Abstract 2810: NSC 743380 induces apoptosis in sensitive cell lines through activation of tyrosine kinase 2 (Tyk2)

Abstract

Abstract Several indole-3-carbinol analogs are under preclinical evaluation at the National Cancer Institute. One analog, NSC743380 (1-[(3-chlorophenyl)-methyl]-1H-indole-3-carbinol), is selectively toxic to a subset of NCI 60 cell lines in vitro and has undergone extensive in vivo testing. In vivo, NSC743380 produced complete regressions in A498 renal xenograft models at doses as low as 45 mg/kg when administered intraperitoneally. Additional studies demonstrated that NSC743380 is orally bioavailable. To extend knowledge regarding the mechanism of action, two pairs of resistant and sensitive cell lines [A498/ACHN (renal) and NCI-H226/A549 (NSCLC), sensitive/resistant, respectively] were used. Results showed that 5-10 min following NSC743380 treatment, phosphorylation of p38 and JNK were enhanced in sensitive but not resistant lines. Two hrs of treatment resulted in an inhibition of transcription and translation (45% and 75% inhibition, respectively) and by 4 hrs NSC743380 induced caspase-dependent apoptosis with loss of c-FLIP. Pathway-specific inhibitors were then used to gain mechanistic insight. Diverse antioxidants and NSAIDs, inhibitors of JNK, RAS/RAF, PPAR, lipid 2nd messenger signaling, transcription and actin polymerization were shown to completely inhibit NSC743380 activity. Microarray analysis of three sensitive cell lines using Affymetrix U133 Plus 2 chipset identified several trends in the transcriptome notably the upregulation of diverse immediate-early genes (IEGs) including; transcriptional regulators [EGR1, FOS/FOSB, HES1, MAFF and SOX9], secreted factors [HBEGF, IL-8 and GDF-15] and several general IEGs [ARC, ERRFI1, GADD45A/B, GEM and IER2]. A second noteworthy trend involved increased expression of the IL-6 family members, IL-6, IL-11 and LIF. These data led to an exploration as to whether enhanced JAK/STAT signaling was responsible for the above effects. Short time course lysates from 3 sensitive (A498, CAKI-1 and NCI-H226) cell lines and 1 resistant (A549) cell line showed that in sensitive lines only, NSC743380 rapidly (5 minutes) enhanced phosphorylation of the JAK family member Tyk2 at Y1054/55 and STAT3 at Y705. Additionally several phosphostate changes were observed in a subset of sensitive cell lines such as JAK1 phosphorylation in A498 cells and transient increase in pSTAT1 in A498 and CAKI-1 cell lines. Furthermore, two inhibitors of JAK/STAT signaling, AG490 and resveratrol, completely inhibited NSC743380 activity and p38/JNK phosphorylation. Subsequent siRNA knockdown experiments showed that Tyk2 siRNA inhibited NSC743380 activity whereas STAT3 and JAK1 siRNA along with scrambled siRNA had no effect. These data suggest that signaling through the JAK family member Tyk2 contributes towards NSC743380 activity and the data are being evaluated to determine how best to move forward. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2810. doi:1538-7445.AM2012-2810