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Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. The intracellular level of PGE2 is regulated by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This enzyme catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2. Down-regulation of 15-PGDH was observed in various malignancies and overexpression of 15-PGDH has been known to suppress the carcinogenic processes. However, mechanisms underlying anti-carcinogenic effects and tumor suppressive functions of 15-keto PGE2 are poorly understood in various cancer. H-Ras transformed mammary epithelial MCF10A-ras cells show enhanced expression of cyclooxygenase-2 and activation of signal transducer and activator of transcription 3 (STAT3) and down-regulation of 15-PGDH compared to normal mammary epithelial MCF10A cells. STAT 3 plays a key role in growth of cancer cells as well as inflammation-associated carcinogenesis. This prompted us to investigate the effect of 15-keto PGE2 on STAT3 activation in MCF10A-ras cells. 15-Keto PGE2 suppressed the phosphorylation of STAT3 in MCF10A-ras cells in concentration- and time-dependent manners as determined by Western blot and immunocytochemical analyses. 15-Keto PGE2 also inhibited the dimerization of STAT3 which led to suppression of nuclear translocation and transcriptional activity of STAT3 as determined by Western blot analysis and the luciferase assay, respectively. 15-keto PGE2 suppressed the expression of cyclin D1. In addition, 15-keto PGE2 inhibited the growth of MCF10A-ras cells as determined by anchorage-independent growth and colony forming assays. However, a non-electrophilic analogue 13,14-dihydro-15-keto PGE2 which lacks the α,β-unsaturated carbonyl moiety failed to inhibit the phosphorylation, dimerization, and nuclear translocation of STAT3. Moreover, 13,14-dihydro-15-keto PGE2 did not affect the growth of MCF10A ras cells. These findings suggest that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. STAT3 has several cysteine residues in its activation sites. We observed that reducing agents N-acetyl-L-cysteine and dithiothreitol abrogated the suppressive effect of 15-keto PGE2 on STAT3 phosphorylation, suggesting that the involvement of the thiol modification. Biotinylated 15-keto PGE2 covalently bound to STAT3 as determined by immunoprecipitation with STAT3 followed by immunoblotted with streptavidin. Molecular docking analysis predicted Cys251 and Cys259 residues of STAT3 as putative binding sites of 15-keto PGE2. In conclusion, 15-keto PGE2 inhibits STAT3 signaling through cysteine thiol modification, thereby suppressing MCF10A-ras cell growth and proliferation. Thus, tumor suppressor function of 15-PGDH is attributable, at least in part, to inactivation of oncogenic STAT3 signaling by its catalytic product 15-keto PGE2. Citation Format: Eun-Ji Lee, Young-Il Hahn, Su-Jung Kim, Young-Joon Surh, Hye-Kyung Na. 15-Keto prostaglandin E2 inhibits STAT3 signaling in H-Ras transformed mammary epithelial cells [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 2231. doi:10.1158/1538-7445.AM2017-2231

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin, a natural compound isolated from the traditional Indian spice turmeric (Curcuma longa.,Zingiberaceae), has anti-tumor, anti-proliferative and anti-inflammatory properties. This compound has been extensively used in traditional herbal medicine over the centuries mainly due to its pronounced anti-inflammatory effects. Many studies suggest that curcumin regulates various cancer-related intracellular signaling pathways without damaging normal cells and tissues. Here, we report that curcumin effectively inhibits activation of STAT3 in dose- and time-dependent manners and causes apoptosis of H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 as a putative target of curcumin. We found that the α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its direct binding to STAT3. Tetrahydrocurcumin that lacks the electrophilic α,β-unsaturated carbonyl moiety failed to inhibit STAT3 activation and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate persistently activated STAT3 signaling through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis. Citation Format: Young-Il Hahn, Su-Jung Kim, Bu-Young Choi, Hye-Kyung Na, Young-Joon Surh. Curcumin induces apoptosis in H-Ras transformed human mammary epithelial cells: Cysteine 259 of STAT3 as a putative target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2015-1892