Abstract 1723: Effect and mechanism of PF299804 alone and in combination with STAT3 inhibitor in human sarcoma cell lines

Abstract

Abstract Background: Curative treatment of sarcomas is achieved by surgical resection, supplemented with neoadjuvant chemotherapy and postoperative radiation. Approximately 50% of patients will eventually die. We previously reported that 78% (36/46) of our cohort of patients with soft tissue sarcoma were positive for total EGFR/HER1. The EGFR/HER1 pathway is an important molecular target; its inhibition is now well established clinically in several epithelial-origin tumors. No pan-HER inhibitors have yet been tested in sarcomas. The principal aim of this project was to investigate the effect and mechanism of PF299804 (an irreversible pan-HER inhibitor) therapy alone and in combination with STAT3 inhibitor in human sarcoma cell lines. Methods: PF299804 mono-therapy and in combination with STAT3 inhibitor (S3I-201) were investigated in a panel of 12 human sarcoma cell lines. Crystal-violet colorimetric and clonogenic assays were used to measure drug effects. Soft agar colony formation assay was used to evaluate anchorage-independent growth (the ability to evade anoikis). For assessing potential mechanisms, we investigated expression and activity of EGFR signalling by Western blot. Results: HER family receptors (EGFR/HER1, HER2 and HER3) were positively expressed in the sarcoma cell lines representing liposarcoma (778 and 449b), fibrosarcoma (SW684 and HT1080), synovial sarcoma (SW980), fibrous histocytoma (GCT) and osteosarcoma (143B, HOS, MG63, SJSA, U2-OS and Saos-2). IC50 values of PF299804 were more than 1µM in these cell lines, which was about 1000-fold higher than the sensitive control lung cell line PC9. Mechanism studies by Western blot assay found that 200nM PF299804 (emulating total plasma exposure measured in human clinical trial) dramatically suppressed the activation of both EGFR/HER1 and HER2, as well as their representative downstream signalling factors Erk1/2, p38MAPK and SAPK/JNK in ras/raf/MAPK and AKT in PI3K/AKT pathways, but not of STAT3. Combination therapy using PF299804 and STAT3 inhibitor S3I-201 achieved synergistic anti-proliferation in 3 out of 4 sarcoma cell lines (449B, 778, 143B and HOS), representing different subtype, grade, recurrence and metastasis: 449B and 778 are primary well-differentiated and its recurrent liposarcoma cell lines; HOS and 143B are low-metastatic and high- metastatic (HOS with k-ras oncogene transformation) osteosarcoma cell lines, respectively. This synergistic effect was associated with additional down-regulation of phosphorylated STAT3. Conclusion: Although the second generation irreversible pan-HER inhibitor PF299804 inactivated HER family and downstream pathways, it encountered resistance at least partially due to the STAT3 activation. Therefore, addition of STAT3 inhibitor in PF299804 therapy may block STAT3 activation and achieve synergistic anti-proliferation in sarcoma cell lines. Citation Format: Xiaochun Wang, David Goldstein, Philip Crowe, Jia-Lin Yang. Effect and mechanism of PF299804 alone and in combination with STAT3 inhibitor in human sarcoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1723. doi:10.1158/1538-7445.AM2014-1723