J Cancer Prev 2021; 26(3): 207-217
Published online September 30, 2021
© Korean Society of Cancer Prevention
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 2Korea Chemical Bank, Korea Research Institute of Chemical Technology, Daejeon, 3Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science, Seoul National University, 4Cancer Research Institute, Seoul National University, Seoul, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: Breast neoplasms, Cyclopentenone prostaglandin, 15-Deoxy-&Delta,12,14-prostaglandin J2, STAT3
Breast cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related deaths among women worldwide [1-3]. Breast cancer prevalence is increasing worldwide every year, especially in young women. Therefore, development of efficient therapeutic strategies as well as identification of novel targets for prevention and treatment of breast cancer is clinically important .
15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a cyclopentenone prostaglandin (PG) that can act as an endogenous ligand for PPARg [4,5]. 15d-PGJ2 is characterized by the presence of an α,β-unsaturated carbonyl group in the cyclopentenone ring. Because of this electrophilic moiety, 15d-PGJ2 is capable of forming covalent adducts with some target proteins, thereby altering their structures and biological functions, independently of PPARγ activation [6-9].
Janus kinase (JAK)-STAT axis plays a major role in the regulation of cell cycle progression and proliferation . STAT proteins are a family of latent cytoplasmic transcription factors that become phosphorylated by JAK in response to various cytokines and growth factors. The phosphorylated STAT (P-STAT) undergoes dimerization and translocates into the nucleus where it binds to the promoter of its downstream target genes and induces their transcription. At least seven members of mammalian STAT family proteins have been identified: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which are encoded by distinct genes.
All STAT proteins consist of an amino acid domain (NH2), a coiled-coil domain responsible for binding to interactive proteins, a DNA binding domain, a linker domain, a Src homology 2 (SH2) domain required for phosphorylation and dimerization, and a C-terminal transactivation domain . Among STAT family members, persistent activation of STAT3 is frequently observed in many different types of human malignancy, which contributes to the growth and survival of cancer cells [12-15]. The knockdown of the STAT3 by antisense RNA or siRNA resulted in the induction of cancer cell apoptosis and tumor regression [16,17]. Therefore, targeting aberrant STAT3 signaling provides a universal therapeutic strategy for treating a wide variety of human tumors that harbor abnormal STAT3 activity.
15d-PGJ2 has potent tumor suppressive effects against breast, prostate and colorectal cancer [18-20]. It induces apoptosis by reactive oxygen species-mediated inactivation of Akt in colorectal cancer cells . 15d-PGJ2 inhibits constitutive NF-κB activities in chemotherapy-resistant estrogen receptor (ER)-negative breast cancer cells, which accounts for induction of their apoptosis . Furthermore, 15d-PGJ2 induced apoptosis of cancer cells by modulating expression levels of the Bcl-2 family member proteins, such as Bax and Bcl-2 [23,24]. It also induces apoptosis of macrophages, fibroblasts, and endothelial cells that constitute tumor microenvironment [25-27].
Several studies revealed an association between 15d-PGJ2-mediated STAT3 inactivation and induction in cancer cells. 15d-PGJ2 downregulated expression of genes encoding interleukin-6 (IL-6) and STAT3 while upregulating suppressors of cytokine signaling (SOCS) 3 in TPC-1 human thyroid papillary carcinoma cells . In activated glial cells, 15d-PGJ2 induces the transcription of SOCS 1 and 3, which in turn inhibits JAK activity [28,29]. We recently reported that 15d-PGJ2-mediated inactivation of STAT3 through covalent modification which led to apoptosis in H-
15d-PGJ2 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s nutrient mixture F-12 (1:1) and horse serum were obtained from Gibco BRL (Grand Island, NY, USA). Cholera toxin, hydrocortisone, insulin, human EGF), and MTT were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). P-STAT3-TA-Luc plasmid was supplied from BD Biosciences Clontech (Palo Alto, CA, USA).
The immortalized human mammary epithelial MCF10A and MCF10A cells transformed by an active Ha-
Cells were plated at a density of 3 × 104 cells/300 μL in 48-well plates, and the cell viability was determined by the MTT reduction assay. After incubation, cells were treated with the MTT solution (final concentration, 1 mg/mL) for 2 hours. The dark blue formazan crystals formed in intact cells were dissolved with dimethyl sulfoxide, and the absorbance at 570 nm was read using a microplate reader. Results were expressed as the percentage of MTT reduction obtained in the treated cells, assuming that the absorbance of control cells was 100%.
PC-3 cells were seeded in a six-well dish at a density of 2 × 105 cells per well and grown to 60% to 80% confluence in the complete growth medium. The cells were transfected with plasmid construct harboring the STAT3 wild type (WT) or a mutant form of STAT3 with cysteine 251 (Cys251) or Cys 259 replaced by alanine (C251A/C259A) using WelFect-M GOLD transfection reagent (WelGENE, Gyeongsan, Korea), and the transfection was carried out according to the instructions supplied by the manufacture.
Male BALB/c (nu/nu) mice, 6 weeks of age, were purchased from Central Lab Animal Inc. (Seoul, Korea) and were housed in a 12 hours light/12 hours dark cycle and fed rodent chow and water ad libitum. PC-3 cells (1 × 107 in 100 μL PBS) were injected subcutaneously on the right hind flank. Tumor volume (length × width × depth × 0.52) was measured three times a week. Tumor volume was regularly measured with digital calipers and calculated according to the formula: V = 0.5 ab2, where ‘a’ is the longest and ‘b’ is the shortest perpendicular diameters. After mice were killed, xenograft tumors were excised and fixed in formalin for immunofluorescence or immunohistochemical analysis. All protocols for animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (authorization number: SNU-170810-2-6).
Breast cancer cells were lysed in lysis buffer [250 mmol/L sucrose, 50 mmol/L Tris-HCl (pH 8.0), 25 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EDTA, 2 mmol/L NaF, 2 mmol/L sodium orthovanadate, and 1 mmol/L phenylmethylsulfonylfluoride] for 15 minutes on ice followed by centrifugation at 12,000 ×
For co-immunoprecipitation experiments, cells were lysed in immunoprecipitation buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin and protease inhibitors] (Cell Signaling Technology). On the following day, samples were added to 50 μL A/G-agarose beads (Santa Cruz Biotechnology) and rotated for 2 hours at 4°C. Protein A/G-agarose beads were washed four times with buffer A (10 mM HEPES pH 7.9, 60 mM KCl, 2 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 1 mM dithiothreitol, and protease and phosphatase inhibitors). Immunocomplexes were resolved on SDS PAGE, transferred to polyvinylidene fluoride membranes and analyzed with immunoblotting.
A molecular docking study was carried out using Schrodinger packages v9.5 (Schrödinger, LLC, New York, NY, USA). STAT3 crystal structures were retrieved from PDB bank (PDB code: 1BG1) [31,32]. Protein structures were prepared with the standard procedure of the Protein Preparation Wizard module. STAT3 protein was protonated at neutral pH, and hydrogen atoms were refined by energy minimization. The LigPrep module was used from the Maestro suite to generate a 3D structure of the ligands by adding hydrogen atoms and removing salt and ionizing at pH 7.4. The grid box was automatically determined within 5.0 Å of the Cys259 residue. The reaction type for covalent bonding was set to Michael addition. Binding energy on the 10 STAT3-15d-PGJ2 complexes was calculated after minimization of residues within 3 Å of ligand using Prime MM-GBSA. A binding pose with low Prime ΔGbind was selected for analysis.
Kaplan–Meier analysis of the STAT3 genes in 902 breast cancer patient database (including all molecular subtypes or ER-positive or ER-negative) was performed using Kaplan–Meier Plotter (http://kmplot.com/analysis/index.php?p=service&default=true).
Data sets of breast invasive carcinoma of The Cancer Genome Atlas (TCGA) were downloaded using TCGAbiolink  in R. Eight hundred seventy four primary BRCA breast invasive carcinoma samples were categorized into two groups, namely a high and low-expression group based on the 75% cut-off value of The P-STAT3 level from protein expression data [reverse phase protein array (RPPA)]. Normalized mRNA expression data of each 218 sample in high and low groups were retrieved. GSEA was conducted to explore potential biological processes and pathways by using the MSigDB database. Gene set enrichment was performed by the difference of class for the categorical phenotype to calculate fold change. Other options were set to default. Gene sets less than 0.01 of a nominal
In necessary, data were expressed as means ± SDs of at least three independent experiments, and statistical analysis for a single comparison was performed using the Student’s
To determine the role for STAT3 in breast cancer, we first analyzed the expression of STAT3 in invasive ductal carcinoma and non-tumor tissues from the tissue array. We found that STAT3 was significantly upregulated in breast cancer tissues, compared to adjacent normal tissues (Fig. 1A). GSEA was conducted using RNA-seq and RPPA data set of TCGA databases to explore potential biological processes and pathways in STAT3 overactivating breast cancers. The 75% cut-off value of P-STAT3 levels in RPPA was used to categorize patients with breast invasive carcinoma into low- and high-level groups. GSEA reveals that enriched pathways positively associated with STAT3 activation were KRAS signaling, hypoxia, and inflammatory responses, while the negatively correlated pathways include DNA repair and G2M-checkpoint (Fig. 1B). We further explored the prognostic implication of STAT3 in breast cancer. Kaplan–Meier survival analysis revealed that patients with a high level of STAT3 expression had significantly shorter recurrence-free survival time (Fig. 1C) compared with those with a low level of STAT3 expression in ER-negative breast cancer.
We also compared the STAT3 signaling in representative human breast cancer cell lines with different ER status. As illustrated in Figure 2A, ER-negative MDA-MB-468 and MDA-MB-231 cells displayed a greater extent of STAT3 activation than the ER-positive MCF-7 cells as evidenced by enhanced STAT3 tyrosine 705 (Tyr705) phosphorylation. MCF10A-
We further examined whether the anti-proliferative and proapoptotic activities of 15d-PGJ2 were associated with suppression of STAT3 activation in MDA-MB-231 and MDA-MB-468 cells. Treatment of these cell lines with 15d-PGJ2 concentration dependently reduced P-STAT3 levels (Fig. 3A and 3B). In addition, 15d-PGJ2 also effectively inhibited IL-6-induced phosphorylation of STAT3 and the STAT3 target protein, cyclin D1 in the LNCaP prostate cancer cell line expressing a relatively low level of constitutively active STAT3 (Fig. 3C). Consistent with these observations, IL-6-induced STAT3 transcriptional activity measured by the luciferase reporter gene assay was diminished by 15d-PGJ2 treatment (Fig. 3D). As STAT3 Tyr705 phosphorylation is known to facilitate STAT3 dimerization, we analyzed whether 15d-PGJ2 inhibited STAT3 dimerization by using the co-immunoprecipitation assay. For this experiment, we transfected PC-3 cells with hemagglutinin (HA)- and Myc-tagged STAT3. As shown in Figure 3E, 15d-PGJ2 markedly suppressed the dimerization of exogenous STAT3 (Fig. 3E).
The electrophilic β-carbon in an α,β-unsaturated carbonyl group can react with electron-rich nucleophiles, forming a covalent bond via the Michael addition reaction. 15d-PGJ2 harboring such reactive moiety can covalently bind to nucleophilic cysteinyl thiol group(s) of diverse cellular proteins . The STAT3 protein is composed of 6 functional domains (Fig. 4A), among which the coiled-coil domain is the most critical region for STAT3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding [35,36]. Notably, a short region in the first α-helix of the coiled-coil domain and a portion of the DNA-binding domain of STAT3 interact with another transcription factor, c-Jun, which facilitates the IL-6-inducible a2-macroglobulin gene transcription .
As part of our initial studies to identify potential binding sites of 15d-PGJ2 for STAT3, we conducted molecular docking simulation to predict the mode of interaction between 15d-PGJ2 and selected cysteine residues of STAT3. Covalent docking results show that 15d-PGJ2 may stably bind to the interface of the DNA binding domain and the coiled-coil domain of STAT3 near cysteine 251 and cysteine 259 residues with –44.81 kcal/mol and –42.64 kcal/mol, respectively of binding free energy calculated with Molecular Mechanics with Generalized Born and Surface Area (MM-GBSA ΔG). On docking poses of 15d-PGJ2 with Cys251 and Cys259 residues, its carboxyl group is predicted to form hydrogen bonds with Arg325 and Ile258 residues, respectively and an aliphatic chain lies and occupies a cavity of binding pocket (Fig. 4B).
To find out which of the two putative cysteines are likely to be preferred for covalent interaction with 15d-PGJ2, we mutated both amino acids to alanine and analyzed the effects of these mutations on the 15d-PGJ2-induced PARP cleavage. The PC-3 cell line bears a STAT3 whole gene deletion mutation on chromosome 17. Therefore, this STAT3-null cell line is useful for studying the effects of ectopic overexpression of STAT3 and its mutant constructs, especially by excluding the effects of endogenous STAT3. As breast cancer cells we used in this study express endogenous STAT3, we utilized this PC-3 cells as a tool to more precisely assess the comparative effects of ectopically overexpressed STAT3 and its cysteine-alanine mutant forms.
In PC-3 cells transiently transfected with cysteine 251-mutated STAT3 (STAT3C251A), 15d-PGJ2-induced PARP cleavage was almost equivalent to that observed in the cells expressing wild type STAT3 (STAT3WT) (Fig. 4C). However, substitution of Cys259 with Ala (STAT3C259A) resulted in marked reduction in the PARP cleavage following 15d-PGJ2 treatment (Fig. 4C).
EMT is an essential feature of invasive and metastatic cancer. During EMT, cells undergo morphological changes with enhancement of motility [38,39]. Loss of epithelial marker expression and a concomitant increase in the expression of mesenchymal markers are the characteristic events of EMT [38,40]. The contribution of intracellular signaling components to the induction of EMT through activation of the JAK/STAT3 signaling in cancer progression has been reported .
Enforced expression of STAT3C259A caused a shift in cell morphology from mesenchymal to a more epithelial phenotype, with increased cell-to-cell contact and loss of cell spreading (Fig. 5A). Further, we showed that cysteine 259-mutated STAT3 cells expressed a higher level of the typical epithelial marker, E-cadherin and a lower level of the mesenchymal marker, N-cadherin than did the WT STAT3 cells (Fig. 5B).
The critical role of Cys259 in oncogenic functions of STAT3 was also assessed in vivo by using a mouse xenograft breast tumor model in which green fluorescent protein (GFP)-tagged STAT3WT- or STATC259A-overexpressing PC-3 cells were transplanted into athymic nude mice. While tumors derived from wild type cells were continuously growing for 27 days, those from cells harboring STAT3C259A showed a slower growth rate (Fig. 5C). Moreover, the infiltrating number of STAT3-expressing cells in tumor tissues was significantly reduced by Cys259 mutation (Fig. 5D). Immunohistochemical analysis revealed that tyrosine P-STAT3Y705 are downregulated in STAT3C259A compared with STAT3WTexpressing tumors (Fig. 5E).
The above findings, taken all together, indicate that the Cys259 residue of STAT3 is essential for its function as a transcription factor, and suggest that covalent modification of this amino acid by 15d-PGJ2 mimics mutation at the same site which may provoke a similar impact.
PPARγ activation has been linked to cell growth inhibition and induction of apoptosis in several types of human cancer [18-20]. 15d-PGJ2, initially discovered as an endogenous ligand for PPARγ, has anti-cancer properties . 15d-PGJ2 induces apoptosis of some cancer cells [20-24,28,43,44]. However, its tumor suppressive effects are not necessarily mediated through activation of PPARγ. For instance, 15d-PGJ2 enhanced the apoptosis induced by TNF-related apoptosis-inducing ligands (TRAIL), which was not blocked by a PPARγ inhibitor . In another study,15d-PGJ2 significantly reduced the growth of oral squamous cell carcinoma, which was mainly attributed to the induction of apoptosis . Under the same experimental conditions, rosiglitazone and ciglitazone, two prototypic PPARγ activators, failed to exert a growth inhibitory effect . 15d-PGJ2 was shown to inhibit the migrative ability of mouse mammary adenocarcinoma cells, and these effects were independent of PPARγ . 15d-PGJ2 can also affect the activities/expression of some redox-sensitive transcription factors such as NF-κB [6,7], AP-1 , STAT [44,45], p53 , HIF-1α , and Nrf2  as well as their regulators, independently of PPARγ activation.
The role for the IL-6/STAT3 axis in tumor growth and progression as well as tumor-promoting inflammation has been well documented . Persistent activation of IL-6/STAT3 signaling is involved in various inflammation-associated malignancies, especially, breast cancer [45,50]. Thus, aberrant overactivation of STAT3 increases cancer cell proliferation, survival, and invasion while it represses antitumor immunity . Herein, we investigated whether anti-tumor effects of 15d-PGJ2 could be mediated by targeting STAT3 in breast cancer cells. 15d-PGJ2 treatment induced the apoptosis as evidenced by production of cleaved PARP in MDA-MB-231 and MDA-MB-468 cells with constitutively activated STAT3, but only weakly in ER-positive MCF-7 cells. In parallel with induction of apoptosis, phosphorylation of STAT3 Tyr705 was reduced in MDA-MB-231 and MDA-MB-468 cells in the presence of 15d-PGJ2. These results suggest P-STAT3 as the Achilles’ heel of ER-negative breast cancer. Likewise, 15d-PGJ2, but not rosiglitazone and ciglitazone, inhibited STAT3 phosphorylation and induced apoptosis in human oral squamous cell carcinoma cells .
Many studies have shown that 15d-PGJ2 structurally modifies the redox-sensitive transcriptional factors and thereby affects their functions through alkylation of cysteinyl thiol groups . Canonical STAT3 activation relies on the phosphorylation of Tyr705 by JAKs, which facilitates the SH2 domain-mediated STAT3 dimerization. However, the coiled-coil domain is also considered important for dimerization and subsequent nuclear translocation of STAT3 [52-55]. Two cysteine residues (Cys251 and Cys259) located in the coiled-coil domain appear to be involved in STAT3 dimerization via disulfide linkage . Computer-based docking analysis suggested Cys251 or Cys259 as putative binding sites of 15-PGJ2 on STAT3. To test the validity of this in silico prediction, we conducted site-directed mutagenesis studies in which the aforementioned cysteines were replaced by alanine. PC-3 cells were transfected with wild type or cysteine 251- or 259-mutated STAT3 followed by treatment with 15-PGJ2. The result shows that C259A mutation markedly attenuated the 15d-PGJ2-induced apoptosis, but C251A mutation had a marginal effect. This is in agreement with our recent finding that 15d-PGJ2 covalently modify Cys259 of human recombinant STAT3 as analyzed by mass spectrometry .
Until now, several thiol modifiers have been reported to inhibit STAT3 activity and functions. Stattic, alkylates Cys251, Cys259, Cys367, and Cys426 in STAT3 . The CDDO-Me inhibits STAT3 dimerization by binding to Cys259 . S3I-201 non-specifically modifies five cysteines (Cys108, Cys259, Cys367, Cys542, and Cys687) in the STAT3 protein . Furthermore, 15-keto-PGE2 interacts directly with Cys251 and Cys259 . Notably, mutation of STAT3 Cys259 significantly retarded the growth of PC-3 prostate cancer cells, which resembles an effect achieved by covalent modification of the same amino acid by 15d-PGJ2 .
Intensive efforts have been devoted to developing STAT3 inhibitors as anti-cancer drugs. There are two strategies for inhibiting the STAT3 signaling pathway. One approach would be targeting an upstream kinase responsible for STAT3 activation. Although the STAT3 pathway can be effectively suppressed by pharmacologic inhibition of JAK, JAK inhibitors have limitations often encountered in clinical trials, including off-target toxicities. Direct inactivation of STAT3 is less likely to cause unintentional inhibition of additional signaling pathways than targeting upstream molecules. In conclusion, the present study provides compelling evidence that 15d-PGJ2 is a potential compound capable of directly antagonizing STAT3 though covalent modification of this transcription factor (Fig. 6). Therefore, 15d-PGJ2 hence has a therapeutic value for the treatment of breast cancer presenting aberrant STAT3 regulation.
This work was supported by the Basic Science Research Program grant (No. 2021R1I1A1A01046540 to S.-J. K) and the BK21 FOUR Program (5120200513755) from the National Research Foundation, Republic of Korea.
No potential conflicts of interest were disclosed.
Eun-Ryeong Hahm, Su-Hyeong Kim, Sivapar V. Mathan, Rana P. Singh, Shivendra V. SinghJ Cancer Prev 2021; 26(2): 128-136 https://doi.org/10.15430/JCP.2021.26.2.128
Kyunghwa Cho, Hee Geum Lee, Juan-Yu Piao, Su-Jung Kim, Hye-Kyung Na, Young-Joon SurhJ Cancer Prev 2021; 26(2): 118-127 https://doi.org/10.15430/JCP.2021.26.2.118
Wondimeneh Shibabaw Shiferaw, Yared Asmare Aynalem, Tadesse Yirga Akalu, Tefera Mulugeta DemelewJ Cancer Prev 2020; 25(2): 111-118 https://doi.org/10.15430/JCP.2020.25.2.111