Elsevier

European Journal of Cancer

Volume 49, Issue 18, December 2013, Pages 3936-3944
European Journal of Cancer

Synergistic inhibition of ovarian cancer cell growth by combining selective PI3K/mTOR and RAS/ERK pathway inhibitors

https://doi.org/10.1016/j.ejca.2013.08.007Get rights and content

Abstract

Background

Ovarian cancer is the major cause of death from gynaecological malignancy with a 5 year survival of only ∼30% due to resistance to platinum and paclitaxel-based first line therapy. Dysregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and RAS/extracellular signal-regulated kinase (ERK) pathways is common in ovarian cancer, providing potential new targets for 2nd line therapy.

Methods

We determined the inhibition of proliferation of an extensive panel of ovarian cancer cell lines, encompassing all the major histotypes, by the dual PI3K/mTOR inhibitor PF-04691502 and a MEK inhibitor, PD-0325901. In addition, we analysed global gene expression, mutation status of key PI3K/mTOR and RAS/ERK pathway members and pathway activation to identify predictors of drug response.

Results

PF-04691502 inhibits proliferation of the majority of cell lines with potencies that correlate with the extent of pathway inhibition. Resistant cell lines were characterised by activation of the RAS/ERK pathway as indicated by differential gene expression profiles and pathway activity analysis. PD-0325901 suppressed growth of a subset of cell lines that were characterised by high basal RAS/ERK signalling. Strikingly, using PF-04691502 and PD-0325901 in combination resulted in synergistic growth inhibition in 5/6 of PF-04691502 resistant cell lines and two cell lines resistant to both single agents showed robust synergistic growth arrest. Xenograft studies confirm the utility of combination therapy to synergistically inhibit tumour growth of PF-04691502-resistant tumours in vivo.

Conclusions

These studies identify dual targeted inhibitors of PI3K/mTOR in combination with inhibitors of RAS/ERK signalling as a potentially effective new approach to treating ovarian cancer.

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).

References (40)

  • I. Hers et al.

    Akt signalling in health and disease

    Cell Signal

    (2011)
  • S. Wullschleger et al.

    TOR signaling in growth and metabolism

    Cell

    (2006)
  • O.J. Shah et al.

    Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies

    Curr Biol

    (2004)
  • R. Siegel et al.

    Cancer statistics, 2012

    CA Cancer J Clin

    (2012)
  • Integrated genomic analyses of ovarian carcinoma

    Nature

    (2011)
  • G. Singer et al.

    Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma

    J Natl Cancer Inst

    (2003)
  • V. Auner et al.

    KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

    BMC Cancer

    (2009)
  • R.C. Bast et al.

    The biology of ovarian cancer: new opportunities for translation

    Nat Rev Cancer

    (2009)
  • K.M. Hannan et al.

    MTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF

    Mol Cell Biol

    (2003)
  • J. Downward et al.

    Signalling pathways in cancer therapy

    Nat Rev Cancer

    (2003)
  • B.B. Friday et al.

    Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy

    Clin Cancer Res

    (2008)
  • M.S. Chapman et al.

    Novel mitogen-activated protein kinase kinase inhibitors

    Expert Opin Investig Drugs

    (2011)
  • K.E. Sheppard et al.

    Targeting PI3Kinase/AKT/mTOR signaling in cancer

    Crit Rev Oncog

    (2012)
  • H. Cheng et al.

    Discovery of the highly potent PI3K/mTOR dual inhibitor PF-04691502 through structure based drug design

    Med Chem Commun

    (2010)
  • ...
  • P.M. LoRusso et al.

    Phase I pharmacokinetic and pharmacodynamic study of the oral MAPK/ERK kinase inhibitor PD-0325901 in patients with advanced cancers

    Clin Cancer Res

    (2010)
  • E.M. Wallace et al.

    Progress towards therapeutic small molecule MEK inhibitors for use in cancer therapy

    Curr Top Med Chem

    (2005)
  • A.P. Brown et al.

    Pharmacodynamic and toxicokinetic evaluation of the novel MEK inhibitor, PD0325901, in the rat following oral and intravenous administration

    Cancer Chemother Pharmacol

    (2007)
  • T.C. Chou

    Drug combination studies and their synergy quantification using the Chou-Talalay method

    Cancer Res

    (2010)
  • G.K. Smyth et al.

    Use of within-array replicate spots for assessing differential expression in microarray experiments

    Bioinformatics

    (2005)
  • Cited by (68)

    • A BAG's life: Every connection matters in cancer

      2020, Pharmacology and Therapeutics
      Citation Excerpt :

      Moreover, learning from currently available data, BAG1 targeting could potentially be beneficial in additional malignancies, such as ovarian cancer, in which resistance to the first line therapy with platinum and paclitaxel is a serious obstacle to a successful cure. In fact, in resistant cancer cells, the deregulation of PI3K/AKT/mTOR and MAPK signaling are common events; thus, BAG1 involvement might be expected (Sheppard et al., 2013). In colorectal cancer cells in which the Rb protein promotes tumor development and determines cells' sensitivity to toxic insults, BAG1 was found to be a key executor for the regulation of DNA damage-induced apoptosis.

    • Role of the PI3K/AKT/mTOR signaling pathway in ovarian cancer: Biological and therapeutic significance

      2019, Seminars in Cancer Biology
      Citation Excerpt :

      In another study, Santiskulvong and co-workers [118] demonstrated that OC cell lines with PI3K activating mutations or PTEN deletions (SKOV-3, A2780, MOVCAR18, OAW42, and IGROV1) are more sensitive towards dactolisib than OC cells lacking PI3K mutations or PTEN deletions (OVCAR5, HEYC2, OV207, and OV167). Sheppard et al. [119] evaluated growth inhibitory effects of PF-04691502, a dual pan-PI3K, and mTORC1/mTORC2 inhibitor, in 30 OC cell lines. Results of the study revealed that majority of the OC cell lines tested were sensitive to PF-04691502.

    • Low-dose triple drug combination targeting the PI3K/AKT/mTOR pathway and the MAPK pathway is an effective approach in ovarian clear cell carcinoma

      2019, Cancer Letters
      Citation Excerpt :

      Considering that single-targeted kinase inhibition results in kinome rewiring and resistance in various tumor models, more cancers are expected to benefit from combined inhibition of mTORC1/2, PI3K and MEK1/2 [37,38]. Synergistic combinations of PI3K-mTORC1/2 inhibition and MEK1/2 inhibition have indeed been successfully used preclinically in multiple cancer types (serous and mucinous ovarian cancer, endometrial cancer and melanoma) with PI3K/AKT/mTOR pathway and MAPK pathway alterations [39–42]. Moreover, single long-dose treatment might result in more resistant clonal tumor cells [43,44], which can be prevented by combination treatment.

    View all citing articles on Scopus
    View full text