Stabilization of C/EBPß through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells.

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/EBPß (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPß 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/EBPß cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPß 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/EBPß for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPß was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPß, thereby promoting cancer-associated inflammation.

STAT3 as a Potential Target for Tumor Suppressive Effects of 15-Deoxy-Δ12,14-prostaglandin J2 in Triple Negative Breast Cancer.

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.

15-Deoxy-Δ12,14 -prostaglandin J2 binds and inactivates STAT3 via covalent modification of cysteine 259 in H-Ras-transformed human breast epithelial cells.

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 Stabilizes IKKα Protein through Direct Interaction in Transformed and Cancerous Human Breast Epithelial Cells.

Signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NF-κB) 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-κB pathway as well as the classical one. The α subunit of the inhibitor of the κB kinase (IKK) complex, IKKα, is involved in both classical and non-classical activation of NF-κB. Although the cross-talk between STAT3 and NF-κB has been suggested in several studies, the interplay between STAT3 and the regulators of NF-κB including IKKα has not been fully clarified yet. In this study, we observed overexpression and co-localization of IKKα 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 IKKα, but not IKKß or IKKγ, in these cells. This was attributable to direct binding to and subsequent stabilization of IKKα protein by blocking the ubiquitin-proteasome system. Notably, we identified the lysine 44 residue of IKKα as a putative binding site for STAT3. Moreover, siRNA knockdown of IKKα 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-κB activation by STAT3 through stabilization of IKKα, which contributes to breast cancer promotion and progression. Thus, breaking the STAT3-IKKα alliance can be an alternative therapeutic strategy for the treatment of breast cancer.

15-Keto prostaglandin E2 suppresses STAT3 signaling and inhibits breast cancer cell growth and progression.

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,ß-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.

Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells.

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,ß-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.

15-Deoxy-Δ12,14-Prostaglandin J2 Exerts Proresolving Effects Through Nuclear Factor E2-Related Factor 2-Induced Expression of CD36 and Heme Oxygenase-1.

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) was enhanced along with increased accumulation of the nuclear factor E2-related factor 2 (Nrf2) in the nucleus. Knockdown of Nrf2 abolished 15d-PGJ2-induced expression of CD36 and HO-1, and silencing of CD36 and HO-1 attenuated 15d-PGJ2-induced efferocytosis. Moreover, peritoneal macrophages isolated from Nrf2-null mice failed to upregulate 15d-PGJ2-induced expression of CD36 and HO-1 and to mediate efferocytosis. Unlike 15d-PGJ2, its nonelectrophilic analog 9,10-dihydro-15d-PGJ2 lacking the α,ß-unsaturated carbonyl group could not induce CD36 expression and efferocytosis. INNOVATION: 15d-PGJ2, as one of the terminal products of cyclooxygenase-2, exerts proresolving effects through induction of efferocytosis. The results of this study suggest that 15d-PGJ2 possesses a therapeutic value in the management of inflammatory disorders. CONCLUSION: 15d-PGJ2 facilitates resolution of inflammation by inducing Nrf2-induced expression of CD36 and HO-1 in macrophages. Antioxid. Redox Signal. 27, 1412-1431.