assessment-resistance-to-tyrosine-kinase-inhibitors-an-interrogation

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

University of Southern California

Mutations or deletions in exons 18-21 in the ) are present in approximately 15% of tumors in patients with non-small cell lung cancer (NSCLC). They lead to activation of the EGFR kinase domain and sensitivity to molecularly targeted therapeutics aimed at this domain (gefitinib or erlotinib). These drugs have demonstrated objective clinical response in many of these patients; however, invariably, all patients acquire resistance. To examine the molecular origins of resistance, we derived a set of gefitinib-resistant cells by exposing lung adenocarcinoma cell line, HCC827, with an activating mutation in the EGFR tyrosine kinase domain, to increasing gefitinib concentrations. Gefitinib-resistant cells acquired an increased expression and activation of JUN, a known oncogene involved in cancer progression. Ectopic overexpression of JUN in HCC827 cells increased gefitinib IC from 49 nmol/L to 8 μmol/L ( < 0.001). Downregulation of JUN expression through shRNA resensitized HCC827 cells to gefitinib (IC from 49 nmol/L to 2 nmol/L;  < 0.01). Inhibitors targeting JUN were 3-fold more effective in the gefitinib-resistant cells than in the parental cell line ( < 0.01). Analysis of gene expression in patient tumors with EGFR-activating mutations and poor response to erlotinib revealed a similar pattern as the top 260 differentially expressed genes in the gefitinib-resistant cells (Spearman correlation coefficient of 0.78,  < 0.01). These findings suggest that increased JUN expression and activity may contribute to gefitinib resistance in NSCLC and that JUN pathway therapeutics merit investigation as an alternate treatment strategy. .

JUN-Mediated Downregulation of EGFR Signaling Is Associated with Resistance to Gefitinib in EGFR-mutant NSCLC Cell Lines.

The cancer-associated protein Anterior Gradient 2 (AGR2) has been described, predominantly in adenocarcinomas. Increased levels of extracellular AGR2 (eAGR2) have been correlated with poor prognosis in cancer patients, making it a potential biomarker. Additionally, neutralizing AGR2 antibodies showed preclinical effectiveness in murine cancer models suggesting eAGR2 may be a therapeutic target. We set out to identify a peptide by mRNA display that would serve as a theranostic tool targeting AGR2. This method enables the selection of peptides from a complex (>10) library and incorporates a protease incubation step that filters the selection for serum stable peptides. We performed six successive rounds of enrichment using a 10-amino acid mRNA display library and identified several AGR2 binding peptides. One of these peptides (H10), demonstrated high affinity binding to AGR2 with a binding constant (K) of 6.4 nM. We developed an AGR2 ELISA with the H10 peptide as the capture reagent. Our H10-based ELISA detected eAGR2 from cancer cell spent media with a detection limit of (20-50 ng/ml). Furthermore, we investigated the therapeutic utility of H10 and discovered that it inhibited cell viability at IC (9-12 μmoles/L) in cancer cell lines. We also determined that 10 μg/ml of H10 was sufficient to inhibit cancer cell migration in breast and prostate cancer cell lines. A control peptide did not show any appreciable activity in these cells. The H10 peptide showed promise as both a novel diagnostic and a potential therapeutic peptide.

Katrin Tiemann

Lawrence J. Ellison Institute for Transformative Medicine,
Center for Applied Molecular Medicine,
Lawrence J. Ellison Institute for Transformative Medicine, Center for Applied Molecular Medicine

University of Southern California

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Katrin Tiemann

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University of Southern California

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Lawrence J. Ellison Institute for Transformative Medicine,
Center for Applied Molecular Medicine,
Lawrence J. Ellison Institute for Transformative Medicine, Center for Applied Molecular Medicine