Proximity proteomic analysis of the NRF family reveals the Parkinson's disease protein ZNF746/PARIS as a co-complexed repressor of NRF2.

The nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor activates cytoprotective and metabolic gene expression in response to various electrophilic stressors. Constitutive NRF2 activity promotes cancer progression, whereas decreased NRF2 function contributes to neurodegenerative diseases. We used proximity proteomic analysis to define protein networks for NRF2 and its family members NRF1, NRF3, and the NRF2 heterodimer MAFG. A functional screen of co-complexed proteins revealed previously uncharacterized regulators of NRF2 transcriptional activity. We found that ZNF746 (also known as PARIS), a zinc finger transcription factor implicated in Parkinson's disease, physically associated with NRF2 and MAFG, resulting in suppression of NRF2-driven transcription. ZNF746 overexpression increased oxidative stress and apoptosis in a neuronal cell model of Parkinson's disease, phenotypes that were reversed by chemical and genetic hyperactivation of NRF2. This study presents a functionally annotated proximity network for NRF2 and suggests a link between ZNF746 overexpression in Parkinson's disease and inhibition of NRF2-driven neuroprotection.

Oxygen-independent stabilization of HIF-2α in breast cancer through direct interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer.

Overactivated NRF2 induces pseudohypoxia in hepatocellular carcinoma by stabilizing HIF-1α.

Hypoxia-inducible factor-1α (HIF-1α) is highly expressed/activated in most hypoxic tumors including hepatocellular carcinoma (HCC). Another key transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), is also constitutively overactivated in HCC. In an attempt to determine whether HIF-1α and NRF2 could play complementary roles in HCC growth and progression, we investigated the crosstalk between these two transcription factors and underlying molecular mechanisms in cultured HCC cells and experimentally induced hepatocarcinogenesis as well as clinical settings. While silencing of HIF-1α in HepG2 human hepatoma cells did not alter the protein expression of NRF2, NRF2 knockdown markedly reduced the nuclear accumulation of HIF-1α without influencing its mRNA expression. In diethylnitrosamine-induced hepatocarcinogenesis in wild type mice, there was elevated NRF2 expression with concomitant upregulation of HIF-1α. However, this was abolished in Nrf2 knockout mice. NRF2 and HIF-1α co-localized and physically interacted with each other as assessed by in situ proximity ligation and immunoprecipitation assays. In addition, the interaction between NRF2 and HIF-1α as well as their overexpression was found in tumor specimens obtained from HCC patients. In normoxia, HIF-1α undergoes hydroxylation by a specific HIF-prolyl hydroxylase domain protein (PHD), which facilitates ubiquitination and proteasomal degradation of HIF-1α. NRF2 contributes to pseudohypoxia, by directly binding to the oxygen-dependent degradation (ODD) domain of HIF-1α, which hampers the PHD2-mediated hydroxylation, concomitant recruitment of von-Hippel-Lindau and ubiquitination of HIF-1α.

Tumor Promoting Effects of Sulforaphane on Diethylnitrosamine-Induced Murine Hepatocarcinogenesis.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor involved in protection against initiation of carcinogenesis in normal cells. Notably, recent studies have demonstrated that aberrant activation of NRF2 accelerates the proliferation and progression of cancer cells. The differential effects of NRF2 on multi-stage carcinogenesis have raised a concern about the validity of NRF2 activators for chemoprevention. This prompted us to assess the effects of sulforaphane (SFN), a prototypic NRF2 activating chemopreventive phytochemical, on experimentally induced carcinogenesis. In the present study, SFN was daily injected intraperitoneally (25 mg/kg) for 3 months to male C57BL/6 mice at 6 months after single intraperitoneal administration of a hepatocarcinogen, diethylnitrosamine (DEN). The liver to body weight ratio, tumor growth, and the number and the size of hepatomas measured at 9 months after DEN administration were significantly higher in SFN-treated mice than those in vehicle-treated mice. Moreover, the expression of NRF2, its target protein NAD(P)H:quinone oxidoreductase 1, and the cell proliferation marker, proliferating cell nuclear antigen was further elevated in DEN plus SFN-treated mice. These results suggest that once hepatocarcinogenesis is initiated, SFN may stimulate tumor progression.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

Heregulin-ß1 Activates NF-E2-related Factor 2 and Induces Manganese Superoxide Dismutase Expression in Human Breast Cancer Cells via Protein Kinase B and Extracellular Signal-regulated Protein Kinase Signaling Pathways.

Heregulin-ß1, a ligand of ErbB-2 and ErbB-3/4 receptors, has been reported to potentiate oncogenicity and metastatic potential of breast cancer cells. In the present work, treatment of human mammary cancer (MCF-7) cells with heregulin-ß1 resulted in enhanced cell migration and expression of manganese superoxide dismutase (MnSOD) and its mRNA transcript. Silencing of MnSOD abrogated clonogenicity and migrative ability of MCF-7 cells. Heregulin-ß1 treatment also increased nuclear translocation, antioxidant response element binding and transcriptional activity of NF-E2-related factor 2 (Nrf2). A dominant-negative mutant of Nrf2 abrogated heregulin-ß1-induced MnSOD expression. Treatment with heregulin-ß1 caused activation of protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK). The pharmacological inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase 1/2, which are upstream of Akt and ERK, respectively, attenuated heregulin-ß1-induced MnSOD expression and nuclear localization of Nrf2. In conclusion, heregulin-1 induces upregulation of MnSOD and activation of Nrf2 via the Akt and ERK signaling in MCF-7 cells, which may confer metastatic potential and invasiveness of these cells.

Reprograming of Tumor-Associated Macrophages in Breast Tumor-Bearing Mice under Chemotherapy by Targeting Heme Oxygenase-1.

Tumor-associated macrophages (TAMs) represent one of the most abundant components of the tumor microenvironment and play important roles in tumor development and progression. TAMs display plasticity and functional heterogeneity as reflected by distinct phenotypic subsets. TAMs with an M1 phenotype have proinflammatory and anti-tumoral properties whereas M2-like TAMs exert anti-inflammatory and pro-tumoral functions. Tumor cell debris generated during chemotherapy can stimulate primary tumor growth and recurrence. According to our previous study, phagocytic engulfment of breast tumor cell debris by TAMs attenuated chemotherapeutic efficacy through the upregulation of heme oxygenase-1 (HO-1). To verify the impact of HO-1 upregulation on the profile of macrophage polarization during cytotoxic therapy, we utilized a syngeneic murine breast cancer (4T1) model in which tumor bearing mice were treated with paclitaxel (PTX). PTX treatment markedly downregulated the surface expression of the M1 marker CD86 in infiltrated TAMs. Notably, there were significantly more cytotoxic CD8+ T cells in tumors of mice treated with PTX plus the HO-1 inhibitor, zinc protophorphyrin IX (ZnPP) than in mice treated with PTX alone. Interestingly, the tumor-inhibiting efficacy of PTX and ZnPP co-treatment was abrogated when macrophages were depleted by clodronate liposomes. Macrophage depletion also decreased the intratumoral CD8+ T cell population and downregulated the expression of Cxcl9 and Cxcl10. The expression of the M1 phenotype marker, CD86 was higher in mice injected with PTX plus ZnPP than that in mice treated with PTX alone. Conversely, the PTX-induced upregulation of the M2 marker gene, Il10 in CD11b+ myeloid cells from 4T1 tumor-bearing mice treated was dramatically reduced by the administration of the HO-1 inhibitor. Genetic ablation of HO-1 abolished the inhibitory effect of 4T1 tumor cell debris on expression of M1 marker genes, Tnf and Il12b, in LPS-stimulated BMDMs. HO-1-deficient BMDMs exposed to tumor cell debris also exhibited a diminished expression of the M2 macrophage marker, CD206. These findings, taken all together, provide strong evidence that HO-1 plays a pivotal role in the transition of tumor-inhibiting M1-like TAMs to tumor-promoting M2-like ones during chemotherapy.

Resolvin D1 suppresses inflammation-associated tumorigenesis in the colon by inhibiting IL-6-induced mitotic spindle abnormality.

While failure in resolution of inflammation is considered to increase the risk of tumorigenesis, there is paucity of experimental as well as clinical evidence supporting this association. Resolvin D1 (RvD1) is a representative pro-resolving lipid mediator that is endogenously generated from docosahexaenoic acid for the resolution of inflammation. Here, we report a decreased level of RvD1 in the blood from colorectal cancer patients and mice having inflammation-induced colon cancer, suggesting plasma RvD1 as a potential biomarker for monitoring colorectal cancer. Administration of RvD1 attenuated dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM) plus DSS-induced colorectal carcinogenesis by suppressing the production of interleukin-6 (IL-6) and IL-6-mediated chromosomal instability. The protective effect of RvD1 against chromosomal instability is associated with downregulation of IL-6-induced Cyclin D1 expression, which appears to be mediated by blocking the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) axis. RvD1 inhibited the STAT3 signaling pathway by interfering with the binding of IL-6 to its receptor (IL-6R), suggesting the novel function of RvD1 as a putative IL-6R antagonist. Together, our findings suggest that RvD1-mediated blockade of IL-6 signal transmission may contribute to inhibition of chromosomal instability and tumorigenesis.

Alternative regulation of HIF-1α stability through Phosphorylation on Ser451.

The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.

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.

Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression.

Persistent activation of STAT3 and Nrf2 is considered to stimulate the aggressive behavior of basal-like breast cancer (BLBC). However, the precise mechanism underlying sustained overactivation of these transcription factors and their roles in breast cancer progression remain elusive. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and Nrf2 mRNA were correlated with elevated expression of P-STAT3Y705 and Nrf2 target proteins in breast cancer patients. Our present study demonstrates a unique interaction between Nrf2 and STAT3 in the maintenance and progression of BLBC. RNA sequencing analysis identified the gene encoding IL-23A upregulated by concurrent binding of STAT3 and Nrf2 to its promoter. IL-23A depletion also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, the STAT3-Nrf2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer.

The peptidyl prolyl isomerase, PIN1 induces angiogenesis through direct interaction with HIF-2α.

PIN1, the peptidyl-prolyl isomerase (PPIase), is an enzyme that changes the conformation of phosphoproteins. The conformational change induced by PIN1 alters the function and stability of the target proteins. PIN1 is overexpressed in many different types of malignancies, including breast, lung, cervical, brain and colorectal tumors. PIN1 overexpression has been associated with activation of multiple oncogenic signaling pathways during tumor development. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor activated in hypoxia, plays a role in erythropoiesis, glycolysis, tissue invasion, metastasis and angiogenesis. In this study, we found the direct interaction between HIF-2α and PIN1 in colorectal cancer HCT116 cells. Notably, serine 16 and lysine 63 residues of PIN1 were critical for its interaction with HIF-2α. When PIN1 protein was silenced by transient transfection of PIN1 short interfering RNA, the expression of HIF-2α was attenuated under a hypoxic condition. Moreover, genetic and pharmacologic inhibition of PIN1 abrogated the expression of vascular endothelial growth factor and angiogenesis. The cycloheximide chase experiment revealed the stabilization of HIF-2α by PIN1. Both WW and PPIase domains of PIN1 appear to be critical for its interaction with HIF-2α.

Breast cancer cell debris diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor-associated macrophages.

Chemotherapy is commonly used as a major therapeutic option for breast cancer treatment, but its efficacy is often diminished by disruption of patient's anti-tumor immunity. Chemotherapy-generated tumor cell debris could hijack accumulated tumor-associated macrophages (TAMs), provoking tumor recurrence. Therefore, reprogramming TAMs to acquire an immunocompetent phenotype is a promising strategy to potentiate therapeutic efficacy. In this study, we analyzed the proportion of immune cells in the breast cancer patients who received chemotherapy. To validate our findings in vivo, we used a syngeneic murine breast cancer (4T1) model. Chemotherapy generates an immunosuppressive tumor microenvironment in breast cancer. Here, we show that phagocytic engulfment of tumor cell debris by TAMs reduces chemotherapeutic efficacy in a 4T1 breast cancer model. Specifically, the engulfment of tumor cell debris by macrophages reduced M1-like polarization through heme oxygenase-1 (HO-1) upregulation. Conversely, genetic or pharmacologic inhibition of HO-1 in TAMs restored the M1-like polarization. Our results demonstrate that tumor cell debris-induced HO-1 expression in macrophages regulates their polarization. Inhibition of HO-1 overexpression in TAMs may provoke a robust anti-tumor immune response, thereby potentiating the efficacy of chemotherapy.

H-Ras induces Nrf2-Pin1 interaction: Implications for breast cancer progression.

Aberrant activation of H-Ras is often associated with tumor aggressiveness in breast cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1  (Pin1) is a unique enzyme that interacts with phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue. Pin1 is prevalently overexpressed in human cancers, and its overexpression correlates with poor prognosis. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of cellular redox homeostasis. The sustained activation/accumulation of Nrf2 has been observed in many different types of human malignancies, conferring an advantage for growth and survival of cancer cells. The activated form of H-Ras (GTP-H-Ras) is highly overexpressed in human breast cancer tissues. In our present study, silencing of H-Ras decreased the invasiveness of MDA-MB-231 human breast cancer cells and abrogated the interaction between Pin1 and Nrf2 in these cells. Pin1 knockdown blocked the accumulation of Nrf2, thereby suppressing proliferation and clonogenicity of MCF10A-Ras human mammary epithelial cells. We found that Pin1 binds to Nrf2 which stabilizes this transcription factor by hampering proteasomal degradation. In conclusion, H-Ras activation in cooperation with the Pin1-Nrf2 complex represents a novel mechanism underlying breast cancer progression and constitutive activation of Nrf2 and can be exploited as a therapeutic target.

The positive feedback loop between Nrf2 and phosphogluconate dehydrogenase stimulates proliferation and clonogenicity of human hepatoma cells.

Recent studies report that nuclear factor-erythroid-2-related factor 2 (Nrf2) facilitates tumor progression through metabolic reprogramming in cancer cells. However, the molecular mechanism underlying the oncogenic functions of Nrf2 is not yet well understood. Some of the pentose phosphate pathway (PPP) enzymes are considered to play a role in the cancer progression. The present study was intended to explore the potential role of phosphogluconate dehydrogenase (PGD), one of the PPP enzymes, in the proliferation and migration of human hepatoma HepG2 cells. Genetic ablation of Nrf2 attenuated the expression of PGD at both transcriptional and translational levels. Notably, Nrf2 regulates the transcription of PGD through direct binding to the antioxidant response element in its promoter region. Nrf2 overexpression in HepG2 cells led to increased proliferation, survival, and migration, and these events were suppressed by silencing PGD. Interestingly, knockdown of the gene encoding this enzyme not only attenuated the proliferation and clonogenicity of HepG2 cells but also downregulated the expression of Nrf2. Thus, there seems to exist a positive feedback loop between Nrf2 and PGD which is exploited by hepatoma cells for their proliferation and survival. Treatment of HepG2 cells with ribulose-5-phosphate, a catalytic product of PGD, gave rise to a concentration-dependent upregulation of Nrf2. Collectively, the current study shows that Nrf2 promotes hepatoma cell growth and progression, partly through induction of PGD transcription.

Interaction between Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1 and GTP-H-Ras: Implications for Aggressiveness of Human Mammary Epithelial Cells and Drug Resistance.

Aberrant activation of Ras has been implicated in aggressiveness of breast cancer. Among Ras isoforms (H-, K-, and N-), H-Ras has been known to be primarily responsible for invasion and metastasis of breast cancer cells. Phosphorylation of serine (Ser) or threonine (Thr) is a key regulatory mechanism responsible for controlling activities and functions of various proteins involved in intracellular signal transduction. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, Pin1 changes the conformation of a subset of proteins phosphorylated on Ser/Thr that precedes proline (Pro). In this study we have found that Pin1 is highly overexpressed in human breast tumor tissues and H-Ras transformed human mammary epithelial (H-Ras MCF10A) and MDA-MB-231 breast cancer cells. Notably, Pin1 directly bound to the activated form of H-Ras harbouring a Ser/Thr-Pro motif. Pharmacologic inhibition of Pin1 reduced clonogenicity of MDA-MB-231 human breast cancer cells. Paclitaxel accelerates apoptosis in Pin1 silenced H-Ras MCF10A cells. MDR genes (MDR1 and MRP4) were significantly downregulated in MDA-MB-231 cells stably silenced for Pin1. We speculate that Pin1 interacts with GTP-H-Ras, thereby upregulating the expression of drug resistance genes, which confers survival advantage and aggressiveness of breast cancer cells under chemotherapy.

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.

Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells.

Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression.