Abstract 4665: Induction of receptor tyrosine kinase signaling by sera from patients with lung adenocarcinoma
Authors: Lobato Gabriela C. , Fialkoff Jared D. , Tarhoni Imad , Lund Nicholas , Chukkapalli Vineela , Sayidine Selina , Fhied Cristina L. , Basu Sanjib , Lie Wen-Rong , Liptay Michael J. , Bonomi Philip , Borgia Jeffrey A. 
Published: July 2017

Background: The objective of this study was to evaluate disease stage-associated differences in receptor tyrosine kinase (RTK) signaling in A549 cells using pretreatment sera from cases of pathologically-confirmed lung adenocarcinoma or control cases derived from a lung cancer screening study. This was accomplished as a means to explore the hypothesis that circulating concentrations of decoy receptors and/or ligands, as well as disease-specific ligand degradation all regulate RTK signaling in vivo.

Methods: Pretreatment peripheral blood were prepared from either from non-cancer control patients (n=30) or those with lung adenocarcinoma, consisting of stage I (T1-2N0M0, n=25); locoregionally progressed (T1-2N1-2M0, n=36) or disseminated, stage IV disease (n=43). All patients were enrolled with written informed consent to our IRB-approved, institutional biorepository. Confluent cultures of A549 cells were stimulated for 10 minutes with patient sera diluted 1:1 in RPMI-1640 followed by immediate whole cell lysate preparation. After protein determinations by BCA, 8.5 µg of each sample was interrogated using the Human RTK (phosphoprotein) kit from MilliporeSigma according to manufacturer’s instructions. After being normalized to the kit-supplied (stimulated) HeLa cell lysates, all resulting data were evaluated by one-way ANOVA (LSD and Tukey post-hoc) for categorical comparisons.

Results: The most commonly observed differences in RTK signaling was noted upon comparing cohorts with locoregional (T1-2N1-2M0) progression and disseminated (stage IV) disease. Levels of in c-Met and insulin receptor (IR) signaling were higher in patients with stage IV disease relative to those with locoregional disease by 24% and 10%, respectively (both p&lt;0.01); whereas induction of c-Kit, VEGFR3, and Tie-1 were reduced by 12.8%, 6.67%, and 17.3%, respectively (all p&lt;0.05). Circulating levels of HGF were previously observed by our group to be 25% higher in stage IV patients relative to those with locoregional progression (p=0.005) - agreeing with our observation in direction of change, though not scale. This is in contrast to levels of VEGF-C and VEGF-D previously being observed elevated 51% and 452% (both p&lt;0.001) in these same cohorts. Also of note was the 1.9 fold decrease in ability of sera from stage I patients to stimulate IGF-1R signaling relative to control patients (p=0.002). We have previously reported a 12% increase in IGF-1 levels between these groups (p=0.002), though we observed no changes in IGF binding proteins that could account for the observed differences in IGF-1R signaling.

Conclusions: We observed an apparent discoupling of certain circulating ligands in patient sera to stimulate their respective RTKs in a disease stage-specific manner. We are currently evaluating levels of circulating ligands, potential disease stage-associated (partial) proteolytic degradation, and decoy receptors to help explain these observations.

Citation Format: Gabriela C. Lobato, Jared D. Fialkoff, Imad Tarhoni, Nicholas Lund, Vineela Chukkapalli, Selina Sayidine, Cristina L. Fhied, Sanjib Basu, Wen-Rong Lie, Michael J. Liptay, Philip Bonomi, Jeffrey A. Borgia. Induction of receptor tyrosine kinase signaling by sera from patients with lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4665. doi:10.1158/1538-7445.AM2017-4665