Abstract A113: Mechanisms of gefitinib alone and in combination therapy in soft tissue sarcoma cell lines.
Authors: Wang Xiaochun , Goldstein David , Crowe Philip , Yang Jia-Lin 
Published: August 2014

Background: Use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib is now a part of standard care in biologically appropriate subsets in cancers of lung (1). However, a Phase II trial of single agent gefitinib in synovial sarcoma showed a low response (2). Our preliminary data (3) showed that a panel of seven soft tissue sarcoma (STS) cell lines were resistant to gefitinib with IC50 of more than 10µM, and combination therapy with a STAT3 inhibitor achieved synergistic anti-proliferative effect in 6/7 STS cell lines. The principal aim of this study is to investigate the potential mechanisms behind gefitinib mono-therapy and combination with STAT3 inhibitor S3I-201 in STS cell lines.

Methods: We investigated expression and phosphorylation of EGFR and its signal transducers (AKT, Erk1/2, P38 MAPK, SAPK/JNK, STAT1 and STAT3) by Western blot before and after treatment. We further examined the effect of combination therapy on apoptosis (cleaved caspase 3, 7 and cleaved PARP).

Results: We confirmed synergy in additional experiments with drug reduction index for combined therapy of gefitinib ranged from 2.5 to 6.0 and for S3I-201 from 4.5 to 15.7. Western blot showed that EGF induced EGFR phosphorylation (pEGFR) in all seven STS cell lines, while pEGFR was undetectable or weak in the normal cell culture conditions (without supplemental EGF). Furthermore, the EGF-induced pEGFR was completed blocked by gefitinib monotherpy in all these cell lines. We then examined the interaction between EGFR inhibition (in the presence or absence of EGF) and activity of different EGF-EGFR downstream signalling pathways including PI3K/AKT (AKT), Ras/Erk (Erk1/2, p38 MAPK and SAPK/JNK) and JAK/STAT (STAT3 and STAT1) pathways. pAKT was significantly inhibited in all 7 cell lines, pErk1/2 in 6 out of 7 cell lines, pJNK in 5/7, while pP38 was decreased in 2 out of 7 cell lines. In addition, while gefitinib inhibited both pSTAT1 and pSTAT3, the ratio of pSTAT3/pSTAT1 was increased in most (5/7) of STS cell lines. pSTAT3 was further down-regulated after combination therapy with gefitinib and STAT3 inhibitor S3I-201. Cleaved caspase 3, 7 and PARP were further increased in combination therapy compared to gefitinib monotherapy in synergistic cell lines.

Conclusion: Our research suggests gefitinib alone can inhibit both Ras/Erk and PI3K/AKT pathways, but is incomplete for the JAK/STAT pathway, which may be one mechanism for gefitinib resistance in soft tissue sarcoma cell lines. Combination therapy targeting both EGFR and STAT3 further inhibited active STAT3 and induced apoptosis. Therefore concurrent application of EGFR and STAT3 inhibitors is a worthwhile treatment to pursue in anti-sarcoma therapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A113.

Citation Format: Xiaochun Wang, David Goldstein, Philip Crowe, Jia-Lin Yang. Mechanisms of gefitinib alone and in combination therapy in soft tissue sarcoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A113.