The Mammalian Target of Rapamycin Inhibitors in Breast Cancer: Current Evidence and Future Directions

Mammalian Target of Rapamycin (mTOR) Inhibitor Pathway

The mTOR inhibitor is a critical regulator of several normal cell processes in numerous cell types, including cells of the breast. Several other proteins including, phosphatidylinositol-3-kinase (PI3-kinase)/protein kinase B (AKT), and phosphatase and tensin homologue (PTEN) play roles in mTOR signaling. PI3K is an enzyme that phospohorylates certain components of the cell membrane. Once these components become phosphorylated, they bind the protein AKT which becomes phosphorilated and activated. This triggers the activation of several downstream signaling pathways, which increase cell survival, proliferation and cell growth (1, 2).

One important player in the cell growth and proliferation pathway is mTOR. When activated by AKT, mTOR promotes cell growth and proliferation by stimulating protein synthesis; in addition to receiving signals from AKT, mTOR monitors the environment of the cell for the presence of growth factors and nutrients. The signaling pathway that includes mTOR is highly active in many cancer cells. This can be explained as the result of amplification or mutation of the PI3K gene, the amplification or mutation of the AKT gene, or the loss of function of phosphatase and tensin homolog (PTEN). Increased activity of some growth factor receptors can also enhance the activity of the pathway.

mTOR pathway activation is associated with poor prognosis in many types of cancer including breast cancer and is linked to resistance to many types of therapy (3-5).

A bi-directional cross talk between estrogenic receptor (ER) and growth factor receptors (e.g. HER2) mediate signaling via PI3K/AKT and Mitogen-activated protein kinase (MAPK) pathways. These two pathways can directly phosphorylate genomic ER, resulting in enhanced estrogen-regulated gene transcription.

Hormone Receptor (HR) - positive Breast Cancer: Mechanisms of Resistance to Anti-estrogenic Treatment

Three-quarters of all invasive breast cancer are ER- and/or progesterone receptor (PR)-positive, including at least half of all these in premenopausal women. The natural history of HR-positive disease differs from that of HR-negative in terms of aggressiveness of the disease.

The mechanism of action of ER is its nuclear function, also referred to as genomic activity, which is to alter the expression of genes, which are key for the normal cellular activity, tumor growth and survival. The ER signaling pathway is also regulated by a membrane receptor, human epithelial growth factor receptor-2 (HER2), and insulin-like growth factor receptor (IGF-1R). This activation of ER by the growth factor receptor signaling is referred to as ligand-independent receptor activation. These membrane kinases activate signaling pathways that eventually result in phosphorylation of ER, as well as its co-activators and co-repressors at multiple sites to influence their specific functions.

De novo and acquired resistance to endocrine therapy is a major clinical problem in the treatment of breast cancer. Different mechanisms are involved when cells adapt to allow their escape from manipulations blocking ER signaling, which includes EGFR/HER2, MAPK, extracellular signal-regulated kinase (ERK) 1/2, and AKT pathways. Estrogen-independent growth properties are mediated, at least in part, through the PI3K/AKT/mTOR pathway and hyperactivation of this pathway accounts for survival of cells, despite the presence of continued endocrine blockade (6-8).

Direct blockade of the mTOR pathway is a new and intriguing area in breast cancer therapy, with the potential to modulate growth factor- and estrogen-dependent and estrogen-independent pathways, which contribute to the pathogenesis and progression of the tumor (9, 10).

Anti-HER2 and Breast Cancer

Amplification of HER2 is observed in approximately 20% of cases of invasive breast cancer, and portends a poor prognosis, with an increased risk for disease progression and a reduced overall survival. The HER2 gene encodes a transmembrane tyrosine kinase receptor that belongs to the EGFR family. This family of receptors includes four members (EGFR/HER1, HER2, HER3 and HER4) that function by stimulating growth factor signaling pathways such as the PI3K–AKT–mTOR pathway. Receptors of this family contain an extracellular ligand-binding domain, a lipophilic transmembrane domain, and an intracellular tyrosine kinase domain. Activation of receptor kinase function occurs predominantly via ligand-mediated hetero- or homodimerization (11). In the case of HER2, activation is also thought to occur in a ligand-independent manner, particularly when the receptor is found to be mutated or overexpressed. Overexpression of HER2 enables constitutive activation of growth factor signaling pathways and thereby serves as an oncogenic driver in breast cancer (12, 13). Through both genetic and pharmacological approaches it has been determined that HER2 was both necessary and sufficient for tumor formation and maintenance in models of HER2-amplified breast cells. Trastuzumab (Herceptin®), a humanized, recombinant monoclonal antibody that binds to the extracellular domain of HER2, has been shown to selectively exert antitumor effects in patients with HER2-amplified breast cancer, and not in tumors with normal HER2 expression (14). The benefits of this drug have been defined exclusively in HER2 overexpressing-amplified disease. Trastuzumab improves overall survival when given in combination with chemotherapy for metastatic breast cancer and reduces the risk of disease recurrence and death when given in the adjuvant setting, making the drug the foundation for systemic therapy of HER2-overexpressing tumors (15).

Mechanisms of Resistance

Despite the clinical benefit seen with trastuzumab administration, both de novo and acquired clinical resistance have been increasingly recognized. Trastuzumab monotherapy in the metastatic setting results in response rates of 11-26% (clinical benefit rate: 48%), implying that many cases of HER2-amplified metastatic breast cancer will not respond to monotherapy. In addition, the duration of response to trastuzumab-based therapy ranges from 5 to 9 months, suggesting that acquired resistance often develops (16, 17). Elucidating the molecular mechanisms of trastuzumab resistance has been difficult given the number of mechanisms of action of trastuzumab. Nevertheless, a detailed molecular understanding of clinical resistance to trastuzumab, might greatly aid in the development of more effective targeted therapies, and has thus gained significant attention. Recently, several models of resistance have been described, although final validation with analyses of human tumor samples has been limited (18, 19).

Up-regulation of PI3K pathway. Persistent activation of the PI3K–AKT–mTOR signaling pathway drives aberrant cell growth and proliferation in a variety of tumor types. Recent work has demonstrated a strong association between mutational activation of this pathway and resistance to therapies targeted against the HER kinases such as trastuzumab (Figure 1). Constitutive activation of PI3K most frequently occurs via two mechanisms: loss-of-function of PTEN, or via activating mutations in the gene encoding the catalytic subunit of PI3K (PIK3CA) (20). As a negative regulator of the PI3K pathway, loss of PTEN function through mutational inactivation or down-regulation of expression results in activation of PI3K–AKT signaling and prevents trastuzumab-mediated growth arrest of HER2-amplified breast cancer cells (21, 22). Dysregulation of the mTOR pathway creates a favorable environment for the development and progression of many types of cancer, including breast cancer, and is associated with the development of resistance to endocrine therapy and to the HER2 monoclonal antibody. Therefore, the addition of mTOR inhibitors to conventional therapy for breast cancer can enhance therapeutic efficacy and/or overcome innate or acquired resistance (23).

Controversies in breast cancer and mTOR resistance. The PI3K/AKT/mTOR pathway can be activated in breast cancer by different mechanisms, such as amplification of the PI3K p110-alpha catalytic subunit or AKT; mutation of the PI3K p85-beta regulatory subunit or p70S6K; loss of the PTEN, which inhibits PI3K-dependent activation of AKT and terminates PI3K-mediated signaling; and, more often, sustained activation or overexpression of cell surface growth factor receptors, such as HER1, HER2 or IGF-1R (24). The presence of compensatory pathways is not surprising in such a complicated network. One negative feedback loop involves p70S6K, IGF-1R substrate-1 and IGF-1R proteins. IGF-1R substrate-1 is a docking protein for the IGF-1R and serves to activate the regulatory subunit of PI3K. Phosphorylation of p70S6K by activated mTOR leads to the degradation of IGF-1R substrate-1, and consequently to suppression of PI3K/AKT signaling. The inhibition of p70S6K through mTOR-targeted agents, therefore interrupts this negative feedback, resulting in sustained activation of the IGF-1R signaling (25). Taking advantage of this compensatory pathway, combined therapies designed to block both mTOR and IGF-1R pathways, or to block both mTOR and PI3K/AKT pathways may provide a synergistic effect.

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Figure 1.

Mammalian target of rapamycin (mTOR), mechanisms of resistance to trastuzumab. Resistance to trastuzumab due to phosphatase and tensin homolog (PTEN) deficiency. HER2=human epidermal growth factor receptor 2; PI3K=phosphatidylinositol-3-kinase; PIP3=phosphatidylinositol (3,4,5)-triphosphate; mTOR=mammalian target of rapamycin; Src=v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian).

Other studies have demonstrated that inhibition of mTOR complex-1 (mTORC1) leads to MAPK pathway activation through a PI3K-dependent feedback loop, and that prolonged exposure to rapamycin may lead to AKT inhibition through depletion of mTORC2 (mTOR-rictor complex), which is normally located upstream and activates AKT (26, 27).

Two key regulatory loops have been described that may limit the effectiveness of drugs that have been developed to target mTOR in cancer. The mTOR-activated kinase S6K1 phosphorylates and destabilizes the insulin-receptor substrate 1 and 2 (IRS1 and IRS2) proteins in IGF-responsive cells. mTOR inhibition can block the negative feedback on IGF-1R signaling interfering in AKT/PI3K signaling (28, 29). The result is an increase in AKT phosphorylation, protein kinase activity, and downstream signaling, which could potentially counteract the inhibition of mTOR. Thus, concern has been raised that loss of this negative feedback loop may overcome the antitumor effectiveness of mTOR blockade and limit their effectiveness. In addition, a positive regulatory loop exists involving the mTORC2 that is activated directly by growth factors. In contrast to mTORC1, the mTORC2 phosphorylates AKT directly, and this is thought to be required for full activation of the AKT pathway by mitogenic signals. As such, mTORC2 functions as an upstream regulator of AKT and delivers an additional stimulatory signal to mTOR1. However, rapamycin analogs that target mTOR proteins appear to specifically block only the mTORC1 and do not inhibit the mTORC2 (30).

The Clinical Evidence

Clinical trials are currently underway with PI3K, AKT and mTOR inhibitors. NVP-BEZ235 belongs to the class of imidazoquinolines, and potently and reversibly inhibits PI3K catalytic activity by competing at its ATP-binding site (31). Other agents include the dual PI3K/mTOR inhibitor XL765, and the pure PI3K inhibitor XL147.

Initial data from phase I studies suggest that these compounds are safe and that PI3K signaling inhibition is achievable (32). The mTOR inhibitors temsirolimus and everolimus are further ahead in development for use either as a single-agents or in combination with endocrine therapy (Table I).

Everolimus. This mTOR inhibitor has been shown to reverse AKT-induced resistance to hormonal therapy and trastuzumab. Phase I and II clinical trials have demonstrated that everolimus has promising clinical activity in women with HER2-positive, HER2-negative, and ER-positive breast cancer when combined with HER2-targeted therapy, cytotoxic chemotherapy, and hormonal therapy, respectively (33-35).

Temsirolimus. The initial phase II study of letrozole-alone and in combination with the other mTOR inhibitor temsirolimus in patients with metastatic breast cancer showed benefit from the combination in terms of median progression-free survival (18.0 months vs. 9.5 months). The successive large, phase III, randomized trial of letrozole-alone and in combination with temsirolimus in 992 postmenopausal women was terminated early, however, after an interim analysis demonstrated a lack of benefit from the combination. This negative result is not ascribed to the lack of efficacy of this combinatorial strategy, but rather to the suboptimal inhibition of the mTOR pathway with the dose of temsirolimus implemented (30% of patients discontinued treatment), to the study population (phase II was camed out on heavily pretreated patients with endocrine-resistant disease, whereas phase III was up front), and to the unselected patient population, with the inability to identify patients in whom the tumors exhibited dependence on PI3K/mTOR activation (36, 37).

Neoadjuvant treatment. A phase II, double-blind, randomized study of everolimus in combination with letrozole vs. placebo and letrozole in the neoadjuvant setting, proved to be superior over letrozole and placebo with a higher significant response rate (68% vs. 59%). This study incorporated carefully conducted pre-study and on-study tumor biopsies and pharmacodynamic studies and demonstrated a near doubling of the cell-cycle response rate by decreases in Ki67 in the everolimus group, the patients with a PI3K mutation being those with the greatest benefit (38).

Metastatic breast cancer. TAMRAD is a phase II trial that enrolled 111 patients with HR-positive HER2-negative metastatic breast cancer who had previously received adjuvant therapy with an aromatase inhibitor (AI). Patients were stratified by time-to-progression after prior AI treatment and randomized 1:1 to receive either tamoxifen-alone or everolimus plus tamoxifene. The primary objective was to estimate the clinical benefit rate and secondary end-points included safety and time-to-progression (TTP). The clinical benefit was 42% for the tamoxifen treated group and 61% (p=0.045) for the association arm. Similarly, TTP was favored in the combination group (4.5 vs. 8.6 months; hazard ratio, HR=0.54, p=0.0021), as was overall survival (OS) (HR=0.45, p=0.007). Clinical benefit was particularly increased in patients with secondary hormone resistance (44% for tamoxifen vs. 74% for everolimus/tamoxifene). Looking at TTP as a function of intrinsic hormone resistance, TTP was 3.8 months for those treated with tamoxifene and 5.4 months for those treated with the combination (HR=0.70, p>0.05). Among those with secondary hormone resistance, TTP was 5.5 months for those treated with tamoxifen and 14.8 months for those treated with the combined arm (HR=0.46, p=0.0087). OS was significantly better among patients with secondary resistance (HR=0.73, p=0.41 vs. HR=0.21, p=0.002) (35).

BOLERO-2 phase III trial. BOLERO-2 is a phase III that enrolled 724 women postmenopausal women with advanced ER-positive HER2-negative refractory advanced breast cancer (with recurrence or progression following prior therapy with letrozole or anastrozole). Patients were randomly allocated in a 2:1 ratio to receive everolimus at 10 mg daily or placebo, with both arms receiving exemestane. The primary end-point was median progression-free survival (PFS). No crossover after disease progression was allowed. Previous therapies included antiestrogenic therapy (tamoxifen, fulvestrant), and one chemotherapy regimen. The trial was stopped early after the February 2011 pre-specified interim analysis found a significantly better PFS for the association arm. On July 20, 2012, the U.S. Food and Drug Administration approved everolimus tablets for the treatment of post-menopausal women with advanced HR-positive, HER2-negative breast cancer in combination with exemestane, after failure of treatment with letrozole or anastrozole. The PFS by investigator assessment was 7.8 and 3.2 months in the everolimus and placebo arms, respectively [HR=0.45 (95% CI=0.38-0.54), p<0.0001]. PFS results were also consistent across the subgroups of age, race, presence and extent of visceral metastases, and sensitivity to prior hormonal therapy. The objective response rates were 12.6% and 1.7% in the everolimus and placebo arms, respectively. An interim analysis of OS conducted at 46% of expected events was not statistically significant [HR=0.77(95% CI=0.57-1.04)]. The final analysis of OS is expected to take place in June 2014. The most common grade 3-4 laboratory abnormalities (≥ 3%) were lymphopenia, hyperglycemia, anemia, decreased potassium, increased aspartatoaminotransferase (AST), increased alaninoaminotransferase (ALT), and thrombocytopenia. Fatal adverse reactions occurred in 2% of patients on the everolimus arm compared to 0.4% of patients on the placebo arm. Adverse reactions resulting in permanent discontinuation occurred in 24% and 5% of patients in the everolimus and placebo arms, respectively. Dose interruptions or reductions were necessary in 63% of patients on the everolimus arm compared to 14% on the placebo arm (39).

Forty percent of patients on the everolimus arm were ≥65 years of age and 15% were ≥75 years of age. No overall differences in efficacy were observed between elderly and younger patients. The incidence of deaths due to any cause within 28 days of the last everolimus dose was 6% in patients ≥65 years compared to 2% in patients <65 years. Adverse reactions leading to permanent treatment discontinuation occurred in 33% of patients ≥65 years of age compared to 17% of patients <65 years of age (40-42).

NCT00426556 phase II trial is ongoing to investigate the efficacy and safety of 10 mg everolimus daily combined with weekly trastuzumab and paclitaxel in patients with HER-2-overexpressing metastatic breast cancer (43, 44).

BOLERO-1 phase III trial is ongoing to assess everolimus plus trastuzumab and paclitaxel in the treatment of HER2-positive locally advanced or metastatic breast cancer. This trial is enrolling 717 patients from the US (45).

BOLERO-3 (NCT01007942) phase III trial is assessing the effect of the combination of everolimus, vinorelbine and trastuzumab on PFS and OS in HER2-positive women with locally advanced or metastatic breast cancer, resistant to trastuzumab and who have been pre-treated with a taxane. The initiation of these trials was based on results from two phase I trials of everolimus plus vinorelbine and trastuzumab (NCT00426530) and everolimus in combination with trastuzumab plus paclitaxel (NCT00426556) in patients with HER2-overexpressing metastatic breast cancer. In these trials, everolimus, vinorelbine trastuzumab combination halted tumor growth in 62% of patients and everolimus, paclitaxel plus trastuzumab combination halted tumor growth in 77% of patients. The data showed that everolimus may overcome tumor resistance to trastuzumab. The primary end-point of this study is PFS. The study is due to complete in December 2012 (46).

Perspectives

The discordant results between the temsirolimus and everolimus trials are not well-understood. One reason that might explain the findings is that the population was different in both studies: the temsirolimus trial included only endocrine treatment-naive patients, while the everolimus population was composed of patients with disease refractory to previous treatment with AI.

Based on BOLERO-2, mTOR inhibition in combination with endocrine therapy will be considered a new therapeutic strategy for women with previously AI-treated advanced breast cancer, but it is critical to identify predictive biomarkers to select patients most likely to benefit from these therapies.

One important area of study will be the use of a total targeted approach with anti-estrogen and mTOR in comparison to chemotherapy in the metastatic setting: a combined approach has produced promising results in pre-treated post-menopausal patients with metastatic breast cancer and in patients harboring PI3K mutations who were treated with endocrine therapy.

Studies are ongoing to assess the efficacy of mTOR inhibitors with chemotherapy and HER2-targeted agents to overcome resistance to the monoclonal antibody to HER2 trastuzumab. Consistent with a mechanistic-based approach, studies are currently ongoing with mTOR and IGF-1R inhibitors and with mTOR and PI3K inhibitors to prevent the negative feedback loops secondary to mTOR inhibition.

Adjuvant study of mTOR inhibitors is greatly needed based on the results seen in the advanced setting. While HR-positive breast cancer represents more than half of early-stage cases, there are only a handful of trials looking at improving the outcome of these patients. Tamoxifen and, more recently, AIs, have significantly improved the outcome of patients with early-stage disease, but still about 15%–20% of these patients will experience relapse. Careful study of mTOR agents in the early-stage setting is needed in selected patient populations. Patients with luminal-B tumors, characterized by higher proliferation and more frequently relapses than luminal-A cancers, might derive particular benefit from this therapeutic strategy.

In the neoadjuvant setting, a phase II study of everolimus in combination with letrozole vs. placebo and letrozole proved to be superior over letrozole and placebo with a higher significant response rate and a decrease in Ki67 expression in the everolimus-treated group. This is potentially important, since a Ki67 drop in the neoadjuvant setting has recently been demonstrated to correlate with long-term outcome.