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Abstract

Mechanistic target of rapamycin controls cell growth, metabolism, and aging in response to nutrients, cellular energy stage, and growth factors. In cancers including breast cancer, mechanistic target of rapamycin is frequently upregulated. Blocking mechanistic target of rapamycin with rapamycin, first-generation and second-generation mechanistic target of rapamycin inhibitors, called rapalogs, have shown potent reduction of breast cancer tumor growth in preclinical models and clinical trials. In this review, we summarize the fundamental role of the mechanistic target of rapamycin pathway in driving breast tumors. Moreover, we also review key molecules involved with aberrant mechanistic target of rapamycin pathway activation in breast cancer and current efforts to target these components for therapeutic gain. Further development of predictive biomarkers will be useful in the selection of patients who will benefit from inhibition of the mechanistic target of rapamycin pathway.

Keywords

Mechanistic target of rapamycin mechanistic target of rapamycin inhibitors breast cancer diagnostic biomarkers clinical trials

Introduction

Breast cancer (BC) is the most common cancer for females and the leading cause of cancer-related death in females globally and represents the high incidence rate and mortality.¹ It is a heterogeneous disease often characterized by its hormone receptor status and expression of human epidermal growth factor receptor 2 (HER2).² These molecular markers used to classify BC subtypes also typically predict response to targeted therapies. Thus, identifying novel BC targets and developing diagnostic biomarkers in order to provide early and effective therapy remain of paramount importance.

To date, a number of therapies have been developed to specifically inhibit crucial oncogenic targets in a variety of cancers. For BC in particular, endocrine therapies such as the selective estrogen receptor (ER) modulator, tamoxifen, antagonize ER in estrogen receptor–positive (ER+) cancers while the humanized monoclonal antibody, trastuzumab, inhibits receptor signaling in HER2− amplified/overexpressing cancers.^3–7 Each therapy achieves a relatively high response rate in their respective patient populations, but an equally and discouragingly high number of patients become refractory to the treatment over time. Mechanistic target of rapamycin (mTOR) is an evolutionary well-conserved serine/threonine protein kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. mTOR has a broad range of action and is involved in the regulation of cell growth, aging, and metabolism.⁸ Recently, biological studies point to aberrant PI3K/AKT/mTOR signaling pathway (hereafter referred to as mTOR signaling pathway) activation as central for cancer growth, survival, and motility as well as targeted therapy resistance mechanisms.^9–12 Consequently, heavy emphasis on mTOR signaling research has led to the development of numerous pathway inhibitors.¹³ Here, we will review the recent progress in pharmacological intervention of the mTOR pathway including its rationale, limitations, and methods of patient stratification.

The mTOR pathway

mTOR as a potential macromolecular target is a macrolide produced by Streptomyces hygroscopicus bacteria and that first gained attention due to its broad antiproliferative properties.^14,15 Shortly afterwards, biochemical approaches in mammals resulted in purification of mTOR and its discovery as the physical target of rapamycin.^16–18 mTOR comprised two structurally and functionally distinct complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2; for more details, see Figure 1).⁸ mTORC1 is composed of mTOR, mammalian lethal with SEC13 protein 8 (mLST8), DEP domain-containing mTOR-interacting protein (DEPTOR), regulatory-associated protein of mTOR (RAPTOR), and proline-rich AKT1 substrate 1(AKT1S1). mTORC2 consists of mTOR, mLST8, DEPTOR, PROTOR, rapamycin-insensitive companion of mTOR (RICTOR), and target of rapamycin complex 2 subunit MAPKAP1 (MAPKAP1).¹⁹ mTORC1 is activated by a myriad of inputs such as growth factors, energy status, proinflammatory cytokines, oxygen levels, amino acids, and the canonical wingless type (Wnt) pathway.⁸ Growth factors, insulin and insulin-like growth factor 1 (IGF1), exert their action on mTORC1 through receptor tyrosine kinases (RTKs) and the well-characterized PI3K/AKT and Ras/Raf/Mek/Erk signaling pathways. These pathways activate mTORC1 by phosphorylating and thereby inhibiting the tumor suppressor tuberous sclerosis 1 and 2 (TSC1/2) complex. The TSC1/2 complex is a key regulator of mTORC1 and functions as a GTPase-activating protein that negatively regulates guanosine triphosphate (GTP)-binding protein Rheb (Rheb) by converting it into its inactive guanosine diphosphate (GDP)-bound state.^20,21 In contrast, downregulation of mTORC1 is accomplished via activation of the TSC1/2 complex by AMP-activated protein kinase (AMPK), liver kinase B1 (LKB1), and DNA damage-inducible transcript 4 (DDIT4) protein in situations of low energy (high AMP), low oxygen levels,²² and DNA damage.²³ Much less is known about the later discovered mTORC2 signaling pathway. mTORC2 is insensitive to nutrients but does respond to growth factors such as insulin in association with ribosomes.²⁴ Besides its initial described role in actin cytoskeleton organization, mTORC2 activates cell metabolism, survival, and growth. mTORC2–ribosome interaction may be a conserved mechanism of mTORC2 activation that is physiologically related to both normal and cancer cells.

Figure 1.

Schematic overview of the mTOR signaling pathway with the most important factors and their action. Critical inputs regulating mTORC1 include growth factors and RTK. When activated, promoting protein synthesis, lipogenesis, and energy metabolism, mTORC1 inhibits autophagy and lysosome biogenesis. Alternatively, when activated by growth factors, mTORC2 regulates cytoskeletal organization and cell survival/metabolism.

The effects of rapamycin on mTOR signaling are much more complex than originally anticipated. However, obviously, rapamycin with the intracellular 12-kDa FK506-binding protein (FKBP12) forms a gain-of-function complex. This complex directly interacts with mTOR and inhibits it, when it is part of mTORC1 but not mTORC2. Many mTORC1 functions are highly sensitive to rapamycin, whereas exactly how the binding of FKBP12-rapamycin to mTORC1 inhibits its activity is just unknown. Rapamycin is likely to compromise the structural integrity of mTORC1 as well as allosterically reduce the specific activity of its kinase domain.^25–27

Involvement of mTOR pathway in BC

Given its importance in tumor growth, progression, and metabolism, it is not surprising that mTOR plays a pivotal role in BC. Encoded by oncogenes or tumor suppressor genes, many of the protein components of the mTOR pathway depend on whether they activate or suppress pathway signaling. In BC, activation of the mTOR pathway has been estimated to be as frequent as 70% of BC overall, and pathway activation promotes tumor growth and progression.^28–31 A similar upregulation is observed in other common cancer types such as hepatocellular, colon, and lung carcinomas.³²

Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) is activated upstream by the binding of a growth factor or ligand to its cognate growth factor RTK, which include members of the human epidermal growth factor receptor (HER) family and the insulin and IGF1 receptor, among others.^33,34 PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol 3,4,5 trisphosphate (PIP3), which in turn leads to phosphorylation of AKT. PIP3 acts as a docking site for AKT, which is the central mediator of the PI3K pathway and phosphoinositide-dependent kinase 1 (PDK1). Phosphorylation of AKT stimulates protein synthesis and cell growth by activating mTOR via effects on the intermediary TSC1/2. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a tumor suppressor, which has inhibitory effects on the pathway by dephosphorylating PIP3 to PIP2. PIP3 levels are hence closely regulated by the opposing activities of PTEN and PI3K.³⁵ The role of inositol polyphosphate 4-phosphatase type II (INPP4B), another tumor suppressor, is increasingly recognized. INPP4B is also involved in dephosphorylation of PIP3 to PIP26. Its loss has been reported as a marker of aggressive basal-like BC.³⁶

mTOR is a downstream effector of PI3K and AKT. mTORC1 is the target of rapamycin and rapamycin analogues, such as everolimus, and leads to cell anabolic growth by promoting messenger RNA (mRNA) translocation and protein synthesis^37,38 and also has roles in glucose metabolism and lipid synthesis. Its downstream substrate S6 kinase 1 can phosphorylate the activation function domain 1 of the ER, which is responsible for ligand-independent receptor activation.^39,40 However, mTORC2 organizes the cellular actin cytoskeleton and regulates AKT phosphorylation.⁴¹ Rapalogues exert their effect mainly on mTORC1, and the incomplete inhibition can lead to feedback loops causing paradoxical activation of AKT and proliferative effects via other downstream targets. LKB1, a tumor suppressor, is a serine–threonine kinase upstream of AMPK, which in turn serves to negatively regulate mTOR signaling via TSC1/2. LKB1 is also known as serine/threonine kinase,¹¹ with germline mutations in LKB1/SKT11 causing the Peutz-Jeghers tumor predisposition syndrome. Inactivation of the LKB1/AMPK pathway has been implicated in BC.^42,43 Furthermore, alterations in the genes encode several nodes of the mTOR pathway, which are frequently found in BC. These included activating mutations or amplifications in the genes encoding insulin-like growth factor 1 receptor (IGF1R),^44,45 PIK3CA,^46,47 PDK1,⁴⁸ HER2,^49,50 RAC-alpha serine/threonine protein kinase (AKT1),^51,52 and fibroblast growth factor receptor 1 (FGFR1)⁵³ and loss of function or reduced expression of the genes encoding PTEN,⁴⁷ INPP4B,^54,55 and LKB1⁵⁶ (for more details, see Table 1).

Table 1.

Common mTOR pathway alterations in BC.

Gene	Effect on signaling	Frequency (%)
Gene	Effect on signaling	HR+	HER2+	TNBC
Activating mutations or amplifications
PIK3CA	Activation of PI3K signaling	28–47	23–33	8
AKT1	Activation of AKT signaling	2.6–3.8	–	–
PDK1	Activation of AKT signaling	22	22	38
ERBB2	Activation of ERBB2 signaling	10	100	–
IGF1R	Activation of IGF1R signaling	41–48	18–64	42
FGFR1	Activation of FGFR signaling	8.6–11.6	5.4	5.6
Loss-of-function mutations or under-expression
PTEN	Activation of PI3K signaling	29–44	22	67
INPP4B	Activation of PI3K signaling	8.4–37.7	38	88
LKB1	Loss of regulation of AKT signaling	4.3–8.6	–	–

mTOR: mammalian target of rapamycin; BC: breast cancer; HR+: hormone receptor–positive; TNBC: triple-negative breast cancer; HER2+: human epidermal growth factor receptor 2 positive; PI3K: phosphoinositide 3-kinase; IGF1Rl: insulin-like growth factor 1 receptor; FGFR: fibroblast growth factor receptor.

Taken together, activation of mTOR plays a central role in BC, and blocking this pathway is an attractive strategy for BC treatment. The main goal of this review is to offer the rationale for the use of mTOR inhibitors in BC and provide an overview of the current and prospective clinical trials with mTOR inhibitors in BC.

mTOR pathway and ER

Approximately 70%–75% of BC is considered ER+, indicating some level of estrogen dependence for tumor growth.⁵⁷ Tamoxifen is a selective estrogen receptor modulator (SERM), which binds to the nuclear ERα to block its binding to estrogen and therefore block receptor activation. A majority of ER+ BC patients often receive drugs like tamoxifen, but resistance to them is a common issue.⁵⁸ While there are multiple mechanisms behind this resistance, mTOR appears to have a major role, with the mTOR pathway activating estrogen-independent ER transcriptional activity, making it hyper sensitive to activation and less likely to bind tamoxifen.⁵⁹ Research has shown that in the long term, BC cells may use the mTOR pathway to escape dependency from ER signaling and thus increase their resistance to tamoxifen.⁶⁰ Given the fundamental role of mTOR pathway signaling in the maintenance of ER+ BC and its emerging role in resistance to endocrine therapy, strategic inhibition of pathway signaling must be considered in the treatment of ER+ BC. This rationalizes the use of endocrine therapy and mTOR pathway inhibition in combination.⁴⁶

mTOR pathway and HER2

The HER2 subgroup was characterized by overexpression of HER2 and other genes pertaining to the HER2 amplicon. The basal-like class was largely characterized by the lack of expression of ER and HER2 and by positive expression of genes characteristic of basal-like cells of the breast and by high proliferative activity.⁶¹ Since HER family receptors can activate mTOR signaling, HER2 expression is important in the overactivation of mTOR signaling in BC. HER2 is amplified in upward of 15%–20% of all BC, which can result in a nearly 100-fold increase of protein expression.⁶² Its status as a key biomarker comes from the fact that HER2 expression correlates with a much poorer prognosis and a generally more aggressive cancer.⁶³ mTOR signaling has been linked with resistance to HER2 therapies in BC, such as with the antibody-based drug trastuzumab,⁶⁴ and the dual epidermal growth factor receptor (EGFR) HER1 and HER2 inhibitor lapatinib.⁶⁵ Activation of mTOR signaling in tumor cells after ErbB inhibition can arise as a result of mutations in the mTOR pathway and the use of other growth factor receptors like IGF1R, contributing to drug resistance.⁶⁶

mTOR pathway inhibitors

mTOR is targeted by rapamycin. The two mTOR-containing complexes have different sensitivities to rapamycin. mTORC1 is inhibited by a complex formed by rapamycin and FKBP12 protein.⁶⁷ In contrast, mTORC2 is generally resistant to rapamycin; however, in certain cell types, mTORC2 may show sensitivity after prolonged rapamycin treatment.⁶⁸ Sirolimus was first approved as an immunosuppressant for the prevention of graft rejection in kidney transplant recipients more than a decade ago.¹⁹ A few years later, sirolimus obtained approval for its use as an anti-restenosis agent following balloon angioplasty in coronary arterial stents. The early success of rapamycin has encouraged the development of derivative compounds with improved bioavailability called rapalogs (for more details, see Figure 2): everolimus, temsirolimus, and ridaforolimus. Due to the important role of mTOR in cell growth and metabolism, the primary interest shifted to anti-cancer therapy, and in 2007, temsirolimus was approved for the treatment of renal cell carcinoma and shortly thereafter for mantle cell lymphoma. Meanwhile, everolimus has received approval for treatment of pancreatic neuroendocrine tumors, subependymal giant cell astrocytoma, renal cell carcinoma, and HER2-BC in combination with exemestane. In general, first-generation mTOR inhibitors are well tolerated.^69,70 The major toxicities include stomatitis, headache, diarrhea, vomiting, and thrombocytopenia. Due to their role in metabolism, they can cause hyperglycemia, hyperlipidemia, and hypophosphatemia. However, the success of rapalogs in cancer therapy in general has not been as impressive as initially hoped. The possible mechanism of resistance to mTORC1 inhibitors came about by the discovery of a negative feedback loop in which mTORC1 inhibition leads to AKT activation through upregulating RTK such as platelet-derived growth factor receptor (PDGFRs) and insulin receptor substrate 1 (IRS-1). Abrogation of the negative feedback loop by mTORC1 inhibitors has only been demonstrated to influence PI3K/AKT signaling, whereas the impact of mTORC1 inhibition in other prosurvival pathways has not been addressed.⁷¹ mTORC1 inhibition increases the levels and activity of the adaptor protein IRS-1.⁷² The mTORC1-mediated IRS-1–negative regulation relies on its target S6K1. This negative feedback loop has been directly related to the indolence of some types of cancers, suggesting that tumors with aberrant mTORC1 activation may in turn display reduced PI3K/AKT activity. Some researches have shown that the activation of mTORC1 regulates the expression of PDGFRs and that rapamycin treatment restores PDGFR levels and therefore PI3K signaling.⁷³ Finally, the proximal mTORC1 activator Rheb has been implicated in the direct interaction and inhibition of serine/threonine protein kinase B-raf (B-Raf) and RAF proto-oncogene serine/threonine protein kinase (Raf1),⁷⁴ which highlights the complexity of the connections between mTORC1, PI3K, and MAPK pathways. In order to overcome the shortcomings and resistance of rapalogs, the second generation of mTOR inhibitors, superior to rapalogs, has been developed that functions as adenosine triphosphate (ATP)-competitive inhibitors of mTOR. Unlike rapalogs, which inhibit only mTORC1, the ATP analogues block the phosphorylation of all known downstream targets of mTORC1 and mTORC2. Furthermore, because of the similarity between the kinase domains of mTOR and PI3Ks, some of these new compounds additionally inhibit PI3K, leading to a broad inhibitory action with blocking of the feedback activation of PI3K/AKT signaling described before. Second-generation mTOR inhibitors can therefore be divided into mTORC1/2 inhibitors and PI3K/mTOR inhibitors. In addition, a series of compounds have been developed that block upstream of the mTOR pathway such as AKT inhibitors and PI3K inhibitors (Figure 2). Numerous PI3K pathway inhibitors have been developed, but the two earliest and most extensively explored are LY294002 and wortmannin. Despite effective inhibition of the PI3K pathway and demonstration of anti-tumor activity in BC cell models, both have been limited to preclinical studies due to poor solubility, instability, and high toxicity.^75,76 Compounds selective for Class I PI3Ks currently in clinical development are GDC-0941,⁷⁷ XL-147,⁷⁸ BKM120,⁷⁹ GSK1059615, CAL-101,⁸⁰ and PX-866.⁸¹ Although a truly p110α-specific inhibitor has yet to be identified, such compounds may be isolated based on initial studies showing that TGX-221,⁸² CAL-101, and AS-252424⁸³ have inhibitory activities specific for p110β, p110δ, and p110γ, respectively. In addition, there is encouraging preclinical data that the novel Class I PI3K inhibitor, CH5132799, is particularly selective for PIK3CA mutations.⁸⁴ Dual PI3K/mTOR inhibitors are generally ATP-competitive compounds that block the activity of all PI3K catalytic isoforms, mTORC1, and mTORC2. The advantage of a dual inhibitor is the ability to effectively turn off the PI3K pathway and overcome feedback inhibition observed with mTORC1 inhibitors. Examples of dual PI3K/mTOR inhibitors in advanced stages of drug development include BEZ235⁸⁵ and BGT226,⁸⁶ PKI-402 and PKI-587,^87,88 XL765,⁸⁹ and SF1126.⁹⁰ Also, GDC-0980 has demonstrated very potent anti-PI3K and anti-mTOR activity across a broad range of cell lines and in xenograft models.^91,92 There have been different strategies for developing AKT inhibitors, including ATP-competitive inhibitors, phosphatidylinositol analogues, and allosteric inhibitors. While pan-AKT, ATP-competitive agents such as GSK690693 and A-443654 have demonstrated anti-tumor activity in preclinical models and are now in phase-I trials, allosteric inhibitors are also in various late stages of development and may have advantages in terms of selectivity and specificity.^93,94 Interacting with the PH domain or hinge region, these compounds promote an inactive form of the enzyme by preventing localization to the membrane or access to the PDK-1-dependent phosphorylation site. MK2206 is one such highly selective, allosteric pan-inhibitor that has entered phase-II clinical trials and shown marked suppression on BC growth with acceptable tolerability.⁹⁵

Figure 2.

Schematic overview of the mTOR signaling pathway with the target position of drugs.

mTOR inhibitors in BC therapy

Rapamycin was the first available mTOR inhibitor. It was initially developed and used as an immunosuppressant in transplant recipients. Temsirolimus was subsequently developed and is approved for the treatment of renal cell carcinoma. Everolimus is an oral mTOR inhibitor which has been approved for use in postmenopausal women with HR+ BC; it is also approved for using in other cancers including renal cell carcinoma, neuroendocrine tumors of the pancreas, and subependymal giant cell astrocytomas. These agents are termed as “rapalogues” and work as allosteric inhibitors of mTORC1. However, in view that they inhibit only the mTORC1 complex, their use has been associated with negative feedback regulatory mechanisms and other mechanisms of resistance,⁹⁶ hence attenuating their efficacy in the single-agent setting. Therefore, developing novel and effective therapies are urgently needed. Combination therapies have been shown to inhibit breast tumor growth in a large number of in vitro and in vivo preclinical studies and have encouraged clinical trials in BC patients (Table 2, information retrieved from www.clinicaltrials.gov).

Table 2.

Summary of clinical trials of mTOR pathway inhibitors in combination with other inhibitors in BC.

NCT number	Patient population	Drug	Phase	Status
NCT01743560	Postmenopausal women with estrogen receptor–positive locally advanced or metastatic breast cancer	Everolimus; exemestane	IV	Active, not recruiting
NCT01231659	Postmenopausal women with locally advanced or metastatic breast cancer	Everolimus; letrozole	IV	Recruiting
NCT00863655	Postmenopausal women with ER+ locally advanced or metastatic breast cancer who are refractory to letrozole or anastrozole	Everolimus; exemestane	III	Completed
NCT00083993	Postmenopausal women with locally advanced or metastatic breast cancer	Temsirolimus; letrozole	III	Terminated
NCT01007942	Women with HER2+ locally advanced or metastatic breast cancer	Everolimus; vinorelbine; trastuzumab	III	Completed
NCT00876395	Women with HER2+ locally advanced or metastatic breast cancer	Everolimus; trastuzumab	III	Active, not recruiting
NCT01626222	Postmenopausal women with ER+ locally advanced or metastatic breast cancer	Everolimus; exemestane	III	Completed
NCT02137837	Postmenopausal stage IV breast cancer	Fulvestrant; anastrozole; everolimus	III	Active, not recruiting
NCT01610284	Postmenopausal women with HR+ and HER2− locally advanced or metastatic breast cancer refractory to aromatase inhibitor	Fulvestrant; BKM120	III	Active, not recruiting
NCT01633060	Postmenopausal women with HR+ and HER2− aromatase inhibitor treated locally advanced or metastatic breast cancer who progressed on or after mTOR inhibitor based treatment	Fulvestrant; BKM120	III	Active, not recruiting
NCT01298713	Women with anti-aromatase-resistant metastatic breast cancer	Tamoxifen; everolimus	II	Active, not recruiting
NCT01698918	Postmenopausal women with ER+ and HER2− metastatic or locally advanced breast cancer	Everolimus; letrozole; exemestane	II	Active, not recruiting
NCT01740336	Women with locally recurrent or metastatic breast cancer	GDC-0941; Paclitaxel	II	Active, not recruiting
NCT01437566	Women with advanced or metastatic breast cancer in patients resistant to aromatase inhibitor therapy	GDC-0980; Fulvestrant	II	Completed
NCT01776008	Premenopausal women with ER+ and HER2− invasive breast cancer	MK2206; Anastrozole	II	Active, not recruiting
NCT01625286	Women with ER+ advanced or metastatic breast cancer	AZD5363; paclitaxel	II	Active, not recruiting
NCT01783444	Advanced breast cancer	Exemestane; everolimus; capecitabine	II	Active, not recruiting
NCT01857193	Postmenopausal women with ER+ and HER2− locally advanced or metastatic breast cancer	Everolimus; exemestane; LEE011	II	Recruiting
NCT01499160	Postmenopausal women with advanced endocrine-resistant breast cancer	Everolimus; letrozole; lapatinib	II	Terminated
NCT00570921	Women with HR+ metastatic breast cancer after failure of aromatase inhibitor therapy	Everolimus; fulvestrant	II	Completed
NCT01797120	Postmenopausal women with HR+ metastatic breast cancer resistant to aromatase inhibitor therapy	Everolimus; fulvestrant	II	Active, not recruiting
NCT01234857	Women with HR+ breast cancer	Ridaforolimus; dalotuzumab	II	Completed
NCT01605396	Postmenopausal women with breast cancer	Ridaforolimus; dalotuzumab; exemestane	II	Active, not recruiting
NCT01272141	Women with triple-negative metastatic or locally advanced breast cancer	Lapatinib; everolimus	II	Terminated
NCT01111825	Women with metastatic HER2− amplified or TNBC	Temsirolimus; neratinib	II	Completed
NCT01520103	Women with advanced breast cancer	Vinorelbine; everolimus	II	Active, not recruiting
NCT02291913	Women with ER+ and HER2− advanced breast cancer	Everolimus; exemestane; tamoxifen; fulvestrant; anastrozole; letrozole; toremifene	II	Active, not recruiting
NCT02520063	Postmenopausal women with ER+ and HER2− breast cancer	Letrozole; everolimus; TRC105	II	Recruiting
NCT01283789	Women with HER2+ metastatic breast cancer	Lapatinib; everolimus	II	Active, not recruiting
NCT02216786	Women with ER+ advanced or metastatic breast cancer	AZD2014; everolimus; fulvestrant	II	Recruiting
NCT01783756	Women with HER2+ breast cancer with metastases in the brain who have progressed on trastuzumab	Lapatinib; everolimus; capecitabine	II	Active, not recruiting
NCT01305941	Women with HER2+ breast cancer brain metastases	Everolimus; vinorelbine; trastuzumab	II	Active, not recruiting
NCT02035813	Women with HER2− metastatic breast cancer and persisting HER2− circulating tumor cells	Everolimus; eribulin	II	Recruiting
NCT00062751	Postmenopausal women with locally advanced or metastatic breast cancer	Letrozole; temsirolimus	II	Completed
NCT00699491	Women with locally recurrent or metastatic breast cancer	Temsirolimus; cixutumumab	II	Active, not recruiting
NCT02258451	Women with HER2− and HR+ breast cancer with bone metastases treated with exemestane and everolimus	Exemestane; everolimus	II	Recruiting
NCT00912340	Women with breast cancer that has not responded to hormone therapy and has spread from where it started to other places in the body	Everolimus; trastuzumab	II	Active, not recruiting
NCT02456857	Women with localized TNBC with tumors predicted insensitive to standard neoadjuvant chemotherapy	Temsirolimus; liposomal doxorubicin; bevacizumab	II	Recruiting
NCT00915603	Women with HER2− metastatic breast cancer	Everolimus; bevacizumab; paclitaxel	II	Completed
NCT01627067	Women who are obese or overweight and postmenopausal with HR+ breast cancer that has spread to other parts of the body	Everolimus; exemestane; metformin	II	Active, not recruiting
NCT01495247	Women that have locally advanced or metastatic HER2− breast cancer	BEZ235; paclitaxel	II	Completed
NCT01430585	Women with ER+ and HER2− early breast cancer	PF-04691502; letrozole	II	Terminated
NCT01344031	Postmenopausal women with breast cancer that has spread to other parts of the body	MK2206; anastrozole; fulvestrant	I	Active, not recruiting
NCT01791478	Postmenopausal women with HR+ metastatic breast cancer	BYL719; letrozole	I	Recruiting
NCT01870505	Women with HR+ locally advanced unresectable or metastatic breast cancer	BYL719; letrozole; exemestane	I	Active, not recruiting
NCT01296555	Postmenopausal women with locally advanced or metastatic human HER2− and HR+ breast cancer	GDC-0032; letrozole; fulvestrant	I	Recruiting
NCT01482156	Women with ER+/HER2− metastatic breast cancer and metastatic renal cell cancer	Everolimus; BEZ235	I	Completed
NCT00317720	Women with HER2 overexpressing, PTEN-deficient metastatic breast cancer progressing on trastuzumab-based therapy	Everolimus; trastuzumab	I	Completed
NCT02123823	Women with locally advanced or metastatic breast cancer	Everolimus; exemestane; BI 836845	I	Active, not recruiting
NCT00426556	Women with HER2 overexpressing metastatic breast cancer	Everolimus; trastuzumab; paclitaxel	I	Completed
NCT02616848	Women with TNBC	Everolimus; eribulin	I	Recruiting
NCT01248494	Postmenopausal women with HR+ metastatic breast cancer	BEZ235; BKM120; letrozole	I	Completed
NCT00574366	Women with metastatic breast cancer	Everolimus; erlotinib	I	Completed
NCT00253318	Women with metastatic breast cancer	Docetaxel; everolimus; dexamethasone	I	Terminated
NCT02077933	Patients with advanced solid tumors, with dose-expansion cohorts in renal cell cancer, pancreatic neuroendocrine tumors, and advanced breast cancer women	Alpelisib; everolimus; exemestane	I	Active, not recruiting
NCT02058381	Premenopausal women with locally advanced or metastatic breast cancer	BKM120; BYL719	I	Active, not recruiting
NCT01220570	Women with breast cancer	Ridaforolimus; dalotuzumab	I	Completed
NCT01471847	Women with HER2+ locally advanced or metastatic breast cancer who failed prior to trastuzumab	BEZ235; trastuzumab	I	Completed

ER+: estrogen receptor–positive; HER2+: human epidermal growth factor receptor 2 positive; HER2−, human epidermal growth factor receptor 2 negative; HR+: hormone receptor–positive; TNBC: triple-negative breast cancer.

mTOR inhibits preclinical research in BC

On tamoxifen-resistant MCF-7 cell, combining the PI3K/mTOR inhibitors BEZ235 with GSK2126458 inhibited AKT signaling but BEZ235 showed greater effects than GSK2126458 on p70S6K and rpS6 signaling with effects resembling those of rapamycin.⁹⁷ Either everolimus or BEZ235 treatment may overcome acquired resistance to letrozole by targeting the PI3K/AKT/mTOR pathway in BC cell clones.⁹⁸ Moreover, BEZ235 targets both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER⁺ BC.⁶⁰ Inhibition of PI3K and mTOR induced long-term estrogen-deprived (LTED) cell apoptosis and prevented the emergence of hormone-independent cells.⁹⁹ Everolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells via G1 arrest due to downregulation of cyclin D1 and p21 and reduction of ER expression and transcriptional activity except the ER chaperone and heat shock protein 90 protein (HSP90).¹⁰⁰ Combination of the rapamycin analogue everolimus and an ATP-competitive mTOR inhibitor BEZ235 showed synthetic lethality in several mTOR-addicted triple-negative breast cancer (TNBC) cell lines.¹⁰⁷

mTOR inhibitors in HR+/HER2− BC

The phase-III trial compared the combination of temsirolimus plus letrozole to placebo plus letrozole in the first-line setting in patients with HR+ advanced BC (Table 3). The study was terminated after an interim analysis showed that combination treatment was associated with more grade 3 or 4 adverse events (37% vs 24%).¹⁰² The BOLERO-2 trial was randomized phase-III trial in advanced BC evaluating an mTOR inhibitor and aromatase inhibitor (AI) combination. It randomized 724 postmenopausal women with HR+ advanced BC who had relapsed or progressed on nonsteroidal AI in a 2:1 ratio to exemestane plus everolimus versus exemestane plus placebo. The addition of everolimus improved the progression-free survival (PFS) at central review to 10.6 months compared to 4.1 months with exemestane alone (p < 0.001), and the PFS at local review to 6.9 versus 2.8 months (p < 0.001).¹⁰³ These results led to the Food and Drug Administration (FDA) approval in August 2012 of everolimus with exemestane in the treatment of postmenopausal women with HR+/HER2− advanced BC after failure of treatment with letrozole or anastrozole.¹⁰⁴ The tamoxifen plus everolimus (TAMRAD) study evaluated the addition of everolimus to tamoxifen in a phase-II randomized study among HR+/HER2− AI-resistant metastatic BC patients. Addition of everolimus significantly improved the clinical benefit rate as well as time to progression and overall survival (OS), although the study sample size was limited to 111 patients.¹⁰⁵

Table 3.

Summary of completed randomized trials of mTOR inhibitors in BC.

Phase	Patient population (N)	Comparison arms	Key findings	References
Phase III	Postmenopausal women with HR+ locally advanced or metastatic breast cancer (n = 1112)	Letrozole + temsirolimus versus letrozole + placebo	Patients age ⩽ 65 years PFS: 9.0 versus 5.6 months (HR = 0.75; 95% CI = 0.60–0.93; p = 0.009); Adverse grade 3 to 4 events (37% vs 24%)	Wolff et al.¹⁰²
Phase III	Women with HR+/HER2− advanced breast cancer that recurred or progressed during/after treatment with nonsteroidal aromatase inhibitors (n = 724)	Everolimus + exemestane versus placebo + exemestane	OS: 31.0 versus 26.6 months (95% CI = 28.0–34.6 versus 22.6–33.1 months; p = 0.14); central PFS: 10.6 versus 4.1 months; HR = 0.36; 95% CI = 0.27–0.47; p < 0.001); local PFS: 6.9 versus 2.8 months (HR = 0.43; 95% CI = 0.35–0.54; p < 0.001).	Baselga et al.¹⁰³ and Piccart et al.¹⁰⁴
Phase II	postmenopausal women with HR+/HER2− AI-resistant metastatic breast cancer (n = 111)	Everolimus + tamoxifen versus tamoxifen	CBR: 61% versus 42% (95% CI = 47–74 versus 29–56); TTP: 8.6 versus 4.5 months (p = 0.002); HR: 0.54 versus 0.45 (95% CI = 0.36–0.81 vs 0.24–0.81); adverse events: fatigue (72% vs 53%), stomatitis (56% vs 7%), rash (44%vs7%), anorexia (43% vs 18%), and diarrhea (39% vs 11%)	Bachelot et al.¹⁰⁵
Phase II	Postmenopausal women with operable ER+ breast cancer (n = 270)	Letrozole + everolimus versus letrozole + placebo	RR: 68.1% versus 59.1%; AR: 57% versus 30%	Baselga et al.¹⁰⁶
Phase III	Postmenopausal women with ER+ advanced/recurrent breast cancer that was refractory to any nonsteroidal aromatase inhibitor (NSAI; n = 106)	Everolimus + exemestane versus placebo + exemestane	PFS: 8.5 versus 4.2 months	Ito et al.¹⁰⁷
Phase III	Women with HER2+, trastuzumab-resistant, advanced breast carcinoma who had previously received taxane therapy (n = 569)	Everolimus + vinorelbine + trastuzumab versus placebo + vinorelibine + trastuzumab	PFS: 7.0 versus 5.8 months (95% CI = 6.74–8.18 versus 5.49–6.90; p = 0.0067); subgroup analysis: PFS improved in HR− cancers but not in HR+ cancers	André et al.¹⁰⁸
Phase III	Women with locally assessed HER2+ advanced breast cancer (n = 719)	Everolimus + paclitaxel + trastuzumab versus placebo + paclitaxel + trastuzumab	In HR−/HER2+ subpopulation, PFS: 20.27 versus 13.08 months (95% CI = 14.95–24.08 versus 95% CI = 10.05–16.56; p = 0.0049)	Hurvitz et al.¹⁰⁹
Phase I	Women with HER2+ metastatic breast cancer pretreated with trastuzumab (n = 50)	Everolimus + vinorelbine + trastuzumab	ORR: 19.1%; DCR: 83.0%; PFS: 30.7 weeks	Jerusalem et al.¹¹⁰

PFS: progression-free survival; HR: hazard ratio; OS: overall survival; CBR: clinical benefit rate; TTP: time to progression; RR: response rate; AR: antiproliferative response; ORR: overall response rate; DCR: disease control rate; CI: confidence interval; HR+: hormone receptor–positive; HER2+: human epidermal growth factor receptor 2 positive; HER2−: human epidermal growth factor receptor 2 negative; ER+: estrogen receptor–positive; AI: aromatase inhibitor.

Phase-II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in 270 postmenopausal women with ER+ early-stage BC for 4 months led to an increase in response rate (68.1% vs 59.1%) and an higher antiproliferative response (57% vs 30%) in comparison to letrozole alone.¹⁰⁶ The role of everolimus addition to endocrine therapy in the adjuvant setting was evaluated by studies, for instance, the UNIRAD and SWOG/NSABP S1207 phase-III randomized studies in patients with early ER+/HER2− BC at high risk of relapse, which possibly provide an evidence base for the use of these agents in patients prior to the development of secondary hormone resistance.^107,111,112 This study will help to determine which lines of therapy are most appropriate for mTOR pathway inhibition plus endocrine therapy, as well as assessing the potential benefit of mTOR inhibition in all lines of treatment, as a mainstay of endocrine therapy.

mTOR inhibitors in HER2+ BC

The BOLERO-3 trial was a phase-III study which randomized patients previously treated with taxane and trastuzumab to everolimus 5 mg or placebo in combination with vinorelbine and trastuzumab, testing the hypothesis that the addition of everolimus could overcome trastuzumab resistance (Table 3). The addition of everolimus improved the PFS from 5.8 to 7.0 months (HR = 0.78; p = 0.0067).¹⁰⁸ The role of everolimus in HER2+ BC, however, remains unclear, especially with the approved indications for trastuzumab, emtansine, lapatinib, and pertuzumab.

The BOLERO-1 study was a phase-III randomized study evaluating everolimus in combination with paclitaxel and trastuzumab in HER2+ advanced BC in the first-line setting, testing the potential for everolimus to circumvent trastuzumab resistance. PFS in the full population was not statistically improved at 14.9 months in the group receiving everolimus compared to 14.5 months in the group receiving placebo (p = 0.1167). In the HR-negative subpopulation, there was a 7.2 months prolongation in PFS with the addition of everolimus (20.27 vs 13.08 months, p = 0.0049), though the protocol-specified significance threshold (p = 0.0044) was not crossed. Although PFS was not significantly different between groups in the full analysis population, the 7.2-month prolongation we noted with the addition of everolimus in the HR−/HER2+ population warrants further investigation, even if it did not meet prespecified criteria for significance. Proactive monitoring and early management of adverse events in patients given everolimus and chemotherapy are crucial.¹⁰⁹ Everolimus combined with weekly vinorelbine and trastuzumab generally was well tolerated and had encouraging anti-tumor activity in heavily pretreated patients with HER2+ metastatic BC that progressed on trastuzumab.¹¹⁰

mTOR inhibitors in HER2− BC

In the metastatic setting, a randomized phase-II trial evaluated the combination of paclitaxel and bevacizumab with or without everolimus in 112 women with untreated metastatic HER2− BC. In a preliminary report, although response rates and median PFS were better with everolimus, the improvement in efficacy did not reach statistical significance, possibly attributed to the higher toxicities and lower dose intensity achieved in the everolimus arm. Possible explanation for these results may include lower dose intensity in the everolimus arm, treatment of less resistant patients, or intrinsic differences in everolimus activity when added to antiestrogen versus chemotherapy.¹¹³

mTOR inhibitors in TNBC

First, a phase-II neoadjuvant study including 50 TNBC patients tested the addition of everolimus to weekly paclitaxel for 12 weeks, followed by fluorouracil, epirubicin, and cyclophosphamide (FEC) every 3 weeks for four cycles. A higher clinical response rate was showed in the arm including everolimus (48% vs 30%), but this did not reach statistical significance.¹¹⁴ The hypothesis of synergism between mTOR inhibitors and taxanes was also under investigation in a phase-III study, where 403 patients with HER2− BC showing no response after four cycles of neoadjuvant epirubicin and cyclophosphamide (with or without bevacizumab) were randomized to receive either paclitaxel alone or paclitaxel plus everolimus. Pathological complete response (pCR) was achieved in 3.6% of patients treated with paclitaxel and everolimus versus 5.6% in the control arm (p = 0.476), making this a negative study.¹¹⁵

Toxicities and adverse event

mTOR inhibitors are associated with certain class-effect toxicities such as hyperglycemia and rash. Stomatitis was the most common adverse event reported in both the TAMRAD and the BOLERO-2 trials. It is clinically distinct from conventional chemotherapy-associated mucositis, being characterized by aphthous ulcerations and grey-white pseudomembranous changes.^116,117 mTOR inhibitors may cause hyperglycemia and hyperlipidemia, with elevations in both low-density lipoprotein cholesterol and triglycerides. Everolimus is contraindicated in patients with uncontrolled diabetes and requires optimization of glycemic control prior to initiation.¹¹⁸ Patients on everolimus can be predisposed to infections, including bacterial, fungal, and viral infections, as well as reactivation of hepatitis B virus. Baseline screening for hepatitis B and other infections such as hepatitis C, HIV, and tuberculosis should be considered before drug initiation for patients at risk of reactivation with prior exposure or certain risk factors. Moreover, everolimus is also associated with increased incidence of fatigue, asthenia, and anorexia.^119,120

Prospect

BC therapy has undergone a remarkable revolution in the past two decades with the advent of truly targeted therapies with remarkable efficacy and tolerable toxicities. mTOR inhibitors together with AKT and PI3K inhibitors still remain an attractive and promising therapeutic option for the treatment of BC, and ongoing as well as future clinical studies will reveal whether they can be used for the therapy of this devastating disease. Ongoing studies will explore the efficacy of the addition of next-generation mTOR pathway inhibitors to existing endocrine treatments and with inhibitors of upstream and downstream effectors. Key considerations to be addressed in future researches include determining which is the optimal treatment combination, whether total blockade or partial inhibition of the mTOR pathway is preferable, and whether isoform-specific or pan-PI3K inhibition will be more beneficial to BC patients. Identifying new methods of pre-selecting patients who will benefit most from treatment will be also paid close attention to in the future, for instance, defining molecular signatures for biomarker screening and large-scale genomic screening. We are looking forward to investigating the additional benefit to patients of mTOR pathway inhibitors treatments in the treatment of BC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Targeting the mTOR pathway in breast cancer

Abstract

Keywords

Introduction

The mTOR pathway

Involvement of mTOR pathway in BC

mTOR pathway and ER

mTOR pathway and HER2

mTOR pathway inhibitors

mTOR inhibitors in BC therapy

mTOR inhibits preclinical research in BC

mTOR inhibitors in HR+/HER2− BC

mTOR inhibitors in HER2+ BC

mTOR inhibitors in HER2− BC

mTOR inhibitors in TNBC

Toxicities and adverse event

Prospect

Footnotes

Declaration of conflicting interests

Funding

References