Synergistic inhibition of ovarian cancer cell growth by combining selective PI3K/mTOR and RAS/ERK pathway inhibitors
Introduction
Ovarian cancer has the highest mortality rate among all gynaecological cancers [1] largely due to the late diagnosis. Most patients respond to debulking surgery and treatment with a combination of taxane and platinum-based therapy, but later develop disease recurrence due to intrinsic and acquired resistance. Thus novel strategies are required to better treat this disease at diagnosis and/or provide an effective second line treatment. Dysregulation of both the phosphoinositide 3-kinase (PI3K) pathway and RAS/extracellular signal-regulated kinase (ERK) pathway are highly prevalent in all histotypes of ovarian cancer and hence targeting these pathways may provide a novel alternative to conventional therapy [2], [3], [4], [5].
PI3K initiates a signalling cascade that activates mammalian target of rapamycin complex 1 (mTORC1) via AKT that also induces subsequent phosphorylation of many factors that impact on cell metabolism, angiogenesis, cell growth, proliferation and survival [6], [7], [8]. RAS signalling via RAF and mitogen-activated protein kinase kinase (MEK) leads to the activation of both ERK1 and ERK2. ERK phosphorylates several cytosolic and nuclear proteins, including transcription factors that regulate the cell cycle [9]. Currently, inhibitors of RAF and MEK are the most advanced in the clinic for blocking ERK signalling [10], [11], whilst for the PI3K pathway there are many agents targeting different members of the pathway (PI3K, AKT, mTORC1 and mTOR) including some that inhibit multiple components (PI3K and mTOR) [12]. The dual PI3K and mTOR inhibitors have shown great promise in preclinical models [13]. PF-04691502 (PF502) is an ATP-competitive inhibitor of PI3K and both mTOR complexes [14] and is currently in several clinical trials [15], PD-0325901 (PD901) is a selective inhibitor of both MEK isoforms (MEK1/MEK2) and thus prevents the activation of ERK and is also currently in a clinical trial [16].
Given the high frequency of activating events in both the PI3K and RAS pathways we sought to determine the efficacy of PF502 and PD901 on a panel of 30 ovarian tumour cell lines. In addition, we performed global mRNA expression profiling, complemented with targeted mutation and pathway activity analysis to identify potential predictive and response biomarkers. These analyses identified RAS signalling as a key mediator of PF502 resistance and established the rationale for combination therapies with PF502 and PD901 in ovarian cancer.
Section snippets
Cell lines
Individuality of ovarian cell lines listed in Supplementary Table S1 was routinely confirmed by a polymerase chain reaction (PCR) based short tandem repeat (STR) analysis using six STR loci.
Therapeutics
2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d] pyrimidin-7(8H)-one (PF-04691502) [14] and N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4iodophenyl)amino]-benzamide (PD-0325901) [17], [18] were obtained from Pfizer Oncology.
Cell proliferation assay
Cells were drug treated
PF502 and PD901 inhibit ovarian cancer cell proliferation
The PF502 concentration, that inhibited proliferation by 50% (GI50) ranged from 16 to 640 nM (Fig. 1A) whereas response to PD901 showed a bimodal pattern, with a subset of cells that were highly sensitive (GI50’s 3–300 nM: Fig. 1B). All cell lines showed sensitivity to at least one of the two agents.
PF502 and PD901 induces cell death
PF502 induced cell death in all cell lines and significantly correlated (Spearman correlation test r = −0.66, p < 0.0001) with the drugs ability to inhibit cell proliferation (Fig. 1C). In contrast, in
Discussion
Both the PI3K/mTOR and RAS/ERK pathways are highly dysregulated through gene amplifications, gene deletions [2] and mutations in all histotypes of ovarian cancer [5]. In this study we analysed the response of an extensive panel of 32 ovarian cancer cell lines to specific inhibitors of PI3K/mTOR (PF502) and RAS/ERK (PD901) signalling which are currently in clinical trials. The majority of cells showed growth inhibition in response to PF502 whilst there was a clear division between PD901
Conflict of interest statement
J. Christensen is an employee and shareholder, Pfizer. Pfizer supplied the two therapeutics PF502 and PD901.
Acknowledgements
The authors thank Jeannette Schreuders, Susan Jackson and Ekaterina Bogatyreva for technical assistance. This work was supported by grants from the NHMRC of Australia to KES and RBP (#I043884), RWJ (#251608 and #566702), GAM (#400120 and#566876), RDH (#166908 and #251688), WAP (#628620), RBP (#509087 and #400116) and from Pfizer Oncology. Researchers were funded by NHMRC (Research Fellowships to RWJ, RDH and RBP) and Cancer Council of Victoria (Sir Edward Weary Dunlop Fellowship to GAM).
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