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STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) functions as an allosteric inhibitor of STAT3. 15d-PGJ2 inhibits phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3 in H-Ras-transformed human mammary epithelial cells (MCF10A-Ras) through the Michael addition reaction at cysteine 259 of STAT3. Comparative studies with 15d-PGJ2 analogues reveal that both C12-C13 and C9-C10 double bonds conjugated to the carbonyl group in the cyclopentenone ring of 15d-PGJ2 are essential for STAT3 binding. Antiproliferative and pro-apoptotic activities of 15d-PGJ2 in MCF10A-Ras cells are attributable to covalent modification of STAT3 on Cys259, and mimic the effects induced by mutation of this amino acid.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBPβ (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPβ is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBPβ cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPβ protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBPβ for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPβ was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPβ, thereby promoting cancer-associated inflammation.

Signal transducer and activator of transcription 3 (STAT3) and nuclear factor-&kappa, B (NF-&kappa, B) are two representative transcription factors that play a critical role in inflammation-associated tumorigenesis through multi-level cooperation. Unlike other types of tumors, breast carcinomas have shown a significant dependency on the non-classical NF-&kappa, B pathway as well as the classical one. The &alpha, subunit of the inhibitor of the &kappa, B kinase (IKK) complex, IKK&alpha, is involved in both classical and non-classical activation of NF-&kappa, B. Although the cross-talk between STAT3 and NF-&kappa, B has been suggested in several studies, the interplay between STAT3 and the regulators of NF-&kappa, B including IKK&alpha, has not been fully clarified yet. In this study, we observed overexpression and co-localization of IKK&alpha, and STAT3 in human breast cancer tissues as well as in H-Ras transformed human breast epithelial (H-Ras MCF-10A) and breast cancer (MDA-MB-231) cells. By utilizing small interfering RNA (siRNA) technology, we were able to demonstrate that STAT3 up-regulated IKK&alpha, but not IKK&beta, or IKK&gamma, in these cells. This was attributable to direct binding to and subsequent stabilization of IKK&alpha, protein by blocking the ubiquitin-proteasome system. Notably, we identified the lysine 44 residue of IKK&alpha, as a putative binding site for STAT3. Moreover, siRNA knockdown of IKK&alpha, attenuated viability, anchorage-independent growth and migratory capabilities of H-Ras MCF-10A cells. Taken together, these findings propose a novel mechanism responsible for NF-&kappa, B activation by STAT3 through stabilization of IKK&alpha, which contributes to breast cancer promotion and progression. Thus, breaking the STAT3-IKK&alpha, alliance can be an alternative therapeutic strategy for the treatment of breast cancer.

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ

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 is an anti-inflammatory natural compound isolated from the turmeric (Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in 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 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.

Aims: 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been shown to rescue cells from inflammatory insults and to participate in the resolution of acute inflammation. In this study, we investigated molecular mechanisms underlying proresolving effects of 15d-PGJ2. Results: 15d-PGJ2 injected into the peritoneum of mice facilitated the resolution of zymosan A-induced peritonitis. 15d-PGJ2 administration reduced the number of total leukocytes and attenuated polymorphonuclear leukocyte infiltration. Furthermore, 15d-PGJ2 increased the proportion of macrophages engulfing apoptotic neutrophils, a process called efferocytosis. In addition, when the thioglycollate-elicited mouse peritoneal macrophages were stimulated with 15d-PGJ2, their efferocytic activity was amplified. In another experiment, RAW264.7 murine macrophages exposed to 15d-PGJ2 conducted phagocytic clearance of apoptotic cells to a greater extent than the control cells. Under these conditions, expression of CD36 and heme oxygenase-1 (HO-1) ...

Overproduction of prostaglandin E2 (PGE2) has been linked to enhanced tumor cell proliferation, invasiveness and metastasis as well as resistance to apoptosis. 15-Keto prostaglandin E2 (15-keto PGE2), a product formed from 15-hydroxyprostaglandin dehydrogenase-catalyzed oxidation of PGE2, has recently been shown to have anti-inflammatory and anticarcinogenic activities. In this study, we observed that 15-keto PGE2 suppressed the phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) in human mammary epithelial cells transfected with H-ras (MCF10A-ras). 15-Keto PGE2 inhibited the migration and clonogenicity of MCF10A-ras cells. In addition, subcutaneous injection of 15-keto PGE2 attenuated xenograft tumor growth and phosphorylation of STAT3 induced by breast cancer MDA-MB-231 cells. However, a non-electrophilic analogue, 13,14-dihydro-15-keto PGE2 failed to inhibit STAT3 signaling and was unable to suppress the growth and transformation 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. We observed that the thiol reducing agent, dithiothreitol abrogated 15-keto PGE2-induced STAT3 inactivation and disrupted the direct interaction between 15-keto PGE2 and STAT3. Furthermore, a molecular docking analysis suggested that Cys251 and Cys259 residues of STAT3 could be preferential binding sites for this lipid mediator. Mass spectral analysis revealed the covalent modification of recombinant STAT3 by 15-keto PGE2 at Cys259. Taken together, thiol modification of STAT3 by 15-keto PGE2 inactivates STAT3 which may account for its suppression of breast cancer cell proliferation and progression., Highlights • 15-Keto-prostaglandin E2 inhibits activation of STAT3 in MCF10A-ras cells. • 15-Keto-prostaglandin E2 suppresses the growth and transformation of MCF10A-ras cells. . • 15-Keto-prostaglandin E2 attenuated xenograft tumor growth and STAT3 phosphorylation. . • 15-Keto PGE2 modulates STAT3 by directly binding to its Cys259 residue.

15-Deoxy-Δ15d-PGJ15d-PGJ15d-PGJ

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