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.