognosis. To date, EGFR expression levels have not predicted the clinical response to the EGFR-targeting therapies. Elucidation of the molecular mechanisms underlying anti-EGFR-induced antitumor effects may shed some light on the mechanisms of HNC resistance to EGFR-targeting therapeutics and provide novel targets for improving the treatment of HNC. Here, we conducted a quantitative proteomics analysis to determine the molecular networks regulated by EGFR levels in HNC by specifically knocking-down EGFR and employing stable isotope labeling with amino acids in cell culture (SILAC). Following data normalization to minimize systematic errors and Western blotting validation, 12 proteins (e.g., p21, stratifin, and maspin) and 24 proteins (e.g., cdc2 and MTA2) were found to be significantly upregulated or downregulated by EGFR knockdown, respectively. Bioinformatic analysis revealed that these proteins were mainly involved in long-chain fatty acid biosynthesis and beta-oxidation, cholesterol biosynthesis, cell proliferation, DNA replication, and apoptosis. Cell cycle analysis confirmed that G(2)/M phase progression was significantly inhibited by EGFR knockdown, a hypothesis generated from network modeling. Further investigation of these molecular networks may not only enhance our understanding of the antitumor mechanisms of EGFR targeting but also improve patient selection and provide novel targets for better therapeutics.

Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion

Though it is well accepted that adipose tissue is central in the regulation of glycemic homeostasis, the molecular mechanisms governing adipocyte glucose uptake remain unclear. Recent studies demonstrate that mitochondrial dynamics (fission and fusion) regulate lipid accumulation and differentiation in adipocytes. However, the role of mitochondrial dynamics in glucose homeostasis has not been explored. The nitric oxide oxidation products nitrite and nitrate are endogenous signaling molecules and dietary constituents that have recently been shown to modulate glucose metabolism, prevent weight gain, and reverse the development of metabolic syndrome in mice. Although the mechanism of this protection is unclear, the mitochondrion is a known subcellular target for nitrite signaling. Thus, we hypothesize that nitrite modulates mitochondrial dynamics and function to regulate glucose uptake in adipocytes. Herein, we demonstrate that nitrite significantly increases glucose uptake in differentiated murine adipocytes through a mechanism dependent on mitochondrial fusion. Specifically, nitrite promotes mitochondrial fusion by increasing the profusion protein mitofusin 1 while concomitantly activating protein kinase A (PKA), which phosphorylates and inhibits the profission protein dynamin-related protein 1 (Drp1). Functionally, this signaling augments cellular respiration, fatty acid oxidation, mitochondrial oxidant production, and glucose uptake. Importantly, inhibition of PKA or Drp1 significantly attenuates nitrite-induced mitochondrial respiration and glucose uptake. These findings demonstrate that mitochondria play an essential metabolic role in adipocytes, show a novel role for both nitrite and mitochondrial fusion in regulating adipocyte glucose homeostasis, and have implications for the potential therapeutic use of nitrite and mitochondrial modulators in glycemic regulation.

Preclinical modeling of EGFR inhibitor resistance in head and neck cancer

The epidermal growth factor receptor (EGFR) is widely expressed in head and neck squamous cell carcinomas (HNSCC) and can activate many growth and survival pathways within tumor cells. Despite ubiquitous EGFR expression, therapies targeting the receptor are only modestly effective in the treatment of HNSCC. A consistent mechanism of resistance to EGFR targeting agents has not yet been identified in HNSCC likely due, in part, to the paucity of preclinical models. We assessed the in vitro and in vivo responses of a panel of 10 genotypically validated HNSCC cell lines to the EGFR inhibitors erlotinib and cetuximab to determine their validity as models of resistance to these agents. We defined a narrow range of response to erlotinib in HNSCC cells in vitro and found a positive correlation between EGFR protein expression and erlotinib response. We observed cross-sensitivity in one HNSCC cell line, 686LN, between erlotinib and cetuximab in vivo. We attempted to generate models of cetuximab resistance in HNSCC cell line-derived xenografts and heterotopic tumorgrafts generated directly from primary patient tumors. While all 10 HNSCC cell line xenografts tested were sensitive to cetuximab in vivo, heterotopic patient tumorgrafts varied in response to cetuximab indicating that these models may be more representative of clinical responses. These studies demonstrate the limitations of using HNSCC cell lines to reflect the heterogeneous clinical responses to erlotinib and cetuximab, and suggest that different approaches including heterotopic tumorgrafts may prove more valuable to elucidate mechanisms of clinical resistance to EGFR inhibitors in HNSCC.

Abstract C47: Dual kinase inhibition of EGFR and HER2 can overcome resistance to an EGFR‐targeting agent

EGFR and HER2 expression are correlated with poor prognosis in several cancers including bladder cancer. The EGFR‐targeting agent cetuximab is currently in phase II clinical trials for the treatment of bladder cancer, but has only been modestly effective as a single agent in most solid tumor types. In colon cancer, lack of cetuximab efficacy has been attributed to compensatory activating mutations in Kras. The incidence of Kras mutations is rare in bladder carcinomas. There is a need for preclinical models with which to study resistance to EGFR‐targeting agents. Here, we generated four cetuximab‐resistant clones of a cetuximab‐sensitive transitional bladder cancer cell line in vivo by exposing cetuximab‐sensitive bladder cancer cell line xenografts to slowly increasing concentrations of cetuximab. We verified this resistance in vivo by demonstrating growth of cetuximab‐resistant xenografts in the presence of high doses of cetuximab, as well as establishing differential sensitivity to cetuximab between sensitive and resistant xenografts in vivo . We validated the cetuximab‐resistant phenotype of these cells in vitro by showing decreased response to cetuximab in an invasion assay. We examined the levels of total and phosphorylated EGFR, HER2, HER3, AKT, MAPK, and the total protein levels of epithelial‐to‐mesenchymal transition markers E‐cadherin and Vimentin in the isogenic cetuximab‐sensitive and cetuximab‐resistant cells. Increased phosphorylation of the ERBB family members HER2 and HER3 was detected in cetuximab‐resistant clones compared with their cetuximab‐sensitive parental cell lines. Treatment of the cetuximab‐resistant cells with an irreversible dual kinase inhibitor targeting EGFR and HER2 (BIBW2992), successfully inhibited their growth. These data suggest that activation of HER2 and HER3 may contribute to cetuximab resistance where targeting these other HER family members may enhance the efficacy of EGFR blockade. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C47.