Cancer Letters

Cancer Letters

Volume 347, Issue 2, 1 June 2014, Pages 212-224
Cancer Letters

Differential outcome of MEK1/2 inhibitor-platinum combinations in platinum-sensitive and -resistant ovarian carcinoma cells

https://doi.org/10.1016/j.canlet.2014.02.016Get rights and content

Abstract

Deregulated pro-survival signalling plays a role in ovarian carcinoma drug resistance. Here, we show that cisplatin or oxaliplatin in combination with the MEK1/2 inhibitor CI-1040 resulted in a synergistic effect associated with enhanced apoptotic response in platinum-sensitive cells. The drug combinations were additive in platinum-resistant cells exhibiting increased phospho-ERK1/2, down-regulation of apoptosis-related factors (BAX, PUMA, FOXO1) and of phosphatases inhibiting ERK1/2 (DUSP5, DUSP6). Consistently, FOXO1 knockdown in sensitive cells reduced the efficacy of the combination treatment. Pharmacological targeting of ERK1/2 pathway increases cell sensitivity to platinum compounds by interfering with multiple events, ultimately favouring apoptosis induction in selected molecular backgrounds.

Introduction

Ovarian carcinoma is the leading cause of death for gynaecological malignancies in the western world. Although surgery and platinum-based chemotherapy can be effective [1], [2], there is an urgent need for novel therapeutic strategies. The reduced efficacy of platinum-based therapies has been linked to several factors, including specific molecular alterations in tumour cells [3]. Cellular response to drug treatment can be influenced by the activation of survival pathways, which is a common feature of cancer cells [4]. Indeed, the activation of pathways promoting cell growth and survival, such as the PI3K/Akt and the canonical MAPK (i.e., ERK1/2) cascades, plays a role in resistance to platinum compounds in ovarian carcinoma models [5], [6]. The activation of the ERK1/2 cascade is modulated by a number of inhibitory proteins, including the dual-specificity phosphatases (DUSP) 5 and 6, which dephosphorylate and inactivate ERK1/2 in the nucleus and in the cytoplasm, respectively [7]. Since members of the ERK1/2 cascade are frequently altered in human tumours [5], targeting of the canonical MAPK cascade members has been regarded as a promising therapeutic strategy. In this context, a number of inhibitors have been designed that are being tested in preclinical and clinical settings [8]. Among those, MEK inhibitors have been recently recognized as promising agents to be used primarily in combination therapies [9]. Thus, a better knowledge of the MAPK pathway in ovarian carcinoma cell response to drug treatments appears to be crucial to devise effective therapeutic approaches based on MEK inhibition.

Cytotoxic agents, including platinum drugs, have been shown to activate ERK1/2 [10], although the significance of such an activation has been debated. In most cases, ERK1/2 activation has been documented to provide tumour cells with a survival advantage, even if there are circumstances where it appears to favour cell death induction [11]. ERK1/2 may play a pro-survival role in ovarian carcinoma, as increased activation can occur in platinum-resistant cells [12], [13]. In fact, cells selected for resistance to platinum drugs, endowed with increased phospho-ERK1/2 and Akt levels, displayed reduced sensitivity to the epidermal growth factor receptor (EGFR) inhibitor gefitinib; of note, sensitivity to gefitinib was restored upon MEK1/2 inhibition [12]. Moreover, EGFR inhibition was reported to counteract cisplatin-induced activation of survival kinases in ovarian carcinoma cell lines [14], [15].

Among the mechanisms of ERK1/2-mediated drug resistance, blockade of apoptosis induction has been well documented. ERK1/2 have been reported to inactivate proteins implicated in apoptosis, such as pro-apoptotic BH3-only proteins (BOP) and FOXO transcriptional factors. FOXO1 and FOXO3 can activate the expression of cell cycle progression inhibitors and pro-apoptotic proteins [16]. Interestingly, FOXO protein activity is inhibited by phosphorylation mediated by survival kinases including ERK1/2 [17]. Phosphorylations of specific residues (i.e., Thr24 and Ser256 for FOXO1), mediated by Akt, can inactivate FOXO proteins by shuttling them outside the nucleus [18]. Among the BOP, BAD activity is tightly controlled by phosphorylation mediated by survival kinases including ERK1/2, through the subsequent activation of cytoplasmic RSK [19]. Moreover, PUMA, a BOP whose expression is induced by p53, can be controlled by survival kinases, as FOXO3 has been reported to up-regulate PUMA expression in response to growth factor withdrawal [20], [21].

Based on this background, in the present study we used an experimental system reflecting alterations commonly found in the clinical setting (i.e., p53 alteration, ERK1/2 activation) consisting of a p53 wild-type cisplatin-sensitive ovarian carcinoma cell line, and p53-mutant platinum-resistant variants displaying activation of ERK1/2, to investigate the molecular mechanisms underlying the effect of the combination of platinum compounds and MEK inhibitors.

Section snippets

Experimental models, drugs and reagents

The IGROV-1 cell line, established from a patient with ovarian adenocarcinoma, was obtained as described [22]. The resistant sublines were generated by continuous exposure of parental cells to platinum drugs. In detail IGROV-1/OHP and IGROV-1/Pt1 cells, selected by continuous exposure to oxaliplatin and cisplatin, respectively, harbour mutations in the TP53 gene [12], [23], whereas IGROV-1/CP cells, developed by exposure of IGROV-1 cells to increasing concentrations of cisplatin, exhibit

ERK1/2 activation is associated with DUSP5 and DUSP6 down-regulation in platinum-resistant cells

The IGROV-1/Pt1 and IGROV-1/OHP ovarian carcinoma cell lines characterized by acquired resistance to platinum compounds (Table 1) and displaying increased levels of phospho-ERK1/2 with respect to parental IGROV-1 cells (Fig. 1A) were used to assess sensitivity to MEK inhibitors. The two platinum-resistant variants exhibited reduced sensitivity as shown by the IC50 values of CI-1040 and PD0325901 (Table 1).

Cell sensitivity to CI-1040 was further assessed in cells over-expressing ATP-binding

Discussion

Due to the crucial function of MEK signalling in sustaining survival of tumour cells, understanding of how MEK inhibition impacts on tumour cell sensitivity to drug treatment represents a major aspect of tumour cell biology and therapy. In the present study, using ovarian carcinoma cell lines including platinum-resistant variants characterized by increased phospho-ERK1/2 activation, we found that the combination of cisplatin or oxaliplatin with CI-1040, a potent and selective MEK1/2 inhibitor,

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

We thank Andrea Vecchi of the institutional FACS Core Facility for technical support, Jayne Stommel (The Salk Institute, La Jolla, California) for the p53/GFP plasmid, Ian J. Stratford (University of Manchester, UK) for helpful discussion, and the institutional Functional Genomics Facility for gene expression analysis. G. Cossa was supported by a fellowship by the Fondazione Italiana per la Ricerca sul Cancro, Milan, Italy. This work was partially supported by Fondazione Cariplo, rif. 2011-0490.

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