- Email: [email protected]
Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition?
Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Amna Sheri,1 Lesley-Ann Martin,2 Stephen Johnston1 Abstract The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a critical intracellular signaling cascade that mediates both growth factor–induced r proliferation and cell survival with deregulated signaling through this pathway, a feature of most cancers. Here, we review the role of the PI3K/Akt/mTOR pathway in endocrine-resistant breast cancer and discuss preclinical and clinical studies combining endocrine therapy with mTOR inhibition. Key to the success of such an approach will be a clinical trial design incorporating appropriate tumor selection and validation of biomarkers predicting benefit. Ultimately, a greater understanding of the biology and compensatory mechanisms will allow the partnering of key signal transduction inhibitors together to provide maximal “vertical” or “horizontal” blockade with further preclinical and clinical studies planned to examine possible synergistic combinations with endocrine therapy. Clinical Breast Cancer, Vol. 10, Suppl. 3, S79-S85, 2010; DOI: 10.3816/CBC.2010.s.016 Keywords: Estrogen receptor, Mammalian target of rapamycin, Metastatic breast cancer, PI3K
Introduction
The PI3K/Akt/mTOR Pathway
Endocrine resistance, whether de novo or acquired, remains a key challenge in the treatment of hormone positive breast cancer. Mechanisms for resistance are varied with evidence existing for genomic and non-genomic cross talk between estrogen receptor (ER) and key intracellular signaling pathways.1 A number of growth factor receptor signaling pathways have been implicated in resistance including EGFR/HER1, HER2/HER3 signaling, and insulin-like growth factor 1 signaling acting both through MAPK (mitogen activated protein kinases) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways.2-6 Pharmacological targeting of these pathways potentially provides a mechanism to reverse or block acquired resistance to endocrine therapy.7 The PI3K/Akt/mTOR pathway is a critical intracellular signaling cascade that mediates both growth factor induced proliferation and cell survival with deregulated signaling through this pathway a feature of many cancers with a number of inhibitors of this pathway in preclinical and clinical development.8,9 This article reviews the molecular biology of this pathway, the preclinical and clinical evidence for the use of mTOR inhibitors in improving endocrine responsiveness in breast cancer, along with potential limitations and challenges in taking this approach forward in the clinical setting.
The PI3K/Akt/mTOR pathway is a critical intracellular signaling pathway that is implicated in a number of normal cellular processes involved in proliferation, metabolism, growth, and cell survival. The central component of the pathway is the PI3K heterodimer which comprises the p85 regulatory and p110 catalytic subunit. The pathway is activated following membrane growth factor receptor tyrosine kinase activation, and subsequent phosphorylation of the receptor results in interaction with PI3K either directly or indirectly through adaptor proteins such as insulin related substrate-1 (IRS-1). In addition PI3K can be activated directly by the GTPase protein Ras. Activation of PI3K leads to removal of the inhibitory effect of the regulator unit p85, with subsequent activation of the p110 catalytic subunit which in turn converts membrane related phosphatidylinositol 3, 5-bisphosphonate (PIP2) to the triphosphate form PIP3 (Figure 1). In turn, PIP3 results in phosphorylation of Akt, a serine-threonine kinase which is the major effector of the pathway. Subsequent Akt signaling leads to increased intracellular growth and survival which is characterized by the malignant phenotype.10,11 One important downstream consequence of PI3K/Akt activation is the alleviation of the suppression by the tuberous sclerosis protein complex (TSC1/2) of the mammalian target of rapamycin (mTOR; Figure 1).12 There are at least 2 groups of mTOR proteins, and the key protein involved in transmitting signals from PI3K-Akt is the mTORC1 complex. Activation of Akt inhibits mTORC1’s negative regulator TSC1/2, which then releases its specific inhibition of mTORC1. mTORC1 plays a critical role in the transduction of proliferative signals through phosphorylation of the translational regulator 4E-BP1 (eukaryotic initiation
1Breast
Unit, Royal Marsden Hospital, London Breast Cancer Centre, Institute of Cancer Research, London
2Breakthrough
Submitted: Aug 12, 2010; Revised: Sep 23, 2010; Accepted: Sep 24, 2010 Address for correspondence: Amna Sheri, MD, Royal Marsden Hospital, 123 Old Brompton Road, London, United Kingdom SW7 3RP Fax: 44-207-8082563; email: [email protected]
All of the authors report that they have no relevant relationships to disclose. Supplementary material from this article in the form of slides is available online at cigjournals.com.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Figure 1 Schematic Representation of the PI3K/Akt/mTOR Signaling Pathway Demonstrating the Action of Rapamycin and its Derivatives, Temsirolimus and Everolimus, Which Inhibit mTORC1
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Abbreviations: 4EBPs = 4 eukaryotic binding protein; EIF-4B = eukaryotic initiation factor 4B; EIF-4E = eukaryotic initiation factor 4E; EIF-4G = eukaryotic initiation factor 4G; GβL = G protein beta subunit like protein; IRS1 = insulin receptor substrate 1; PDK1 = pyruvate dehydrogenase kinase isoenzyme 1; PI3K = phosphatidylinositol 3-kinase; PIP2 = phosphatidylinositol 3, 5-bisphosphonate 2; PIP3 = phosphatidylinosotol 3, 5-bisphosphonate 3; PTEN = phosphatase and tensin homolog; mTORC1 = mammalian target of rapamycin complex 1; mTORC2 = mammalian target of rapamycin complex 2; S6 = ribosomal protein S6; TORC1 = TOR complex 1; TORC2 = TOR complex 2; TSC1 = tuberous sclerosis complex 1; TSC2 = tuberous sclerosis complex 2
factor 4E-binding protein), and the ribosomal protein p70s6kk (the 70-kDa S6 kinase).13,14 Activation of 4E-BP1 by mTOR releases the associated protein EIF4E which is closely bound together in quiescent cells, allowing interaction with other proteins to form a multi-subunit EIF4F complex that facilitates translation of mRNAs into proteins. Likewise, activation of the serine-threonine kinase p70 S6K phosphorylates the 40S ribosomal protein S6, which leads to recruitment of the 40s ribosomal subunit into actively translating polysomes that enhance translation of mRNAs. Inhibition of mTOR with rapamycin directly prevents protein translation via these 2 regulatory proteins Deregulated PI3K/Akt/mTOR signaling is a feature of many cancers.15 The pathway is activated by receptor tyrosine kinases as described above, and by the genetic mutation and amplification of key pathway components.16 Several genetic abnormalities are known to activate PI3K/Akt/mTOR signaling. Genetic alterations in the tumor suppressor gene PTEN, N an endogenous inhibitor of the PI3K/Akt/mTOR pathway, are among the most frequently noted somatic mutations in cancer. The protein encoded by PTEN is a phosphatase which serves to remove phosphate groups from key intracellular phosphoinositide signaling molecules.17 Mutation or epigenetic modification of PTEN N leads to accumulation of PIP3
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with deregulation signaling though the Akt/TSC1-2/mTORC1 pathway described above. Furthermore, approximately 15%-35% of breast cancers demonstrate reduced expression of PTEN, N 18 and PTEN N loss has also been associated with poor prognosis in patients with ER positive breast cancer treated with tamoxifen.19 Mutations in the catalytic domain of PI3K have also been identified in around 20%-25% of breast cancers20,21 and have been implicated in breast tumor initiation.22 Most mutations arise in either the kinase or helical domain and have been associated with breast cancers that are hormone receptor positive or over express HER2.18 An understanding of the role of the PI3K/Akt/MTOR pathway in cancer cell growth and survival has been exploited in the development of a number of inhibitors of the pathway as anti-cancer therapies. To date, the most clinically developed agents in this group for the treatment of breast cancer are the mTOR inhibitors with Akt, PI3K and dual mTOR/PI3K inhibitors also in development.
The original inhibitor of mTOR is rapamycin, a lipophilic macrolide identified in the 1970s during antibiotic screening off Streptomyces hygroscopicus found in the soil of Easter Island. It was found to be a potent fungicide with derivatives displaying potent immunosuppressive properties. Subsequent work showed that these drugs had antiproliferative effects in a range of experimental tumors,23 with an ability to block protein translation and halt G1-S cell cycle progression. Despite encouraging preclinical data, rapamycins poor solubility and chemical instability limited clinical development. Rapamycin analogs with a more favorable pharmacological profile were synthesized including CCI-779 (temsirolimus), RAD-001 (everolimus), and AP23573 (ridaforolimus). In particular, the breast cancer cell lines BT-474, SK BR-3, and MCF-7 demonstrated specific sensitivity to temsirolimus with IC50 values ranging from 0.0006 to 0.001μM.24 In each of these cell lines, the PI3K-Akt pathway appears to be constitutively overactive because of either ErbB2, ER activation or PTEN deletions, whereas breast cancer cells resistant to temsirolimus lacked any of these features. Everolimus is an oral hydroxyethyl ether of rapamycin which demonstrated impressive antiproliferative activity against a wide variety of tumor models in vivo.25 Both temsirolimus and everolimus have been used in combination with endocrine therapy both in preclinical models of endocrine-resistant breast cancer and in the clinical setting as outlined below.
Role of the PI3K/Akt/mTOR Pathway in Endocrine Resistance of Breast Cancer It is now clear that the estrogen receptor (ER) can become involved with the PI3K/Akt/mTOR pathway in breast cancer cells,26 with both genomic and non-genomic cross talk between this signaling pathway and ER.1 Because of its role in cell survival, there is evidence that the pathway becomes activated in acquired hormone-resistant breast cancer, and accounts for survival of cells despite the presence of continued endocrine blockade.27,28 As a serine/threonine kinase, Akt promotes cell survival in response to many different external local growth factors, including insulin, insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF),
Amna Sheri et al EGF, heregulin, and VEGF. Akt activates downstream effectors involved in cell survival, including antiapoptotic pathways through phosphorylation of substrates that directly regulate the apoptotic machinery (ie, BAD, caspase-9). Breast cancer cell lines with activated Akt (eg, via loss of the regulatory PTEN N tumor suppressor gene) have been shown to be especially sensitive to mTOR antagonism.24 Growth factor mediated activation of MAPK or Akt can potentiate E2 mediated ER classic transcriptional activity by directly phosphorylating ER. Importantly, both MAPK and Akt have been shown to phosphorylate ER within AF-1, at serine 118 and serine 167 respectively, in the absence of E2, thereby contributing to ligand-independent ER transactivation.4,29,30 Conversely, in addition to its effects on estrogen-regulated gene transcription, ligand bound ER has non-genomic effects via membrane interaction with growth factor receptors.31,32 This may result in rapid activation of EGFR,31 IGF1R,32 HER233 or the cleavage of membrane-bound growth factor receptor ligands such as EGF or TGF-α.34 Other data supports a role of PI3K/Akt pathway in the development of endocrine resistance, as IGF-1 has been shown to down regulate the progesterone receptor via a transcriptional mechanism that involves the PI3K/Akt pathway that is independent of ER.35 These bidirectional interactions between hormonal and growth factor pathways create self-reinforcing synergistic loops that potentiate pro-survival signals, and may allow breast cancer cells to escape normal endocrine responsiveness. Recent laboratory data have confirmed a role for enhanced growth factor activation and increased Akt phosphorylation in ER+ breast cancer cells that develop acquired resistance to long term estrogen deprivation5 or tamoxifen.36 In particular, tamoxifenresistant (TamR-1) cells expressed elevated levels of phosphorylated Akt and ERK1/2 compared with the parental MCF-7 cells, with increased phosphorylation of ER at Ser167. Colocalization studies revealed an association between HER2 and ER in the TamR1 cells, but not the parental MCF-7 cells. In addition, ER was redistributed to extranuclear sites in the TamR-1 cells and was less transcriptionally active. Tamoxifen resistance could be overcome by the combined use of a HER2 tyrosine kinase inhibitor (TKI) and tamoxifen, which was associated with redistribution of ER to the nucleus and loss of phosphorylated Akt. Therefore, these data support the concept of ER cross-talk with the PI3K/Akt pathway in endocrine resistance, and provide a rationale for combining signaling therapies against this pathway with continued endocrine therapy in an attempt to treat or overcome resistance. Preclinical models of ER+ hormone sensitive and resistant breast cancer have been used to examine the effects of combining mTOR antagonists with endocrine therapy.37,38 Boulay et al demonstrated that the estrogen-dependent growth of both wild-type MCF-7 and aromatase-expressing (MCF-7/Aro) breast cancer cells could be inhibited in a dose-dependent manner by the mTOR antagonist everolimus (RAD-001), suggesting that mTOR signaling is required for the estrogen-dependent proliferation of these cells. In subsequent experiments with the MCF-7/Aro cells, both the aromatase inhibitor letrozole and the mTOR inhibitor everolimus inhibited androstenedione-induced cell proliferation. However, the combination of letrozole and everolimus produced maximal growth inhibition, with clear evidence for additive/synergistic effects.37
In particular, increased activity of the letrozole-everolimus combination correlated with a greater effect on G1 progression, and a significant decrease in cell viability and apoptosis. As indicated above, evidence has emerged from several groups that activation of Akt/PKB and the downstream mTOR pathway can cause resistance to tamoxifen. In particular, deGraffenried et al demonstrated that MCF-7 cells expressing a constitutively active Akt were able to proliferate under reduced estrogen conditions, and were resistant to the growth inhibitory effects of tamoxifen, both in vitro and in vivo in xenograft models.38 However, co-treatment with temsirolimus inhibited mTOR activity and restored sensitivity to tamoxifen, primarily through induction of apoptosis, thus suggesting that Akt-induced tamoxifen resistance may, in part, be mediated by signaling through the mTOR pathway. Though the mechanism for the synergy is unclear, estrogen response element (ERE) reporter gene assays showed that mTOR inhibition managed to block ER-induced gene activation. Therefore, these laboratory data support the role of the PI3K/ mTOR/Akt pathway in endocrine resistance in ER positive breast cancer and provide evidence for cross-talk between ER and various components of the signaling cascade. Targeting a downstream element of the pathway such as mTOR has been shown to restore endocrine sensitivity in both cell lines and xenograft models and thus provides a rationale for combining endocrine therapy with mTOR inhibition in the clinical setting.
Clinical Trials in Metastatic Breast Cancer With mTOR Antagonists The first mTOR inhibitor to be developed for use in breast cancer was CCI-779 (temsirolimus), with early phase I studies using the traditional approach of studying the maximum tolerated dose (MTD). Because of the concern that continuous administration may lead to immunosuppression intermittent dose scheduling was selected for clinical development. In a phase I study of 24 patients with advanced solid tumors, temsirolimus was administered over a wide range off doses (7.5-220 mg/m2) once weekly as a 30 minute infusion.39 The most frequent drug related adverse events were asthenia and thrombocytopenia (grade 3 or 4 in 8% of patients), followed by dermatological toxicities and mucositis (grade 3 or 4 in 4% of patients). Elevations in total cholesterol and triglycerides were also reported. Two partial responses (PRs) were observed, 1 in a patient with renal cancer and 1 in a patient with metastatic breast cancer. Similar toxicities were also observed in a phase I study of the oral mTOR inhibitor RAD001 (everolimus)40 although 1 patient also developed grade 3 pneumonitis and another patients reported grade 3 pneumonia. Based on early encouraging phase I data that included efficacy in breast cancer, a multi center phase II trial of 2 doses of temsirolimus (75 mg/m2 or 250 mg/m2 I.V. weekly) was conducted in 109 patients with heavily pretreated patients with locally advanced or metastatic breast cancer.41 The objective response rate to temsirolimus was 9.2% (10 PR) with median time to tumor progression of 12 weeks. Efficacy was similar between the 2 dose levels, but toxicity was more common at the higher dose level especially grade 3 or 4 depression. Once again, the most common adverse events were mucositis and rash with thrombocytopenia, hyperglycemia, and hypercholesterolemia also observed.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Table 1 Results of Clinical Trials of mTOR Inhibitors in Combination With Aromatase Inhibitors Study
Trial Design
Drug Dose and Schedule
Number of Patients
Results Letrozole ORR, 45% median PFS, 11.6 months
Baslega et al 200543
Randomized, 3-arm, phase II
Letrozole 2.5 mg daily orally or letrozole + temsirolimus intermittent schedule I.V. (30 mg daily for 5 days every 2 weeks) or letrozole + temsirolimus 10 mg I.V. daily.
92
Letrozole/Temsirolimus 10 mg daily ORR, 33% median PFS, 11.5 months Letrozole/Temsirolimus 30 mg intermittent ORR, 40% median PFS, 13.2 months
Chow et al 200645
Awada et al 200742
Randomized, double-blind, placebo-controlled, phase III
Phase Ib, open-label, dose escalation
Neoadjuvant, Baselga et al randomized, placebo52 2009 controlled, phase II
Letrozole 2.5 mg orally daily + placebo (daily for 5 days every 2 weeks) or letrozole + temsirolimus 30 mg orally intermittent (daily for 5 days every 2 weeks)
Letrozole 2.5 mg orally daily + everolimus orally daily (5 or 10 mg)
Letrozole 2.5 mg orally daily + placebo orally daily for 4 months pre-surgery, or letrozole 2.5 mg orally daily + everolimus 10 mg orally daily for 4 months pre-surgery
Letrozole ORR, 24% CBR, 43% median PFS, 9.2 months (95% CI, 7.4-11.1 months) 992
18
Letrozole + Temsirolimus intermittent OEE, 24% CBR, 40% median PFS, 9.2 months (95% CI, 7.4-11.1 months) No PK interaction 1 CR at 10 mg 28% reduction in liver metastases in 1 patient at 10 mg 7 patients received combination therapy for > 6 months Letrozole Clinical ORR, 59% Ultrasound ORR, 47% Mammogram ORR, 39%
270 Letrozole + Everolimus Clinical ORR, 68% Ultrasound ORR, 58% Mammogram ORR, 36%
Safety Profile Most common AEs with letrozole/temsirolimus combination: asthenia (42%-60%) diarrhea (36%-43%) mucositis (43%) Grade 3/4 AEs: hyperglycemia (10 patients) asthenia (3 patients)
Grade 3/4 AEs: hyperglycemia (4%) dyspnoea (3%) neutropenia (3%) liver function abnormalities (3%)
Most common AEs: stomatitis (50%) fatigue (44%) anorexia (44%) diarrhea (33%) headache (33%) rash (33%) Most common AEs with combination therapy: stomatitis (36%) rash (20%) asthenia (17%) thrombocytopenia (18.2%) hyperglycemia (13%) Grade 3/4 AEs: stomatitis (2%) hyperglycemia (5%)
Abbreviations: AE = adverse event; CBR = clinical benefit rate; CR = complete response; I.V. = Intravenous; mTOR = mammalian target of rapamycin; ORR = objective response rate; PFS = progression-free survival; PK = pharmacokinetic
Emerging data from preclinical studies demonstrating possible synergy between mTOR antagonists and endocrine therapy provided a rationale for a number of studies combining aromatase inhibitors with mTOR antagonists.37,38 An initial phase Ib study of letrozole with the mTOR inhibitor everolimus had suggested the combination was well tolerated with very few grade 3 toxicities (Table 1).42 A randomized 3 arm, phase II study of the mTOR inhibitor temsirolimus given intravenously 10 mg daily or 30 mg intermittently (daily for 5 days every 2 weeks), was conducted in 109 patients with hormone receptor positive locally advanced or metastatic breast cancer.43 Patients were eligible if they were receiving first- or second-line endocrine therapy but were excluded if they had received a previous aromatase inhibitor. Results suggested a modest benefit to the combination with intermittent temsirolimus in terms of median progression free survival (PFS; 13.2 months vs. 11.6 months), although the objective response rate was not
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statistically different (40% vs. 45%). Notably, the combination was associated with significant toxicities not observed in the letrozole alone arm: grade 1-4 diarrhea (43%), mucositis (43%), and grade 3-4 hyperglycemia (20%). In particular the toxicity was greatest in the higher dose schedule and resulted in dose delays, dose reductions, or discontinuations, so the protocol was amended to adjust the low dose schedule. Updated efficacy results of this study showed a non-statically significant greater clinical benefit rate of 77% for the combination compared with 60% for the letrozole alone arm.44 With phase II data suggesting a possible benefit for temsirolimus and letrozole combinations, a large multi-center randomized double-blind phase III clinical trial was undertaken. Over 1200 postmenopausal women with hormone receptor positive locally advanced or metastatic breast cancer were randomized to letrozole with either temsirolimus (in an oral formulation 30 mg for 5 days every 2 weeks) or placebo.45 The aim of the study was to
Amna Sheri et al detect a 25% improvement in PFS with the combination. The oral combination was better tolerated than in the phase II study with I.V. temsirolimus that had less than 5% grade 3-4 toxicities, including only 4% hyperglycemia. Unfortunately, the trial was terminated early after an interim analysis demonstrated a lack of benefit for the combination. Results among the first 992 patients showed no diff ference in objective response rates (RR) or PFS.
Limitations to mTOR inhibition Given the preclinical rationale and encouraging phase II data, the negative results from this large randomized phase III trial were disappointing (Table 1). Several explanations for the lack of benefit for the combination have been put forward. At a mechanistic molecular level, 2 key regulatory loops have been described that may limit the effectiveness of mTOR inhibitors in the treatment of cancer. An important negative feedback loop exists downstream in the PI3K/ Akt/mTOR pathway. The mTOR-activated kinase S6K1 phosphorylates and destabilizes the IRS1 and IRS2 proteins in insulin like growth factor (IGF) responsive cells.46 In these cells, mTOR inhibition leads to a reduction in S6K1 activity, which allows IRS1/2 expression to be increased with associated enhanced activation of IGFR-1 dependent Akt activity.47,48 This is supported by the clinical observation that phosphorylated Akt is up regulated in both tumor and skin biopsies of patients treated with everolimus49 and as such, this loss of negative feedback may overcome the anti-tumor effectiveness of the mTOR blockade. Secondly, a positive regulatory loop exists involving the mTORC2 complex which can be activated more directly by growth factors and phosphorylate Akt. Therefore, the inability of rapamycin derivatives to block mTORC2 could result in increased Akt signaling that result in ER phosphorylation on serine 167, negating the effect of aromatase inhibition, and thus, limiting the therapeutic benefit of this combination.50,51 Alternatively, the lack of clinical benefit could, in part, reflect a failure to select patients appropriately whose tumors demonstrated dependence on the PI3K/Akt/mTOR pathway. Tissue sampling at time of progression to determine tumor phenotype (ie, phosphorylated Akt or loss of PTEN may be surrogates for activated pathways in theses tumors) would have proved challenging in a large trial.
Biomarkers and Neoadjuvant Studies Neoadjuvant studies, though not necessarily reflecting the adaptive changes over time that may occur during the course of metastatic disease, may provide predictive biomarker data that can help select appropriate patients for future clinical trials. A recent randomized phase II study in 270 postmenopausal women with ER+ primary operable breast cancer has evaluated the benefit of adding everolimus (10 mg/day) or placebo to neoadjuvant letrozole. (2.5 mg/day).52 The primary endpoint of the study was clinical response by palpation with the addition of everolimus to letrozole for 16 weeks, preoperatively resulting in significantly greater RRs as judged by both clinical (68.1% vs. 59.1%) and radiologic assessments. Core biopsies were obtained at baseline and at day 15. A significantly greater reduction in cell proliff eration measured by change in Ki67 was seen in the letrozole/
everolimus combination arm compared with letrozole. Associative correlative studies were also conducted to determine those tumors most likely to respond to combined mTOR antagonism and aromatase inhibition. At day 15, down regulations in progesterone receptor and cyclin D1 were seen in both treatment arms; whereas large reductions in phosphorylated ribosomal protein S6 were seen in only the everolimus arm. Interestingly, specific mutations in PIK3CA were found to be associated with a greater likelihood of an antiproliferative response to the combination of letrozole plus everolimus. In particular mutations in the allosteric helical domain of exon 9 were associated with a poor antiproliferative response to letrozole alone, but a good response to letrozole plus everolimus. This particular, PIK3CA mutation has been associated with a worse prognosis in breast cancer,53 and a greater likelihood of response to the combination implicates the PI3K/ Akt/mTOR pathway in endocrine resistance. Studies on the effect of PIK3CA mutations, however, have been mixed. A recent analysis of samples from 4 neoadjuvant trials treated with endocrine therapy alone in the neoadjuvant setting demonstrated only a weak negative interaction between PIK3CA mutation status and clinical response to neoadjuvant endocrine treatment and no interaction with changes in Ki-67 based proliferation index.54 In a subset analysis of the effect of PIK3CA mutations and relapse-free survival using samples from the P024 trial (previously used to generate a well-validated preoperative prognostic index: PEPI)55 there were insufficient samples containing the exon 9 mutation to draw conclusions. It’s also possible that the impact of any PI3K mutation may depend on coexpression of other factors such as ER or HER2. A recent study reported that mutations in PI3KCA exon 20 kinase domain were actually associated with better clinical outcomes in ER+/HER2– breast cancer patients but not in ER R– or HER2+ 56 subtypes. These findings may have implications for the future development of PI3K/mTOR inhibitors in breast cancer.
Future Directions The PI3K/Akt/mTOR pathway has an established key role in cell growth and survival with preclinical evidence for cross-talk between this pathway and ER. Clinical benefits from co- targeting these pathways however have been modest to date and key challenges for future research are identifying who may benefit from targeting this particular combination of pathways and understanding how best to overcome limitations to this approach. In order to do this, it is essential to consider the issue of heterogeneity. Future trials will need to ensure that they are enriched with the most appropriate patients ie, those with activation of the relevant target. A key question is how best to identify patients for targeted therapy? Recent neoadjuvant studies have attempted to identify biomarkers for response to combined aromatase and mTOR blockade.52 However, sampling of tumor phenotype at the time of progression remains challenging. It may be possible to better characterize patients based on surrogate clinical markers such as previous therapy and responsiveness.57 The BOLERO-2 (Breast Cancer Trials of Oral Everolimus) study will test the combination of everolimus with steroidal aromatase inhibitor exemestane in postmenopausal women with advanced breast cancer who are refractory to letrozole or anastrozole.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Even when oncogenic addiction to a particular pathway such as PI3K/Akt/mTOR is correctly identified, it must be recognized that blockade of a single protein in a complex signaling cascade, albeit a critical downstream effector of the pathway, is unlikely to provide total or prolonged growth inhibition. As the science of regulatory feedback loops becomes clearer, biologic explanations because of compensatory escape pathways (ie, further stimulations of upstream Akt following mTOR inhibition, which overcomes the block) account for short term clinical remissions often with rebound growth at the time of disease progression. A greater understanding of this biology will allow the partnering of key signal transduction inhibitors together to provide maximal vertical or horizontal blockade. For example, dual vertical inhibition of IGF-1R signaling using either monoclonal antibodies or TKIs (which are in phase II trials for breast cancer) could be used to overcome the rapamycininduced Akt activation loop being activated and give greater efficacy than either approach alone, a concept initially tested with promising results in neuroendocrine tumors where octreotide (which targets IGF-IR signaling) was combined with everolimus. This resulted in 4 patients achieving PR and 19 patients having stable disease (SD) out of 32 treated patients.58 More recently, the mTOR inhibitor ridaforolimus has been used in combination with the IGF-1R antibody, dalotuzumab, in patients with advanced solid tumors.59 The most frequent adverse events were stomatitis, fatigue, and hyperglycemia which were mostly grade 1-2. Interestingly, there were signs of activity in 5 heavily pretreated breast cancer patients with 2 achieving PRs, 1 SD, and 2 demonstrating partial metabolic responses on positron emission tomography scan. All 5 of these patients were ER+ and 4 had high expression of Ki67. Despite certain success with the rapalogs, other agents may prove superior inhibitors of the PI3K/Akt/mTOR pathway either alone or in combination with other targeted agents with several agents in development including mTOR kinase inhibitors which target both mTORC1 and MTORC2, Akt inhibitors, PI3K, and dual mTOR/ PI3K inhibitors. Promisingly, the dual mTOR/PI3K inhibitor NVP-BEZ235 has demonstrated superior antiproliferative activity to everolimus in cell lines.60 Previous neoadjuvant studies have revealed inconclusive results about the role of PI3K mutations in endocrine resistance/responsiveness. However, it’s possible that patients with advanced disease who have previously relapsed on endocrine therapy may be more likely to have tumors with activation of the PI3K/Akt/ mTOR pathway. A phase I study of XL765, a dual inhibitor of PI3K and mTOR, is planned in combination with letrozole in patients who have progressed on previous endocrine therapy with further preclinical studies and early-phase trials planned.
Conclusion The PI3K/Akt/mTOR pathway is a validated target for anticancer therapy with both preclinical and clinical evidence pointing to the possibility of synergistic combinations with endocrine therapy. However, it’s not yet clear who might benefit most from this approach, and clinical gains to date have been modest. There is an urgent need to understand the mechanistic science of regulatory pathways in order to design biologically-rational trials to explore this concept further.
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Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation. J Biol Chem 2003; 278:30458-68. 6. Shou J, Massarweh S, Osborne CK, et al. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004; 96:926-35. 7. Johnston SR, Martin LA, Leary A, Head J, et al., Clinical strategies for rationale combinations of aromatase inhibitors with novel therapies for breast cancer. J Steroid Biochem Mol Biol 2007; 106:180-6. 8. Johnston SR. Role of the mTOR pathway in endocrine resistant breast cancer- opportunities for novel combination strategies. Presented at: the Annual Meeting of the American Society of Clinical Oncology; May 29-June 2, 2009; Orlando, FL. ASCO Educational Book 2009; 20-8. 9. van der Heijden MS, Bernards R. Inhibition of the PI3K pathway: hope we can believe in? Clin Cancer Res 2010; 16:3094-9. 10. Downward J. Mechanisms and consequences of activation of protein kinase B/ Akt. Curr Opin Cell Biol 1998; 10:262-7. 11. Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 2004; 22:2954-63. 12. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005; 37:19-24. 13. Hara K, Yonezawa K, Kozlowski MT, et al. Regulation of eIF-4E BP1 phosphorylation by mTOR. J Biol Chem 1997; 272:26457-63. 14. Chung J, Grammer TC, Lemon KP, et al. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Naturee 1994; 370:71-5. 15. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Naturee 2006; 441:424-30. 16. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer 2009; 9:550-62. 17. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998; 273:13375-8. 18. Saal LH, Holm K, Maurer M, et al. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res 2005; 65:2554-9. 19. Shoman N, Klassen S, McFadden A, et al. Reduced PTEN expression predicts relapse in patients with breast carcinoma treated by tamoxifen. Mod Pathol 2005; 18:250-9. 20. W Wu G, Xing M, Mambo E, et al. Somatic mutation and gain of copy number of PIK3CA in human breast cancer. Breast Cancer Res 2005; 7:R609-16. 21. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther 2004; 3:772-5. 22. Miron A, Varadi M, Carrasco D, et al. PIK3CA mutations in in situ and invasive breast carcinomas. Cancer Res 2010; 70:5674-8. 23. Eng CP, Sehgal SN, Vezina C. Activity of rapamycin (AY-22,989) against transplanted tumors. J Antibiot (Tokyo) 1984; 37:1231-7. 24. Y Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer 2001; 8:249-58. 25. O’Reilly R T, Vaxelaire J, Muller M. In vivo activity of RAD-001, an orally active rapamycin derivative, in experimental tumour models. Poc Am Assoc Cancer Research 2002; 43:71 (abstract 359). 26. Simoncini T, Hafezi-Moghadam A, Brazil DP, et al. Interaction of estrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000; 407:538-41. 27. Clark AS, West K, Streicher S, et al. Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther 2002; 1:707-17. 28. Pietras RJ, Arboleda J, Reese DM, et al. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 1995; 10:2435-46. 29. Kato S, Endoh H, Masuhiro Y, et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995; 270:1491-4. 30. Smith CL. Cross-talk between peptide growth factor and estrogen receptor signaling pathways. Biol Reprod 1998; 58:627-32. 31. Fan P, Wang J, Santen RJ, et al. Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells. Cancer Res 2007; 67:1352-60. 32. Kahlert S, Nuedling S, van Eickels M, et al. Estrogen receptor alpha rapidly acti-
Amna Sheri et al vates the IGF-1 receptor pathway. J Biol Chem 2000; 275:18447-53. 33. Chung YL, Sheu ML, Yang SC, et al. Resistance to tamoxifen-induced apoptosis is associated with direct interaction between Her2/neu and cell membrane estrogen receptor in breast cancer. Int J Cancer 2002; 97:306-12. 34. Knowlden JM, Hutcheson IR, Jones HE, et al. Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 2003; 144:1032-44. 35. Cui X, Zhang P, Deng W, et al. Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/ mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer. Mol Endocrinol 2003; 17:575-88. 36. Pancholi S, Lykkesfeldt AE, Hilmi C, et al. ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2. Endocr Relat Cancer 2008; 15:985-1002. 37. Boulay A, Rudloff J, Ye J, et al. Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer. Clin Cancer Res 2005; 11:5319-28. 38. deGraffenried LA, Friedrichs WE, Russell DH, et al. Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity. Clin Cancer Res 2004; 10:8059-67. 39. Raymond E, Alexandre J, Faivre S, et al. Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer. J Clin Oncol 2004; 22:2336-47. 40. O’Donnell A, Faivre S, Burris HA 3rd, et al. Phase I pharmacokinetic and pharmacodynamic study of the oral mammalian target of rapamycin inhibitor everolimus in patients with advanced solid tumors. J Clin Oncol 2008; 26:1588-95. 41. Chan S, Scheulen ME, Johnston S, et al. Phase II study of temsirolimus (CCI779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol 2005; 23:5314-22. 42. A Awada A, Cardoso F, Fontaine C, et al. The oral mTOR inhibitor RAD001 (everolimus) in combination with letrozole in patients with advanced breast cancer: results of a phase I study with pharmacokinetics. Eur J Cancer 2008; 44:84-91. 43. Baselga J, Fumoleau P, Gil M, et al. Phase II 3- arm study of temsirolimus (CCI-779) in combination with letrozole in postmenopausal women with locally advance or metastatic breast cancer: preliminary results. J Clin Oncol 2004; 22(14 suppl): (abstract 544). 44. Baselga J, Roché H, Fumoleau P, et al. Treatment of postmenopausal women with locally advanced or metastatic breast cancer with letrozole alone or in combination with temsirolimus: a randomized, 3arm phase 2 study. Breast Cancer Res Treat 2005; 94(1 suppl): (abstract 1068). 45. Chow LW, Sun Y, Jassem J, et al, Phase 3 study of temsirolimus with letrozole or letrozole alone in postmenopausal women with locally advanced or metastatic breast cancer. Breast Cancer Res Treat 2006; 100(1 suppl): (abstract 6091).
46. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancerr 2006; 6:729-34. 47. O’Reilly R KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006; 66:1500-8. 48. Sun SY, Rosenberg LM, Wang X, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005; 65:7052-8. 49. T Tabernero J, Rojo F, Calvo E, et al. Dose- and schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced solid tumors. J Clin Oncol 2008; 26:1603-10. 50. Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/ PKB by the rictor-mTOR complex. Science 2005; 307:1098-101. 51. Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004; 6:1122-8. 52. Baselga J, Semiglazov V, van Dam P, et al. Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol 2009; 27:2630-7. 53. Barbareschi M, Buttitta F, Felicioni L, et al. Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas. Clin Cancer Res 2007; 13:6064-9. 54. Ellis MJ, Lin L, Crowder R, et al. Phosphatidyl-inositol-3-kinase alpha catalytic subunit mutation and response to neoadjuvant endocrine therapy for estrogen receptor positive breast cancer. Breast Cancer Res Treat 2010; 119:379-90. 55. Ellis MJ, Tao Y, Luo J, et al. Outcome prediction for estrogen receptor-positive breast cancer based on postneoadjuvant endocrine therapy tumor characteristics. J Natl Cancer Inst 2008; 100:1380-8. 56. Loi S, Haibe-Kains B, Majjaj S, et al. PIK3CA mutations associated with gene signature of low mTORC1 signaling and better outcomes in estrogen receptorpositive breast cancer. Proc Natl Acad Sci U S A 2010; 107:10208-13. 57. Johnston S, Pippen J Jr, Pivot X, et al. Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptorpositive metastatic breast cancer. J Clin Oncol 2009; 27:5538-46. 58. Y Yao JC, Phan AT, Change DZ, et al. Phase II study of RAD001 (everolimus) and depot octreotide (Sandostatin LAR) in patient with advanced low grade neuroendocrine carcinoma (LGNET). J Clin Oncol 2006; 24(18 suppl):189s (abstract 4042). 59. Di Cosimo S, Bendell JC, Cervantes-Ruiperez, A. et al. A phase 1 study of the oral mTOR inhibitor ridaforolimus in combination with the IGF-1R antibody in patients with advanced solid tumours. J Clin Oncol 2010; 28(15 suppl):235s (abstract 3008). 60. Serra V, Markman B, Scaltriti M, et al. NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res 2008; 68:8022-30.
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Introduction
The PI3K/Akt/mTOR Pathway
Endocrine resistance, whether de novo or acquired, remains a key challenge in the treatment of hormone positive breast cancer. Mechanisms for resistance are varied with evidence existing for genomic and non-genomic cross talk between estrogen receptor (ER) and key intracellular signaling pathways.1 A number of growth factor receptor signaling pathways have been implicated in resistance including EGFR/HER1, HER2/HER3 signaling, and insulin-like growth factor 1 signaling acting both through MAPK (mitogen activated protein kinases) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways.2-6 Pharmacological targeting of these pathways potentially provides a mechanism to reverse or block acquired resistance to endocrine therapy.7 The PI3K/Akt/mTOR pathway is a critical intracellular signaling cascade that mediates both growth factor induced proliferation and cell survival with deregulated signaling through this pathway a feature of many cancers with a number of inhibitors of this pathway in preclinical and clinical development.8,9 This article reviews the molecular biology of this pathway, the preclinical and clinical evidence for the use of mTOR inhibitors in improving endocrine responsiveness in breast cancer, along with potential limitations and challenges in taking this approach forward in the clinical setting.
The PI3K/Akt/mTOR pathway is a critical intracellular signaling pathway that is implicated in a number of normal cellular processes involved in proliferation, metabolism, growth, and cell survival. The central component of the pathway is the PI3K heterodimer which comprises the p85 regulatory and p110 catalytic subunit. The pathway is activated following membrane growth factor receptor tyrosine kinase activation, and subsequent phosphorylation of the receptor results in interaction with PI3K either directly or indirectly through adaptor proteins such as insulin related substrate-1 (IRS-1). In addition PI3K can be activated directly by the GTPase protein Ras. Activation of PI3K leads to removal of the inhibitory effect of the regulator unit p85, with subsequent activation of the p110 catalytic subunit which in turn converts membrane related phosphatidylinositol 3, 5-bisphosphonate (PIP2) to the triphosphate form PIP3 (Figure 1). In turn, PIP3 results in phosphorylation of Akt, a serine-threonine kinase which is the major effector of the pathway. Subsequent Akt signaling leads to increased intracellular growth and survival which is characterized by the malignant phenotype.10,11 One important downstream consequence of PI3K/Akt activation is the alleviation of the suppression by the tuberous sclerosis protein complex (TSC1/2) of the mammalian target of rapamycin (mTOR; Figure 1).12 There are at least 2 groups of mTOR proteins, and the key protein involved in transmitting signals from PI3K-Akt is the mTORC1 complex. Activation of Akt inhibits mTORC1’s negative regulator TSC1/2, which then releases its specific inhibition of mTORC1. mTORC1 plays a critical role in the transduction of proliferative signals through phosphorylation of the translational regulator 4E-BP1 (eukaryotic initiation
1Breast
Unit, Royal Marsden Hospital, London Breast Cancer Centre, Institute of Cancer Research, London
2Breakthrough
Submitted: Aug 12, 2010; Revised: Sep 23, 2010; Accepted: Sep 24, 2010 Address for correspondence: Amna Sheri, MD, Royal Marsden Hospital, 123 Old Brompton Road, London, United Kingdom SW7 3RP Fax: 44-207-8082563; email: [email protected]
All of the authors report that they have no relevant relationships to disclose. Supplementary material from this article in the form of slides is available online at cigjournals.com.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Figure 1 Schematic Representation of the PI3K/Akt/mTOR Signaling Pathway Demonstrating the Action of Rapamycin and its Derivatives, Temsirolimus and Everolimus, Which Inhibit mTORC1
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Abbreviations: 4EBPs = 4 eukaryotic binding protein; EIF-4B = eukaryotic initiation factor 4B; EIF-4E = eukaryotic initiation factor 4E; EIF-4G = eukaryotic initiation factor 4G; GβL = G protein beta subunit like protein; IRS1 = insulin receptor substrate 1; PDK1 = pyruvate dehydrogenase kinase isoenzyme 1; PI3K = phosphatidylinositol 3-kinase; PIP2 = phosphatidylinositol 3, 5-bisphosphonate 2; PIP3 = phosphatidylinosotol 3, 5-bisphosphonate 3; PTEN = phosphatase and tensin homolog; mTORC1 = mammalian target of rapamycin complex 1; mTORC2 = mammalian target of rapamycin complex 2; S6 = ribosomal protein S6; TORC1 = TOR complex 1; TORC2 = TOR complex 2; TSC1 = tuberous sclerosis complex 1; TSC2 = tuberous sclerosis complex 2
factor 4E-binding protein), and the ribosomal protein p70s6kk (the 70-kDa S6 kinase).13,14 Activation of 4E-BP1 by mTOR releases the associated protein EIF4E which is closely bound together in quiescent cells, allowing interaction with other proteins to form a multi-subunit EIF4F complex that facilitates translation of mRNAs into proteins. Likewise, activation of the serine-threonine kinase p70 S6K phosphorylates the 40S ribosomal protein S6, which leads to recruitment of the 40s ribosomal subunit into actively translating polysomes that enhance translation of mRNAs. Inhibition of mTOR with rapamycin directly prevents protein translation via these 2 regulatory proteins Deregulated PI3K/Akt/mTOR signaling is a feature of many cancers.15 The pathway is activated by receptor tyrosine kinases as described above, and by the genetic mutation and amplification of key pathway components.16 Several genetic abnormalities are known to activate PI3K/Akt/mTOR signaling. Genetic alterations in the tumor suppressor gene PTEN, N an endogenous inhibitor of the PI3K/Akt/mTOR pathway, are among the most frequently noted somatic mutations in cancer. The protein encoded by PTEN is a phosphatase which serves to remove phosphate groups from key intracellular phosphoinositide signaling molecules.17 Mutation or epigenetic modification of PTEN N leads to accumulation of PIP3
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with deregulation signaling though the Akt/TSC1-2/mTORC1 pathway described above. Furthermore, approximately 15%-35% of breast cancers demonstrate reduced expression of PTEN, N 18 and PTEN N loss has also been associated with poor prognosis in patients with ER positive breast cancer treated with tamoxifen.19 Mutations in the catalytic domain of PI3K have also been identified in around 20%-25% of breast cancers20,21 and have been implicated in breast tumor initiation.22 Most mutations arise in either the kinase or helical domain and have been associated with breast cancers that are hormone receptor positive or over express HER2.18 An understanding of the role of the PI3K/Akt/MTOR pathway in cancer cell growth and survival has been exploited in the development of a number of inhibitors of the pathway as anti-cancer therapies. To date, the most clinically developed agents in this group for the treatment of breast cancer are the mTOR inhibitors with Akt, PI3K and dual mTOR/PI3K inhibitors also in development.
The original inhibitor of mTOR is rapamycin, a lipophilic macrolide identified in the 1970s during antibiotic screening off Streptomyces hygroscopicus found in the soil of Easter Island. It was found to be a potent fungicide with derivatives displaying potent immunosuppressive properties. Subsequent work showed that these drugs had antiproliferative effects in a range of experimental tumors,23 with an ability to block protein translation and halt G1-S cell cycle progression. Despite encouraging preclinical data, rapamycins poor solubility and chemical instability limited clinical development. Rapamycin analogs with a more favorable pharmacological profile were synthesized including CCI-779 (temsirolimus), RAD-001 (everolimus), and AP23573 (ridaforolimus). In particular, the breast cancer cell lines BT-474, SK BR-3, and MCF-7 demonstrated specific sensitivity to temsirolimus with IC50 values ranging from 0.0006 to 0.001μM.24 In each of these cell lines, the PI3K-Akt pathway appears to be constitutively overactive because of either ErbB2, ER activation or PTEN deletions, whereas breast cancer cells resistant to temsirolimus lacked any of these features. Everolimus is an oral hydroxyethyl ether of rapamycin which demonstrated impressive antiproliferative activity against a wide variety of tumor models in vivo.25 Both temsirolimus and everolimus have been used in combination with endocrine therapy both in preclinical models of endocrine-resistant breast cancer and in the clinical setting as outlined below.
Role of the PI3K/Akt/mTOR Pathway in Endocrine Resistance of Breast Cancer It is now clear that the estrogen receptor (ER) can become involved with the PI3K/Akt/mTOR pathway in breast cancer cells,26 with both genomic and non-genomic cross talk between this signaling pathway and ER.1 Because of its role in cell survival, there is evidence that the pathway becomes activated in acquired hormone-resistant breast cancer, and accounts for survival of cells despite the presence of continued endocrine blockade.27,28 As a serine/threonine kinase, Akt promotes cell survival in response to many different external local growth factors, including insulin, insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF),
Amna Sheri et al EGF, heregulin, and VEGF. Akt activates downstream effectors involved in cell survival, including antiapoptotic pathways through phosphorylation of substrates that directly regulate the apoptotic machinery (ie, BAD, caspase-9). Breast cancer cell lines with activated Akt (eg, via loss of the regulatory PTEN N tumor suppressor gene) have been shown to be especially sensitive to mTOR antagonism.24 Growth factor mediated activation of MAPK or Akt can potentiate E2 mediated ER classic transcriptional activity by directly phosphorylating ER. Importantly, both MAPK and Akt have been shown to phosphorylate ER within AF-1, at serine 118 and serine 167 respectively, in the absence of E2, thereby contributing to ligand-independent ER transactivation.4,29,30 Conversely, in addition to its effects on estrogen-regulated gene transcription, ligand bound ER has non-genomic effects via membrane interaction with growth factor receptors.31,32 This may result in rapid activation of EGFR,31 IGF1R,32 HER233 or the cleavage of membrane-bound growth factor receptor ligands such as EGF or TGF-α.34 Other data supports a role of PI3K/Akt pathway in the development of endocrine resistance, as IGF-1 has been shown to down regulate the progesterone receptor via a transcriptional mechanism that involves the PI3K/Akt pathway that is independent of ER.35 These bidirectional interactions between hormonal and growth factor pathways create self-reinforcing synergistic loops that potentiate pro-survival signals, and may allow breast cancer cells to escape normal endocrine responsiveness. Recent laboratory data have confirmed a role for enhanced growth factor activation and increased Akt phosphorylation in ER+ breast cancer cells that develop acquired resistance to long term estrogen deprivation5 or tamoxifen.36 In particular, tamoxifenresistant (TamR-1) cells expressed elevated levels of phosphorylated Akt and ERK1/2 compared with the parental MCF-7 cells, with increased phosphorylation of ER at Ser167. Colocalization studies revealed an association between HER2 and ER in the TamR1 cells, but not the parental MCF-7 cells. In addition, ER was redistributed to extranuclear sites in the TamR-1 cells and was less transcriptionally active. Tamoxifen resistance could be overcome by the combined use of a HER2 tyrosine kinase inhibitor (TKI) and tamoxifen, which was associated with redistribution of ER to the nucleus and loss of phosphorylated Akt. Therefore, these data support the concept of ER cross-talk with the PI3K/Akt pathway in endocrine resistance, and provide a rationale for combining signaling therapies against this pathway with continued endocrine therapy in an attempt to treat or overcome resistance. Preclinical models of ER+ hormone sensitive and resistant breast cancer have been used to examine the effects of combining mTOR antagonists with endocrine therapy.37,38 Boulay et al demonstrated that the estrogen-dependent growth of both wild-type MCF-7 and aromatase-expressing (MCF-7/Aro) breast cancer cells could be inhibited in a dose-dependent manner by the mTOR antagonist everolimus (RAD-001), suggesting that mTOR signaling is required for the estrogen-dependent proliferation of these cells. In subsequent experiments with the MCF-7/Aro cells, both the aromatase inhibitor letrozole and the mTOR inhibitor everolimus inhibited androstenedione-induced cell proliferation. However, the combination of letrozole and everolimus produced maximal growth inhibition, with clear evidence for additive/synergistic effects.37
In particular, increased activity of the letrozole-everolimus combination correlated with a greater effect on G1 progression, and a significant decrease in cell viability and apoptosis. As indicated above, evidence has emerged from several groups that activation of Akt/PKB and the downstream mTOR pathway can cause resistance to tamoxifen. In particular, deGraffenried et al demonstrated that MCF-7 cells expressing a constitutively active Akt were able to proliferate under reduced estrogen conditions, and were resistant to the growth inhibitory effects of tamoxifen, both in vitro and in vivo in xenograft models.38 However, co-treatment with temsirolimus inhibited mTOR activity and restored sensitivity to tamoxifen, primarily through induction of apoptosis, thus suggesting that Akt-induced tamoxifen resistance may, in part, be mediated by signaling through the mTOR pathway. Though the mechanism for the synergy is unclear, estrogen response element (ERE) reporter gene assays showed that mTOR inhibition managed to block ER-induced gene activation. Therefore, these laboratory data support the role of the PI3K/ mTOR/Akt pathway in endocrine resistance in ER positive breast cancer and provide evidence for cross-talk between ER and various components of the signaling cascade. Targeting a downstream element of the pathway such as mTOR has been shown to restore endocrine sensitivity in both cell lines and xenograft models and thus provides a rationale for combining endocrine therapy with mTOR inhibition in the clinical setting.
Clinical Trials in Metastatic Breast Cancer With mTOR Antagonists The first mTOR inhibitor to be developed for use in breast cancer was CCI-779 (temsirolimus), with early phase I studies using the traditional approach of studying the maximum tolerated dose (MTD). Because of the concern that continuous administration may lead to immunosuppression intermittent dose scheduling was selected for clinical development. In a phase I study of 24 patients with advanced solid tumors, temsirolimus was administered over a wide range off doses (7.5-220 mg/m2) once weekly as a 30 minute infusion.39 The most frequent drug related adverse events were asthenia and thrombocytopenia (grade 3 or 4 in 8% of patients), followed by dermatological toxicities and mucositis (grade 3 or 4 in 4% of patients). Elevations in total cholesterol and triglycerides were also reported. Two partial responses (PRs) were observed, 1 in a patient with renal cancer and 1 in a patient with metastatic breast cancer. Similar toxicities were also observed in a phase I study of the oral mTOR inhibitor RAD001 (everolimus)40 although 1 patient also developed grade 3 pneumonitis and another patients reported grade 3 pneumonia. Based on early encouraging phase I data that included efficacy in breast cancer, a multi center phase II trial of 2 doses of temsirolimus (75 mg/m2 or 250 mg/m2 I.V. weekly) was conducted in 109 patients with heavily pretreated patients with locally advanced or metastatic breast cancer.41 The objective response rate to temsirolimus was 9.2% (10 PR) with median time to tumor progression of 12 weeks. Efficacy was similar between the 2 dose levels, but toxicity was more common at the higher dose level especially grade 3 or 4 depression. Once again, the most common adverse events were mucositis and rash with thrombocytopenia, hyperglycemia, and hypercholesterolemia also observed.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Table 1 Results of Clinical Trials of mTOR Inhibitors in Combination With Aromatase Inhibitors Study
Trial Design
Drug Dose and Schedule
Number of Patients
Results Letrozole ORR, 45% median PFS, 11.6 months
Baslega et al 200543
Randomized, 3-arm, phase II
Letrozole 2.5 mg daily orally or letrozole + temsirolimus intermittent schedule I.V. (30 mg daily for 5 days every 2 weeks) or letrozole + temsirolimus 10 mg I.V. daily.
92
Letrozole/Temsirolimus 10 mg daily ORR, 33% median PFS, 11.5 months Letrozole/Temsirolimus 30 mg intermittent ORR, 40% median PFS, 13.2 months
Chow et al 200645
Awada et al 200742
Randomized, double-blind, placebo-controlled, phase III
Phase Ib, open-label, dose escalation
Neoadjuvant, Baselga et al randomized, placebo52 2009 controlled, phase II
Letrozole 2.5 mg orally daily + placebo (daily for 5 days every 2 weeks) or letrozole + temsirolimus 30 mg orally intermittent (daily for 5 days every 2 weeks)
Letrozole 2.5 mg orally daily + everolimus orally daily (5 or 10 mg)
Letrozole 2.5 mg orally daily + placebo orally daily for 4 months pre-surgery, or letrozole 2.5 mg orally daily + everolimus 10 mg orally daily for 4 months pre-surgery
Letrozole ORR, 24% CBR, 43% median PFS, 9.2 months (95% CI, 7.4-11.1 months) 992
18
Letrozole + Temsirolimus intermittent OEE, 24% CBR, 40% median PFS, 9.2 months (95% CI, 7.4-11.1 months) No PK interaction 1 CR at 10 mg 28% reduction in liver metastases in 1 patient at 10 mg 7 patients received combination therapy for > 6 months Letrozole Clinical ORR, 59% Ultrasound ORR, 47% Mammogram ORR, 39%
270 Letrozole + Everolimus Clinical ORR, 68% Ultrasound ORR, 58% Mammogram ORR, 36%
Safety Profile Most common AEs with letrozole/temsirolimus combination: asthenia (42%-60%) diarrhea (36%-43%) mucositis (43%) Grade 3/4 AEs: hyperglycemia (10 patients) asthenia (3 patients)
Grade 3/4 AEs: hyperglycemia (4%) dyspnoea (3%) neutropenia (3%) liver function abnormalities (3%)
Most common AEs: stomatitis (50%) fatigue (44%) anorexia (44%) diarrhea (33%) headache (33%) rash (33%) Most common AEs with combination therapy: stomatitis (36%) rash (20%) asthenia (17%) thrombocytopenia (18.2%) hyperglycemia (13%) Grade 3/4 AEs: stomatitis (2%) hyperglycemia (5%)
Abbreviations: AE = adverse event; CBR = clinical benefit rate; CR = complete response; I.V. = Intravenous; mTOR = mammalian target of rapamycin; ORR = objective response rate; PFS = progression-free survival; PK = pharmacokinetic
Emerging data from preclinical studies demonstrating possible synergy between mTOR antagonists and endocrine therapy provided a rationale for a number of studies combining aromatase inhibitors with mTOR antagonists.37,38 An initial phase Ib study of letrozole with the mTOR inhibitor everolimus had suggested the combination was well tolerated with very few grade 3 toxicities (Table 1).42 A randomized 3 arm, phase II study of the mTOR inhibitor temsirolimus given intravenously 10 mg daily or 30 mg intermittently (daily for 5 days every 2 weeks), was conducted in 109 patients with hormone receptor positive locally advanced or metastatic breast cancer.43 Patients were eligible if they were receiving first- or second-line endocrine therapy but were excluded if they had received a previous aromatase inhibitor. Results suggested a modest benefit to the combination with intermittent temsirolimus in terms of median progression free survival (PFS; 13.2 months vs. 11.6 months), although the objective response rate was not
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statistically different (40% vs. 45%). Notably, the combination was associated with significant toxicities not observed in the letrozole alone arm: grade 1-4 diarrhea (43%), mucositis (43%), and grade 3-4 hyperglycemia (20%). In particular the toxicity was greatest in the higher dose schedule and resulted in dose delays, dose reductions, or discontinuations, so the protocol was amended to adjust the low dose schedule. Updated efficacy results of this study showed a non-statically significant greater clinical benefit rate of 77% for the combination compared with 60% for the letrozole alone arm.44 With phase II data suggesting a possible benefit for temsirolimus and letrozole combinations, a large multi-center randomized double-blind phase III clinical trial was undertaken. Over 1200 postmenopausal women with hormone receptor positive locally advanced or metastatic breast cancer were randomized to letrozole with either temsirolimus (in an oral formulation 30 mg for 5 days every 2 weeks) or placebo.45 The aim of the study was to
Amna Sheri et al detect a 25% improvement in PFS with the combination. The oral combination was better tolerated than in the phase II study with I.V. temsirolimus that had less than 5% grade 3-4 toxicities, including only 4% hyperglycemia. Unfortunately, the trial was terminated early after an interim analysis demonstrated a lack of benefit for the combination. Results among the first 992 patients showed no diff ference in objective response rates (RR) or PFS.
Limitations to mTOR inhibition Given the preclinical rationale and encouraging phase II data, the negative results from this large randomized phase III trial were disappointing (Table 1). Several explanations for the lack of benefit for the combination have been put forward. At a mechanistic molecular level, 2 key regulatory loops have been described that may limit the effectiveness of mTOR inhibitors in the treatment of cancer. An important negative feedback loop exists downstream in the PI3K/ Akt/mTOR pathway. The mTOR-activated kinase S6K1 phosphorylates and destabilizes the IRS1 and IRS2 proteins in insulin like growth factor (IGF) responsive cells.46 In these cells, mTOR inhibition leads to a reduction in S6K1 activity, which allows IRS1/2 expression to be increased with associated enhanced activation of IGFR-1 dependent Akt activity.47,48 This is supported by the clinical observation that phosphorylated Akt is up regulated in both tumor and skin biopsies of patients treated with everolimus49 and as such, this loss of negative feedback may overcome the anti-tumor effectiveness of the mTOR blockade. Secondly, a positive regulatory loop exists involving the mTORC2 complex which can be activated more directly by growth factors and phosphorylate Akt. Therefore, the inability of rapamycin derivatives to block mTORC2 could result in increased Akt signaling that result in ER phosphorylation on serine 167, negating the effect of aromatase inhibition, and thus, limiting the therapeutic benefit of this combination.50,51 Alternatively, the lack of clinical benefit could, in part, reflect a failure to select patients appropriately whose tumors demonstrated dependence on the PI3K/Akt/mTOR pathway. Tissue sampling at time of progression to determine tumor phenotype (ie, phosphorylated Akt or loss of PTEN may be surrogates for activated pathways in theses tumors) would have proved challenging in a large trial.
Biomarkers and Neoadjuvant Studies Neoadjuvant studies, though not necessarily reflecting the adaptive changes over time that may occur during the course of metastatic disease, may provide predictive biomarker data that can help select appropriate patients for future clinical trials. A recent randomized phase II study in 270 postmenopausal women with ER+ primary operable breast cancer has evaluated the benefit of adding everolimus (10 mg/day) or placebo to neoadjuvant letrozole. (2.5 mg/day).52 The primary endpoint of the study was clinical response by palpation with the addition of everolimus to letrozole for 16 weeks, preoperatively resulting in significantly greater RRs as judged by both clinical (68.1% vs. 59.1%) and radiologic assessments. Core biopsies were obtained at baseline and at day 15. A significantly greater reduction in cell proliff eration measured by change in Ki67 was seen in the letrozole/
everolimus combination arm compared with letrozole. Associative correlative studies were also conducted to determine those tumors most likely to respond to combined mTOR antagonism and aromatase inhibition. At day 15, down regulations in progesterone receptor and cyclin D1 were seen in both treatment arms; whereas large reductions in phosphorylated ribosomal protein S6 were seen in only the everolimus arm. Interestingly, specific mutations in PIK3CA were found to be associated with a greater likelihood of an antiproliferative response to the combination of letrozole plus everolimus. In particular mutations in the allosteric helical domain of exon 9 were associated with a poor antiproliferative response to letrozole alone, but a good response to letrozole plus everolimus. This particular, PIK3CA mutation has been associated with a worse prognosis in breast cancer,53 and a greater likelihood of response to the combination implicates the PI3K/ Akt/mTOR pathway in endocrine resistance. Studies on the effect of PIK3CA mutations, however, have been mixed. A recent analysis of samples from 4 neoadjuvant trials treated with endocrine therapy alone in the neoadjuvant setting demonstrated only a weak negative interaction between PIK3CA mutation status and clinical response to neoadjuvant endocrine treatment and no interaction with changes in Ki-67 based proliferation index.54 In a subset analysis of the effect of PIK3CA mutations and relapse-free survival using samples from the P024 trial (previously used to generate a well-validated preoperative prognostic index: PEPI)55 there were insufficient samples containing the exon 9 mutation to draw conclusions. It’s also possible that the impact of any PI3K mutation may depend on coexpression of other factors such as ER or HER2. A recent study reported that mutations in PI3KCA exon 20 kinase domain were actually associated with better clinical outcomes in ER+/HER2– breast cancer patients but not in ER R– or HER2+ 56 subtypes. These findings may have implications for the future development of PI3K/mTOR inhibitors in breast cancer.
Future Directions The PI3K/Akt/mTOR pathway has an established key role in cell growth and survival with preclinical evidence for cross-talk between this pathway and ER. Clinical benefits from co- targeting these pathways however have been modest to date and key challenges for future research are identifying who may benefit from targeting this particular combination of pathways and understanding how best to overcome limitations to this approach. In order to do this, it is essential to consider the issue of heterogeneity. Future trials will need to ensure that they are enriched with the most appropriate patients ie, those with activation of the relevant target. A key question is how best to identify patients for targeted therapy? Recent neoadjuvant studies have attempted to identify biomarkers for response to combined aromatase and mTOR blockade.52 However, sampling of tumor phenotype at the time of progression remains challenging. It may be possible to better characterize patients based on surrogate clinical markers such as previous therapy and responsiveness.57 The BOLERO-2 (Breast Cancer Trials of Oral Everolimus) study will test the combination of everolimus with steroidal aromatase inhibitor exemestane in postmenopausal women with advanced breast cancer who are refractory to letrozole or anastrozole.
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Targeting Endocrine Resistance: Is There a Role for mTOR Inhibition? Even when oncogenic addiction to a particular pathway such as PI3K/Akt/mTOR is correctly identified, it must be recognized that blockade of a single protein in a complex signaling cascade, albeit a critical downstream effector of the pathway, is unlikely to provide total or prolonged growth inhibition. As the science of regulatory feedback loops becomes clearer, biologic explanations because of compensatory escape pathways (ie, further stimulations of upstream Akt following mTOR inhibition, which overcomes the block) account for short term clinical remissions often with rebound growth at the time of disease progression. A greater understanding of this biology will allow the partnering of key signal transduction inhibitors together to provide maximal vertical or horizontal blockade. For example, dual vertical inhibition of IGF-1R signaling using either monoclonal antibodies or TKIs (which are in phase II trials for breast cancer) could be used to overcome the rapamycininduced Akt activation loop being activated and give greater efficacy than either approach alone, a concept initially tested with promising results in neuroendocrine tumors where octreotide (which targets IGF-IR signaling) was combined with everolimus. This resulted in 4 patients achieving PR and 19 patients having stable disease (SD) out of 32 treated patients.58 More recently, the mTOR inhibitor ridaforolimus has been used in combination with the IGF-1R antibody, dalotuzumab, in patients with advanced solid tumors.59 The most frequent adverse events were stomatitis, fatigue, and hyperglycemia which were mostly grade 1-2. Interestingly, there were signs of activity in 5 heavily pretreated breast cancer patients with 2 achieving PRs, 1 SD, and 2 demonstrating partial metabolic responses on positron emission tomography scan. All 5 of these patients were ER+ and 4 had high expression of Ki67. Despite certain success with the rapalogs, other agents may prove superior inhibitors of the PI3K/Akt/mTOR pathway either alone or in combination with other targeted agents with several agents in development including mTOR kinase inhibitors which target both mTORC1 and MTORC2, Akt inhibitors, PI3K, and dual mTOR/ PI3K inhibitors. Promisingly, the dual mTOR/PI3K inhibitor NVP-BEZ235 has demonstrated superior antiproliferative activity to everolimus in cell lines.60 Previous neoadjuvant studies have revealed inconclusive results about the role of PI3K mutations in endocrine resistance/responsiveness. However, it’s possible that patients with advanced disease who have previously relapsed on endocrine therapy may be more likely to have tumors with activation of the PI3K/Akt/ mTOR pathway. A phase I study of XL765, a dual inhibitor of PI3K and mTOR, is planned in combination with letrozole in patients who have progressed on previous endocrine therapy with further preclinical studies and early-phase trials planned.
Conclusion The PI3K/Akt/mTOR pathway is a validated target for anticancer therapy with both preclinical and clinical evidence pointing to the possibility of synergistic combinations with endocrine therapy. However, it’s not yet clear who might benefit most from this approach, and clinical gains to date have been modest. There is an urgent need to understand the mechanistic science of regulatory pathways in order to design biologically-rational trials to explore this concept further.
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