Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis
© Goncharenko-Khaider et al.; licensee BioMed Central Ltd. 2012
Received: 27 August 2012
Accepted: 13 November 2012
Published: 17 November 2012
Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.
Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.
In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.
These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.
Ovarian cancer (OC) is the fifth cause of cancer-related death in women, the second most common gynecological cancer, and the leading cause of death from gynecological malignancies[1–3]. High grade serous OC is the most common subtype of OC and over 70% of these patients present with late stage diseases and dissemination of tumor implants throughout the peritoneal cavity[4, 5]. Despite initial aggressive treatment, the five-year survival of patients with late stage disease remains at < 30%, a figure that has not changed for the past 30 years. This is related, at least in part, to the persistence of minimal residual disease after chemotherapy, which contributes to shorter progression-free survival[6, 7]. The tumor environment is being increasingly recognized as an important contributor of tumor progression[8–10] as it may facilitate the survival[11–14], differentiation and proliferation of tumor cells[15, 16]. Furthermore, ascites create a protective environment for ovarian tumor cells that inhibit drug-induced apoptosis (de novo resistance)[13, 17]. Ascites are heterogenous fluids that display marked differences in their levels of soluble factors but some of these factors can potentially activate an array of signaling pathways[18–24]. The demonstration that ascites with prosurvival properties are associated with a shorter progression-free survival in patient with OC underscores the critical role of ascites in OC progression. The molecular changes in tumor cells induced by ascites that result in resistance have not been well characterized. It is important to define the contribution of each pathway both to fully understand cell survival signaling and to validate individual pathways as therapeutic targets.
Activation of the Raf/MEK/ERK pathway has been often associated with the promotion of cell proliferation but also represents, in addition to the PI3K/Akt pathway, an important survival signaling pathway in many tumor cells. The Raf/MEK/ERK pathway promotes survival through the inhibition of the apoptotic cascade by controlling the expression or the activity of Bcl-2 family members[26, 27]. There is evidence that the ERK pathway activation increases the expression of prosurvival Bcl-2 proteins, notably Mcl-1, by promoting de novo gene expression[26, 28–30]. The relative expression of Mcl-1 in tumor cells can be regulated at the transcriptional level or through post translational modifications by ERK. In addition to the ERK signaling, the PI3K/Akt pathway has been found to be critical for Mcl-1 expression[32–34]. The importance of Mcl-1 in mediating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance has been well documented in different cell types. Overexpression of Mcl-1 can attenuate apoptosis induced by TRAIL. Conversely, downregulation of Mcl-1 by siRNA enhances TRAIL-mediated cell death.
TRAIL belongs to the TNF family of cytokines and has emerged as a promising anticancer agent, because of its ability to selectively induce apoptosis in a broad host of tumor cells[35, 38]. TRAIL binding to its receptors (TRAIL-R1 and TRAIL-R2) initiates the extrinsic pathway, resulting in recruitment of the adapter protein Fas-associated death domain (FADD) and procaspase-8 in the death inducing signaling complex (DISC). In some cells (type I cells), the apoptotic signal from active caspase-8 is sufficient to activate downstream effector caspases and induce apoptosis. However, in other cell types, such as OC cells, the apoptotic signal must be further amplified by engaging the intrinsic (mitochondrial) pathway. In this context, caspase-8 cleaves Bid to generate an active tBid, which in turn activates proapoptotic Bax or Bak proteins, and induces mitochondrial outer membrane permeabilization (MOMP). The mitochondria then releases proapoptotic factors that promote effector caspase activation. Overexpression of antiapoptotic Bcl-2 family members, including Bcl-2, Bcl-XL and Mcl-1 is associated with TRAIL resistance in type II cells, because of their ability to prevent tBid-induced MOMP.
In this study, we demonstrate that transcriptional upregulation of Mcl-1 by OC ascites is mediated by an ERK-dependent activation of the transcription factor Elk-1. Moreover, we demonstrate that upregulation of Mcl-1 has a significant role in ascites-mediated attenuation of TRAIL-induced apoptosis.
OC ascites upregulate Mcl-1 expression
Mcl-1 contributes to ascites-induced attenuation of TRAIL-mediated apoptosis
When CaOV3 and OVCAR3 cells were compared directly, the level of TRAIL-induced apoptosis was higher in CaOV3 cells (Figure2C), consistent with the observation that CaOV3 cells expressed lower basal level of Mcl-1 (Figure1E and1F). To further assess the role of Mcl-1 in TRAIL resistance, CaOV3 cells were transfected with Mcl-1 or control siRNA and expression of Mcl-1 was assessed by immunoblot at 24 h and 48 h post transfection. Mcl-1 protein was efficiently downregulated by Mcl-1 siRNA in CaOV3 cells (Additional file1: Figure S2A). Importantly, transfection of CaOV3 and OVCAR3 cells with Mcl-1 siRNA completely abrogated ascites-induced Mcl-1 upregulation in both CaOV3 (Figure2D) and OVCAR3 cells (Figure2E). Of note, the expression of antiapoptotic protein Bcl-2 and Bcl-XL remained unaffected by Mcl-1 siRNA (Additional file1: Figure S2B). Mcl-1 depletion significantly blocked the prosurvival activity of ascites in CaOV3 and OVCAR3 cells. As shown in Figure2D, TRAIL (50 ng/ml) induced apoptosis in CaOV3 cells whereas the presence of ascites, as expected, significantly inhibited TRAIL-induced apoptosis (P < 0.001). In CaOV3 cells transfected with Mcl-1 siRNA, the protective effect of ascites was almost completely abrogated. The transfection of Mcl-1 siRNA in OVCAR3 cells also significantly (P < 0.01) inhibited the protective effect of ascites albeit to a lesser extend (Figure2D). This could be related to the observation that the Mcl-1 siRNA did not completely block Mcl-1 expression in OVCAR3 cells.
OC ascites upregulate Mcl-1 through ERK1/2 signaling
Ascites activates Elk-1 transcription factor via ERK1/2 pathway
Ascites-dependent Elk-1 activation is responsible for Mcl-1 regulation
Ascites-mediated ERK/Ekl-1 signaling is independent of FAK activation
Activated ERK1/2 correlates with Mcl-1 expression in high grade serous OC (HGSOC)
The development of resistance to chemotherapy remains a major problem with OC. Indeed, the poor prognosis is usually attributed to the occurrence of resistance. Defects in the apoptotic cascade have been commonly associated with resistance in OC cells. Although a number of mechanisms have been proposed for OC cells, most studies were performed in unicellular models and did not take into account the interactions that exist between the host and tumor cells. Unlike most other solid cancers where the stroma surrounding tumor cells constitutes the tumor environment, ascites that develop during OC progression represent a unique form of tumor environment. Indeed, soluble factors in ascites create a proinflammatory environment that promotes de novo resistance[13, 17]. Available evidence suggests that soluble factors in the tumor environment engage cell surface receptors to activate survival pathways. This study extends our previous findings that ascites-induced activation of the Akt pathway attenuates TRAIL-induced apoptosis[13, 17] by showing that ERK1/2/Elk-1 signaling is responsible for the transcriptional increase of Mcl-1, which in turn contributes to ascites-mediated inhibition of TRAIL-induced apoptosis in OC cells. Our results show that ascites induce a rapid activation of Akt and ERK1/2 but only that ERK1/2 activation is associated with Mcl-1 upregulation in tumor cells. Moreover, our results demonstrate that Mcl-1 upregulation is one of the mechanisms by which ascites protect OC cells from against TRAIL-induced apoptosis.
Although we have previously reported that one malignant ascites (COV2) induced the phosphorylation of Akt but not ERK, further works, as shown here and by other groups, have demonstrated that ERK activation by various OC ascites is a common findings. Similar observations have been made for the activation of the Akt pathway by ascites. Many ascites have the ability to activate this pathway[6, 13, 17] but it appears that some OC ascites are unabled to increase Akt phosphorylation in OC cell lines[6, 13]. This is believed to be related to the heterogeneity of OC ascites.
TRAIL cytotoxicity in OC cells relies on the activation of both the extrinsic and the intrinsic apoptotic pathways. These two pathways are interconnected, and in OC cells, the proapoptotic Bcl-2 family member Bid is a critical regulator of TRAIL resistance that connects both pathways by promoting mitochondrial activation. Antiapoptotic Bcl-2 family proteins, such as Bcl-2, Bcl-XL and Mcl-1, have a critical role in regulating the balance between survival and death signals at the mitochondrial level. Although Bcl-XL may promote the survival of OC cells[42, 45], the importance of Mcl-1 in OC survival has not been well established. Higher expression of Mcl-1 in OC compared to adenomas or normal ovaries has been reported[46–48], and was, in some studies, associated with poor prognosis. Our study shows that Mcl-1, but not Bcl-2 nor Bcl-XL, is upregulated by OC ascites. Mcl-1 is a downstream target of activated ERK signaling and is important for survival of OC cells in response to TRAIL since siRNA inhibition of Mcl-1 significantly attenuates ascites-mediated resistance to TRAIL.
Ascites-induced signaling events trigger activation of both the Akt and the ERK1/2 pathways. We have previously shown that ascites-mediated Akt activation attenuates TRAIL-induced apoptosis in CaOV3 cells. Ascites activate Akt, which in turn up-regulate the expression of cFLIPs, a caspase-8 inhibitor. The treatment of CaOV3 cells with PI3K/Akt inhibitors partially blocks ascites-mediated survival. Activation of the PI3K/Akt pathway thus represents one way by which ascites confer resistance to TRAIL-induced apoptosis. The present study suggests that ERK1/2 pathway mediates the transcriptional upregulation of Mcl-1. Unlike inhibition of ERK1/2, blocking Akt pathway did not alter ascites-induced upregulation of Mcl-1. This is evidenced by the lack of effect of Akt downregulation by siRNA and Akt inhibition by LY294002 on Mcl-1 expression. In contrast, U0126-mediated inhibition of ERK1/2 readily decreased Mcl-1 at the transcriptional level, and promoted TRAIL-induced apoptosis in OC cells. These results indicate that ERK1/2, but not Akt pathway, plays a determining role in ascites-induced Mcl-1 expression. The ERK1/2 pathway has been previously reported to regulate Mcl-1 transcription in other cell types[26, 28–30]. In addition, the activation of ERK1/2 in OC has been shown to enhance tumor progression[49, 50]. Activation of the ERK1/2 pathway has also been involved in tumor cell survival by coupling survival stimulus to transcription factors controlling gene expression. For example, higher levels of phospho-ERK1/2 in OVCAR3 cells were associated with increased resistance to cisplatin. In addition, the resistance to paclitaxel can be partially obliterated when ERK1/2 activity is inhibited. The correlation between ERK1/2 activation and Mcl-1 expression in tumor samples from patients with HGSOC suggest that the ERK1/2/Mcl-1 pathway likely exerts a protective anti-apoptotic effect to tumor cells and is biologically relevant.
Our data indicate that the Elk-1 transcription factor is an important regulator of ascites-induced Mcl-1 expression. OC ascites induced a rapid (within 30 min) phosphorylation of Elk-1 in tumor cells. Although other transcription factors such as Stat3 and NF-κB have been reported to regulate Mcl-1 expression, it appears that Elk-1 is critical in OC cells as evidenced by the fact that siRNA inhibition of Elk-1 almost completely abolished ascites-induced Mcl-1 upregulation. In accordance with our results, Elk-1-dependent regulation of Mcl-1 expression has been described with other types of cancer[27, 28]. Additional studies have shown that Elk-1 is directly phosphorylated by ERK1/2 and therefore support our findings that ascites induce phosphorylation of not only ERK1/2 but also Elk-1.
We have previously shown that soluble factors present in OC ascites engage αvβ5 integrin to induce a FAK-dependent Akt activation that contributes to protect cells from TRAIL-induced apoptosis. Here, we demonstrate that ERK1/2 activation, which contributes to decrease TRAIL-induced apoptosis, is independent from ascites-mediated FAK activation as shown by the fact that the knockdown of FAK does not affect ERK1/2 and Elk-1 phosphorylation. Although growth factor receptors such as EGFR and PDGFR can often activate the ERK pathway, and ligands of these receptors are present in OC ascites, we do not believe that the ascites-mediated upregulation of Mcl-1 is dependent on these receptors because we previously shown that the inhibition of EGFR and PDGFR does not alter the prosurvival activity of ascites.
Our findings suggest that OC ascites activate multiple signaling pathways to inhibit TRAIL-induced apoptosis and each pathway may contribute to a different level to ascites-mediated protection from TRAIL depending, at least in part, on the cell context. Although the significance of these in vitro observations in regard to the clinic has yet to be determined, we propose that ascites, by activating different survival pathways in tumor cells, contribute to the persistence of tumor cells during treatment and the occurrence of resistance. This has implication from a therapeutic standpoint. Targeting the tumor environment could be an important strategy to sensitize OC cells to chemotherapy.
Materials and methods
Cell culture and reagents
The human OC cell lines CaOV3 and OVCAR3 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in a humidified 5% CO2 incubator at 37°C. Cells were passaged twice weekly. OVCAR3 cells were maintained in RPMI-1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20% FBS, insulin (10 mg/L), glutamine (2 mM) and antibiotics. CaOV3 cells were cultured in DMEM/F12 (Wisent) supplemented with 10% FBS, 2 mM glutamine and antibiotics. TRAIL was purchased from PeproTech (Rocky Hill, NJ). Acellular ascites fractions OVC415, OVC508, OVC509, OVC551 were obtained at the time of initial cytoreductive surgery from women with advanced serous ovarian carcinomas. Samples were supplied by the Banque d’échantillons biologiques (seins/ovaires) et de données de Sherbrooke as part of the Banque de tissus et de données du Réseau de Recherche en Cancer des Fonds de Recherche en Santé du Québec (FRSQ) affiliated to the Canadian Tumor Repository Network (CTRNet). HRP-conjugated anti-mouse and -rabbit antibodies, Akt, Bcl-XL, Elk-1, phospho-ERK1/2 (Thr202/Tyr204), Mcl-1, FAK, phospho-FAK and phospho-Elk-1 (Ser383) antibodies were purchased from Cell Signaling. Antibodies for phospho-Akt (Ser473) were from Life Technologies (Burlington, ON). Bcl-2 antibody was purchased from Dako (Burlington, ON). ERK antibody was from Santa Cruz Biotech (Santa Cruz, CA). PI3K inhibitor LY294002 and MEK inhibitor U0126 were purchased from EMD (Billerica, MA). Tubulin antibody, actinomycin D and propidium iodide were purchased from Sigma-Aldrich (Oakville, ON). Actinomycin D was dissolved in dimethyl sulfoxide at a concentration of 10 mM and stored at −20°C.
Quantitative real time PCR
Total RNA was extracted from CaOV3 and OVCAR3 cells using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol and subjected to reverse transcription (RT) with oligodT from Promega (Madison, WI) and MMULV reverse transcriptase enzyme. RNA concentrations were quantified by measurement of absorbance at 260 nm. The integrity of the cDNA was assessed with the Taqman gene expression assays (Life Technologies), done on RPLPO housekeeping gene. Each sample was normalized to the housekeeping gene levels. Mcl-1 primers were from Life Technologies (gene expression assay hs03043899, cat # 4331182). Cycle conditions for all PCRs were as follow: an initial incubation of 2 min at 95°C followed by 35 cycles at 94°C 30 s, 55°C 30 s, 72°C 60 s. The amplification occurred for 2 min at 72°C. PCR products quantification was performed as previously described in collaboration with Dr C. Asselin (université de Sherbrooke).
Analysis of apoptosis was performed by quantification of the sub-G1 peak by flow cytometry as previously described. Propidium iodide staining for DNA fragmentation was done by fixing cells and staining them with propidium iodide for DNA analysis content as previously described. A total of 10,000 events were analyzed by flow cytometry and the percentage of hypodiploid cells was measured using a BD FACScalibur flow cytometer (BD Biosciences, ON, Canada).
Western blot analysis
Cells were harvested and washed with ice-cold PBS. Whole cell extracts were prepared in lysing buffer (glycerol 10%, Triton X-100 1%, TRIS 10 mM pH 7.4, NaCl 100 mM, EGTA 1 mM, EDTA 1 mM, SDS 0.1%) containing protease inhibitors (0.1 mM AEBSF, 5 μg/ml pepstatin, 0.5 μg/ml leupeptin and 2 μg/ml aprotinin) and phosphatase inhibitors (Na4P2O7 20 mM, NaF 1 mM, Na3VO4 2 mM). Proteins were separated by 12% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Roche, Laval, Québec, Canada) by electroblotting, and immunoblot analysis was performed as previously described. All primary antibodies were incubated overnight at 4°C in 5% fat-free milk. Proteins were visualized by enhanced chemiluminescence (GE Healthcare, Baie d’Urfé, Québec, Canada).
The Fluorescein-labeled Luciferase GL2 duplex or a non-target (scrambled) siRNAs used as a control were from Dharmacon Research (Lafayette, CO). Cells (6 × 104) were seeded in 6-well plates and allowed to adhere for 24 h. Cells (50% confluent) were transfected with a mixture containing Lipofectamine 2000™ (Life Technologies), optiMEM (Life Technologies) and siRNA (10 μM). The siRNAs/Lipofectamine complex was then added to the media of 6-well plates containing cells. Cells were incubated for 4–6 h at 37°C in a CO2 incubator and medium containing FBS was then added. The Mcl-1 and FAK siRNAs were from Dharmacon Research, Akt siRNA from Cell Signaling and Elk-1 siRNA from Santa Cruz.
TMAs were acquired from the Pan-canadian platform for the development of biomarker-driven subtype specific management of ovarian carcinoma (COEUR study). Sections were deparaffinized in citrate buffer containing 0.05% Tween at 97°C for 20 min, washed with PBS and incubated with 3% peroxide. After treatment, slides were submerged in a citrate buffer (0.01 M citric acid, pH 6.0) for 15 min, and incubated with a protein blocking serum-free reagent (Dako Canada). The TMAs were stained by an immunoperoxidase method using an automated tissue immunostainer (Dako Canada) with DABchromogen. The TMAs counter stained with hematoxilin and were visualized by light microscopy at 20× magnification and scored by two blinded independent observers using the H-score method with an inter-rating > 90%. An intensity score of 0–3 was multiplied by the percentage of tumor cells stained to obtain the H-score. P values were calculated by the Mann–Whitney test.
Statistical comparisons between two groups were performed using the Mann–Whitney or Student’s t-test. The correlation between phosphor-ERK1/2 and Mcl-1 expression in tissue section was determined by the Spearman correlation test. Statistical significance was indicated by P < 0.05.
This work was supported by grants from the Cancer Research Society and the Canadian Institutes of Health Research (MOP-115072) to A.P. We wish to thank the Banque d’échantillons biologiques (seins/ovaires) et de données de Sherbrooke as part of the Banque de tissus et de données du Réseau de Recherche en Cancer des Fonds de Recherche en Santé du Québec (FRSQ) affiliated to the Canadian Tumor Repository Network (CTRNet) for providing ascites samples from women with advanced ovarian cancer. We thank Dr C. Asselin for his assistance with the real-time PCR assays and Dre N. Perrault for her help with immunostaining.
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