Thymoquinone inhibits cell proliferation, migration ...

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by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer. Nashwa Kabil1 ท Recep Bayraktar1 ท Nermin Kahraman1 ...
Breast Cancer Research and Treatment https://doi.org/10.1007/s10549-018-4847-2

PRECLINICAL STUDY

Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer Nashwa Kabil1 · Recep Bayraktar1 · Nermin Kahraman1 · Hamada A. Mokhlis1,2 · George A. Calin1,3 · Gabriel Lopez‑Berestein1,3 · Bulent Ozpolat1,3 Received: 29 December 2017 / Accepted: 30 May 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Background/purpose  Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target. Methods/results  In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice. Conclusion  Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression. Keywords  Thymoquinone · eEF-2K · MiR-603 · Triple-negative breast cancer

Introduction Electronic supplementary material  The online version of this article (https​://doi.org/10.1007/s1054​9-018-4847-2) contains supplementary material, which is available to authorized users. * Bulent Ozpolat [email protected] 1



Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 422, Houston, TX 77030, USA

2



Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

3

Center for RNA Interference and Non‑Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA



Breast cancer (BC) is the most common malignancy among women with more than 250,000 new cases in the U.S. per year [1]. Triple-negative breast cancer (TNBC) is a molecular subtype of BC, accounting for 15–20% of all BC cases, and is characterized by the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2)/Neu receptors [2]. Current treatment options for TNBC are still limited to conventional chemotherapies such as anthracyclines (e.g., doxorubicin) and taxane-based therapeutics [3]. Despite recent advancements in TNBC management, patients continue to have reduced response to conventional chemotherapeutics, with higher relapse rates and poorer outcome compared to other subtypes of BC [3].

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Therefore, identifying novel potential targets and developing effective therapeutic strategies are of critical importance. Recent literature has suggested that eukaryotic elongation factor-2 kinase (eEF-2K) is an important molecular driver of highly aggressive cancers such as TNBC [4, 5], pancreatic cancer [6] and glioblastoma multiform [7]. eEF-2K is a Ca2+/CaM-dependent member of the α-kinase family that functions by phosphorylating/inactivating eEF-2 (at threonine 56), resulting in reduced peptide chain elongation during the mRNA translation process [8, 9]. Increased eEF-2K activity has been observed in many cancers, where it functions by promoting tumor cell survival, growth, and metastasis [10]. Our recent findings have revealed that eEF-2K is highly expressed in TNBC patients and is correlated with poor overall survival and prognosis [11]. Furthermore, other studies have highlighted the critical role of eEF-2K in promoting TNBC tumor growth and progression both in vitro and in orthotopic xenograft mouse models [12]. eEF-2K promotes cell proliferation, migration/invasion, and cell cycle progression by regulating critical pathways such as Src/Fak, PI3K/Akt, cyclin D1, and insulin-like growth factor receptor (IGFR) signaling [4–6, 12]. Overall, studies suggest that eEF-2K is one of the critical drivers of TNBC tumorigenesis and is a potential molecular target. Therefore, the development of therapeutic strategies targeting eEF-2K may prove to be clinically beneficial in TNBC patients. Thymoquinone (TQ; 2-methyl-5-isopropyl-1,4-benzoquinone) is the active ingredient of the Nigella sativa seed, known to have broad therapeutic effects due to its oxidant [13], anti-inflammatory [14], anti-diabetic [15], and immunomodulatory properties [16]. Numerous studies demonstrated that TQ is a potential anti-cancerous agent, in a variety of carcinomas including breast, lung, ovarian, liver, prostate, and colorectal carcinomas [17]. TQ exerts its anticancerous effects through different modes of action, such as by inhibition of cell proliferation and angiogenesis, induction of apoptosis and cell cycle arrest, and generation of reactive oxygen species [17]. TQ regulates many essential targets/pathways in cancer including nuclear factor-kappa B (NF-κB) [18], p53 [19, 20], STAT3 [18], and PPAR-γ [21]. However, the molecular mechanisms by which TQ exerts its effects in TNBC still remain unclear. Given the effects of TQ and the major role of eEF-2K in TNBC cells, we hypothesized that TQ acts through the inhibition of eEF-2K in TNBC. The aim of the current study was to identify the molecular mechanisms of TQ-mediated effects in TNBC. We showed for the first time that TQ reduces TNBC cell proliferation and migration/invasion by partially regulating the NF-κB/miR-603/eEF-2K axis.

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Breast Cancer Research and Treatment

Materials and methods Cell lines, cell culture conditions, and reagents Triple-negative breast cancer (TNBC) cell lines (ER−, PR−, and HER2−) MDA-MB-231, MDA-MB-436, and BT-20, ER+ MCF-7, HER2+ SK-BR-3, and mammary epithelial cells MCF-10A were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). MDAMB-231, MDA-MB-436, BT-20, MCF-7, and SKBR-3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12, supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. MCF-10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, epidermal growth factor, hydrocortisone, insulin, and cholera toxin. TQ and pyrrolidine dithiocarbamate (PDCT) (as NF-κB inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Transfection with siRNA We utilized a previously published eEF-2K siRNA [6, 22, 23] and control nonsilencing siRNA [24]. Cells were transfected with either siRNA, at a final concentration of 50 nM for 72 h, using HiPerFect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer’s protocol.

Overexpression of eEF‑2K in MDA‑MB‑231 cells In order to establish stable clones of MDA-MB-231 overexpressing eEF-2K, the cells were transduced with either pCDH-eEF-2K lentiviral plasmid or empty control vector, followed by clonal selection against puromycin according to our previously published protocol [11]. Subsequently, we verified the eEF-2K expression by Western blotting.

Cell viability and colony formation assays T h e M T S [ 3 - ( 4 , 5 - d i m e t hyl t h i a z o l - 2 - yl ) - 5 - ( 3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay was utilized to determine the viability of TNBC cells (MDA-MB-231, MDA-MB-436, and BT-20), ER+ MCF-7, HER2+ SK-BR3, and mammary epithelial cells MCF-10A as described previously [6]. Cells were treated with increasing doses of TQ (1–50 µM) and viability was determined after 72 h. To detect the effect of TQ/EF-2K on colony formation in TNBC, we performed clonogenic assay. MDAMB-231 and MDA-MB-436 cells were seeded at low density (500 cells/well) in 12-well plates and treated with TQ/ or two sequences of EF-2K siRNA and cultured for 10–14

Breast Cancer Research and Treatment

days. Colonies were stained with crystal violet and quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

Cell motility, migration, and invasion assays In vitro cell motility and migration was detected by woundhealing assay [23]. MDA-MB-231 and MDA-MB-436 cells were treated with TQ for 24 h or siRNA for 72 h. After the designated treatments, a straight scratch was made on the cell layer and cells were imaged at consecutive 12-h time points, using a phase-contrast microscope (Nikon Instruments Inc., Melville, NY, USA). Wound healing was determined at each time point by quantifying the open area of the wound of 5 random images per experiment using ImageJ software. Cell invasion was evaluated by in vitro invasion assay [12] after 24-h TQ treatment. Invaded cells were imaged by a light microscope (Nikon Instruments Inc., Melville, NY, USA), and 5 random regions were quantified by ImageJ software.

Protein extraction and Western blotting Western blot analysis was performed as reported previously [6, 11]. Expression levels of selected proteins were detected by specific antibodies for eEF2K, p-EF2 (Thr56), Src, p-Src(Tyr416), p-AKT (Ser473), AKT (Cell Signaling Technology), FAK, p-FAK (Try397) (R&D Systems), β-actin (Sigma), and their HRP-conjugated secondary antibodies.

RNA extraction and miRNA and mRNA reverse transcription RNA isolation and reverse transcription to cDNA were performed as described previously [11]. miR-603 expression was analyzed with a PerfeCTa® microRNA assay kit (Quanta, Gaithersburg, USA) using Quanta miRNA primers (Quanta Biosciences, USA). Relative expression levels of miR-603 were normalized to SNORD48 (Quanta Biosciences, USA) as an endogenous control. eEF-2K gene expression was analyzed with an iQ SYBR Green Supermix RT-PCR kit (BIO-RAD). eEF-2K gene expression was normalized to GAPDH as an endogenous control.

Orthotopic xenograft TNBC tumor model Nude athymic female mice were obtained from MD Anderson Cancer Center. All studies were conducted according to the experimental protocol approved by the MD Anderson Institutional Animal Care and Use Committee. MDAMB-231 and MDA-MB-436 cells (2 × 106 in 20% Matrigel) were injected into the mammary fat pad of each mouse.

After approximately 2 weeks, liposomal TQ treatment was administered every 3 days, at two different doses (20 and 100  mg/kg) by i.v. injection. Liposomes were prepared based on our previously published method [12]. Tumor tissues were removed after 48-h TQ injection and analyzed by Western blot.

Statistical analysis Data were expressed as mean ± SD of three independent experiments, and statistical analysis was performed using the Student’s t test. p values less than 0.05 were considered statistically significant and indicated by asterisk.

Results TQ inhibits TNBC cell viability and proliferation To determine the effects of TQ on TNBC cell viability, we performed MTS assay on a panel of TNBC cell lines (MDAMB-231, MDA-MB-436, and BT-20) after 72-h treatment of increasing doses of TQ (1–50 µM). MTS assay revealed that MDA-MB-231 and MDA-MB-436 cells were the most sensitive to TQ and showed a significant reduction in the number of viable cells at 7.5–10 µM concentration compared to DMSO-treated cells (Fig.  1a), whereas BT-20 showed reduced cell viability at 15–20 µM concentration (Supplementary Fig. 1). Furthermore, the ER+ MCF-7 and HER2+ SKBR3 cells were less sensitive to TQ, showing a 50% reduction in cell viability at 15–20 and 20–30 µM, respectively (Supplementary Fig. 1). Moreover, the mammary epithelial cell line MCF-10A showed the least sensitivity to TQ, with a reduction in cell viability at 30–50 µM TQ (Supplementary Fig. 1; Table 1). We then determined the effects of TQ on colony formation in MDA-MB-231 and MDA-MB-436 cells. TQ treatment (7.5–10  µM) resulted in a significant reduction in colony formation of both MDA-MB-231 (***p = 0.0004) and MDA-MB-436 (***p = 0.0005) cells (Fig. 1b) compared to DMSO-treated control cells.

TQ decreases the migration and invasion of TNBC cells Next, we investigated the effect of TQ treatment (5–10 µM) on cell motility using the wound healing assay. Percentage wound healing was determined after normalization with DMSO-treated control cells. The analysis revealed that wound healing was significantly decreased by 70% in MDA-MB-231 (p