Abstract 2032: Inhibition of signal transducers and activators of transcription 3 in human endometrial cancer, using a novel STAT3 inhibitor.
Authors: Tierney Brent J. , McCann Georgia A. , Rath Kellie S. , Sudhakar Meryl , Naidu Shan , Kuppusamy Periannan , Cohn David E. , Selvendiran Karuppaiyah 
Published: August 2014

Abstract
OBJECTIVE: Signal transducer and activator of transcription 3 (Stat3) proteins have important roles in cancer cell survival and proliferation. Recent studies show constitutive activation of STAT3 is associated with tumor growth and oncogenic transformation in several human cancers, however, little is known about STAT3 expression in endometrial cancer. The objectives of this study were to examine the expression of the STAT family of proteins in endometrial cancer cell lines and to evaluate the efficacy of HO-3867, a novel STAT3 inhibitor. METHODS: Expression of STAT family proteins, cell cycle and apoptotic proteins were evaluated via western blot on Ishikawa, AN3-CA, RL95-2, HEC1B, HEC-1A, and SK-UT1B human endometrial cancer cell lines. Cell viability and proliferation of these cell lines were analyzed with MTT and nucleo counter assay after treatment with increasing concentrations of HO-3867. Cell cycle analysis and apoptosis were performed using flow cytometry and annexin V staining. An ubiquitination assay was used to examine the inhibition of CDK5 degradation. Finally, STAT3 over-expression or suppression experiments were performed using wild-type STAT3 cDNA. RESULTS: Expression of activated STAT1, 2, 5, and 6 was inconsistent among cell lines tested, while pSTAT3 Ser727 had consistently high expression. HO-3867 decreased cell viability through induced G2/M arrest and apoptosis within 24h of treatment. HO-3867 inhibits pSTAT3 Ser727 with no change in total STAT3. This was associated with an increase in cell cycle related proteins p53 and p21, a decrease in anti-apoptotic proteins Bcl2 and Bcl-XL, as well as increased cleavage of caspases. After 24h of treatment with HO-3867, 55-88% of cells were in apoptosis, as detected by Annexin. The downregulation of Ser727 was, at least in part, due to an accelerated inhibition of ubiquitin-dependent CDK5 degradation, as a positive regulator of STAT3 Ser727. Suppression of STAT3 expression with siRNA significantly reduced cell survival. In addition, we confirmed that over-expression of STAT3 cDNA provides resistance to HO-3867. CONCLUSION: Endometrial cancer cells express high levels of pSTAT3 Ser727. HO-3867 induces cell cycle arrest and apoptosis by targeting pSTAT3 Ser727. Our findings suggest a specific role of serine-phosphorylated STAT3 that is independent of tyrosine phosphorylation. HO-3867 may be a potential adjunct to chemotherapy and radiation in the treatment of endometrial cancer.
Citation Format: Brent J. Tierney, Georgia A. McCann, Kellie S. Rath, Meryl Sudhakar, Shan Naidu, Periannan Kuppusamy, David E. Cohn, Karuppaiyah Selvendiran. Inhibition of signal transducers and activators of transcription 3 in human endometrial cancer, using a novel STAT3 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2013-2032