Down-Regulationof Phosphatidylinositol 3 ¶-Kinase / AKT / Molecular Target of Rapamycin Metabolic Pathway by Primary Letrozole-Based Therapy in Human Breast Cancer

Purpose:The phosphatidylinositol 3¶-kinase (PI3K)/AKT/molecular target of rapamycin (mTOR) pathway is involved in the development of tumor resistance to endocrine therapy in breast cancer cell lines and represents an attractive target for pharmacologic intervention. However, the effects of endocrine therapy with aromatase inhibitors on in vivo expression of this signaling cascade, and its relation to tumor response and patient outcome, is unknown. Experimental Design: PI3K, phospho-AKT (pAKT) and phospho-mTOR were assessed by immunohistochemistry on tumor specimens collected at baseline and after 6months of treatment in113 elderly breast cancer patients consecutively enrolled in a randomizedphase II trial of primary letrozole therapy and letrozole associated with metronomic cyclophosphamide. Results: Basal expression of the pathway was not significantly correlated with response or patient outcome. Both letrozole alone and letrozole with cyclophosphamide resulted in a significant reduction of PI3K expression (P = 0.02 and P < 0.005, respectively) and phosphomTOR expression (P = 0.0001and P = 0.0001, respectively). pAKT showed no change in the letrozole arm, whereas it was significantly decreased in the letrozole plus cyclophosphamide arm (P < 0.005). pAKT expression reduction was associated with a greater response rate (P = 0.05) and greater reduction in Ki67 expression (P = 0.05). Phospho-mTOR expression reduction was associated with a significantly longer disease-free survival in a multivariate analysis (P = 0.02). Conclusions: Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. Changes in these molecules may have prognostic significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor with drugs targeting the PI3K/AKT/mTOR signaling pathway. Endocrine therapies that interfere with estrogen receptor (ER) function have contributed to a dramatic reduction in breast cancer mortality (1). To date, aromatase inhibitors have been shown to be the most effective endocrine treatment in postmenopausal women with ER-positive breast cancer (2, 3). The results obtained with the third-generation aromatase inhibitor letrozole showed an improvement in patient outcome compared with tamoxifen. This benefit translates into diseasefree survival (DFS) improvement for adjuvant treatment (4) and overall survival in patients with metastatic disease (5). However, not all ER-positive breast cancers respond to endocrine manipulation and many initially responding tumors develop resistance. Research into the mechanisms of this endocrine resistance has revealed that cross-talk between estrogen/ER and growth factor signaling through nongenomic interactions enable breast cancer cells to bypass endocrine responsiveness (6). The growth factor family of epidermal growth factor (EGFR and HER2) are recognized to be implicated in such endocrine resistance (7, 8) through activation of mitogen-activated Imaging, Diagnosis, Prognosis Authors’Affiliations: Molecular Oncology Laboratories,Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; Unita' di Patologia Mammaria-Breast Cancer Unit, Centro di Medicina Molecolare ad Indirizzo Oncologico, Anatomia Patologica Azienda Istituti Ospitalieri di Cremona, Cremona, Italy ; Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Australia; and Oncologia Medica, Dipartimento di Scienze Cliniche e Biologiche, Universita' di Torino Azienda Ospedaliera San Luigi di Orbassano, Orbassano, Italy Received 5/1/07; revised10/22/07; accepted12/28/07. Grant support: Victorian Breast Cancer Research Consortium, Australia, the Breast Cancer Campaign, UK and the Leukaemia Research Fund, UK and by the ‘‘Associazione Patologia Oncologica Mammaria’’, Cremona, Italy, by the association of ‘‘Amici dell’ospedale di Cremona’’, by a grant from the Consiglio Nazionale Ricerche, Rome, Italy. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Alfredo Berruti, Oncologia Medica, Dipartimento di Scienze Cliniche e Biologiche, Universita' di Torino, Azienda Ospedaliera San Luigi di Orbassano, Regione Gonzole 10, Orbassano 10043, Italy. Phone: 390119026512; Fax: 39-0119026882; E-mail: alfredo.berruti@gmail.com. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1046 www.aacrjournals. org Clin Cancer Res 2008;14(9)May1, 2008 2673 Cancer Research. on October 15, 2017. © 2008 American Association for clincancerres.aacrjournals. org Downloaded from protein kinase/extracellular signal-related kinase (6, 9, 10) and/ or the phosphatidylinositol 3¶-kinase (PI3K)/AKT/molecular target of rapamycin (mTOR) pathway. PI3K is a lipid kinase which activates AKT through PDK1. AKT (or protein kinase B) is a serine/threonine kinase that promotes cell survival through the regulation of various downstream effectors, including antiapoptotic pathways. mTOR is a downstream effector of the PI3K/AKT signaling pathway that activates p70S6 kinase and 4E-binding protein-1, which mediate the response of cancer cells to external stimuli and promote cell proliferation, regulating transition through the G1-S phase of the cell cycle (6, 8). In vitro studies have shown that after long-term estrogen deprivation, i.e., during long-term aromatase inhibitor administration, breast tumor cells exhibit an activation of the PI3K/ AKT/mTOR pathway as an adaptive phenomenon of breast cancer cells to the low estrogen environment (11). Two studies in human breast cancer patients have shown that AKT activation is a negative predictor of response to hormone therapy in metastatic breast cancer patients (12) and short survival in nonmetastatic breast cancer patients treated with adjuvant tamoxifen (13). The PI3K/AKT/mTOR pathway, which is often up-regulated in breast cancer (14), therefore represents an attractive target for pharmacologic intervention with signal transduction inhibitors. Approaches to targeting these cell survival pathways have included either specific PI3K inhibitors, such as LY294002, or rapamycin analogues targeting mTOR, such as temsirolimus (CCI-779) or everolimus (RAD001), both of which are currently under investigation (15, 16). In vitro studies have shown that breast cancer cells might be particularly sensitive to mTOR inhibitors. In MCF-7 (wt) and MCF-7/Aro (transfected with aromatase gene) breast cancer cell lines, the combination of letrozole and everolimus showed a synergistic interaction in producing a maximal growth inhibition (15). These data suggest that a combined approach may prevent or delay the development of endocrine resistance and could significantly improve the therapeutic efficacy of currently available endocrine options. The efficacy of the combination of aromatase inhibitors with everolimus is currently being tested in several ongoing randomized trials in patients with endocrine-responsive breast cancer (17). However, theoretical reasons suggest a negative interaction between rapamycin analogues and aromatase inhibitors. If the cross-talk between the estrogen receptors and growth factor signal transduction is highly dependent on ligands, then estrogen deprivation may theoretically down-regulate the PI3K/AKT/mTOR pathway. In addition, estrogen deprivation causes the inhibition of insulin-like growth factor-I synthesis (18), another growth factor involved in the stimulation of the PI3K/AKT/mTOR pathway (19). To our knowledge, no studies have addressed this issue in human breast cancers in vivo . To investigate the effect of aromatase inhibitors on the PI3K/AKT/mTOR pathway, we have used a primary systemic therapy approach that has proved be an invaluable method for exploring any effect of particular antineoplastic drugs on tumor biology. We have conducted a randomized phase II trial based on letrozole (LET arm) with or without ‘‘metronomic’’ oral cyclophosphamide. In this trial, the combination of letrozole with cyclophosphamide (LET-CYC arm) resulted in greater tumor shrinkage and greater reduction in either proliferative activity and vascular endothelial growth factor expression compared with letrozole alone (20). In the present study, PI3K, AKT, and mTOR were assessed on tumor specimens collected before and after treatment in patients randomized in this trial. The primary aim was to explore the changes of these molecular targets before and after treatment, secondary aims were to evaluate the relationship between these targets and conventional clinical and biological prognostic variables and to correlate the changes of these targets with clinical response and patient outcome.