Intracellular Signaling Mechanisms of Resistance to EGFR-Targeting Agents

The epidermal growth factor receptor (EGFR) is widely expressed in head and neck squamous cell carcinomas (HNSCC) and activates many growth and survival pathways within tumor cells. EGFR-targeting agents are only modestly effective in treating HNSCC, however, and a consistent mechanism of resistance has not been identified, in part, due to the paucity of preclinical models. This dissertation focuses on generating EGFR-inhibitor resistant preclinical models in order to identify biomarkers that may be predictive of response to these agents. We have assessed the response of a panel of HNSCC cell lines to the EGFR inhibitors erlotinib and cetuximab to determine their relevance as models of resistance to these agents. We defined a narrow range of response to erlotinib in HNSCC cells in vitro. We attempted to generate models of cetuximab resistance in cell line-derived xenografts and heterotopic tumorgrafts directly from primary HNSCC patient tumors. Our studies in HNSCC suggest that heterotopic xenografts are more representative of patient response to cetuximab than cell-line derived xenografts, although we did establish a model of cetuximab resistance from bladder cancer cell line-derived xenografts. A candidate-based approach was used to examine the role of HER2, HER3, and c-Met on mediating EGFR inhibitor resistance. We identified increased phosphorylation of a carboxyl-terminal fragment of HER2 (611-CTF) in cetuximab-resistant cells. Afatinib, an irreversible kinase inhibitor targeting EGFR and HER2, successfully restored cetuximab sensitivity in vitro. When afatinib was combined with cetuximab in vivo, we observed an additive growth inhibitory effect in cetuximab-resistant xenografts. We also show that while c-Met activity is not sufficient to alter cellular response to erlotinib, concomitant inhibition of c-Met and EGFR is required for the deactivation of MAPK in the presence of stimulatory ligands. These data support the proposed role for co-targeting c-Met with EGFR in the treatment of HNSCC. The studies presented here are significant because, in addition to suggesting that 611-CTF may be a novel biomarker for cetuximab resistance, they provide a thorough assessment of modeling EGFR inhibitor resistance in HNSCC and suggest heterotopic tumorgrafts as a plausible new model for examining cetuximab resistance in future studies

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

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

Design of a foundational sciences curriculum: Applying the ICAP framework to pharmacology education in integrated medical curricula

Abstract Expectations for physicians are rapidly changing, as is the environment in which they will practice. In response, preclerkship medical education curricula are adapting to meet these demands, often by reducing the time for foundational sciences. This descriptive study compares preclerkship pharmacology education curricular practices from seven allopathic medical schools across the United States. We compare factors and practices that affect how pharmacology is integrated into the undergraduate medical education curriculum, including teaching techniques, resources, time allocated to pharmacology teaching, and assessment strategies. We use data from seven medical schools in the United States, along with results from a literature survey, to inform the strengths and weaknesses of various approaches and to raise important questions that can guide future research regarding integration of foundational sciences in medical school and health professions’ curricula. In this comparative study, we found that there is significant heterogeneity in the number of hours dedicated to pharmacology in the preclerkship curriculum, whereas there was concordance in the use of active learning pedagogies for content delivery. Applying the ICAP (Interactive, Constructive, Active, Passive) Framework for cognitive engagement, our data showed that pharmacology was presented using more highly engaging pedagogies during sessions that are integrated with other foundational sciences. These findings can serve as a model that can be applied beyond pharmacology to other foundational sciences such as genetics, pathology, microbiology, biochemistry, etc

The importance of collaboratively designing pharmacology education programs

Abstract A grounded knowledge of pharmacology is essential for healthcare providers to improve the quality of patients’ lives, avoid medical errors, and circumvent potentially dangerous drug–drug interactions. One of the greatest tools to achieve this foundational knowledge of pharmacology is the dedicated pharmacology educators who teach in health sciences programs. Too often, the pharmacology educators responsible for teaching this material are left siloed at their own institutions with little room for dialog and collaboration. As scientists, we know that it is through dialog and collaboration that ideas grow, are refined, and improve. More collaborative work is needed to identify and describe best practices for pharmacology education in health sciences programs. While evidence‐based, outcomes‐focused studies are the optimum standard for this work, there is also a place for descriptive studies and innovative reports

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 vectortransfected 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 inHNSCCcell 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. © 2012 AACR.link_to_subscribed_fulltex