Endocrine DOI 10.1007/s12020-014-0380-1

REVIEW

PI3K/Akt/mTOR signaling in medullary thyroid cancer: a promising molecular target for cancer therapy Gloria Irene Manfredi • Alessandra Dicitore Germano Gaudenzi • Michele Caraglia • Luca Persani • Giovanni Vitale

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Received: 21 June 2014 / Accepted: 1 August 2014 Ó Springer Science+Business Media New York 2014

Abstract The phosphatidylinositol 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) pathway is a central hub for the regulation of cell proliferation, apoptosis, cell cycle, metabolism, and angiogenesis. Several studies have recently suggested that the PI3K/Akt/mTOR signaling pathway is implicated in the pathogenesis and progression of neuroendocrine tumors. Medullary thyroid cancer (MTC) is a neuroendocrine tumor developing from the C cells of the thyroid. Mutations in the RET protooncogene are involved in the pathogenesis of several forms of MTC. The deregulation of the PI3K/Akt/mTOR pathway seems to contribute to the tumorigenic activity of RET proto-oncogene mutations. Targeting this pathway through specific inhibitors at simple or multiple sites may represent an attractive potential therapeutic approach for patients with advanced MTCs. The aim of this review is to examine the role of the PI3K/Akt/mTOR pathway in the development and progression of MTC and the new therapeutic options that target this signaling pathway. Keywords Medullary thyroid cancer
mTOR
PI3K/ Akt
Targeted therapy

G. I. Manfredi
G. Gaudenzi
L. Persani
G. Vitale Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy A. Dicitore
L. Persani
G. Vitale (&) Laboratory of Endocrine and Metabolic Research, Istituto Auxologico Italiano IRCCS, University of Milan, Via Zucchi 18, Cusano Milanino (MI), 20095 Milan, Italy e-mail: [email protected] M. Caraglia Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy

Introduction Medullary thyroid cancer (MTC) is a neuroendocrine tumor (NET) developing from the C cells of the thyroid [1, 2]. This tumor accounts for about 5–10 % of all thyroid cancers, and it is characterized by the production of calcitonin [1]. MTC occurs in the sporadic form in about 70–80 % of cases, whereas the remaining 20–30 % is represented by three familial forms: multiple endocrine neoplasia type 2A (MEN 2A), multiple endocrine neoplasia type 2B (MEN 2B), and familial MTC not associated with MEN (FMTC) [3–7]. Surgery is the predominant treatment in MTC, while molecular targeted therapy is recently used in patients with advanced disease. Chemotherapy and radiotherapy play a marginal role in advanced MTC. This tumor is resistant to most of the common chemotherapy drugs. External radiation therapy is currently used only for palliation of locally recurrent tumors or symptomatic bone, central nervous system, and mediastinal metastases [8, 9]. Several studies have recently suggested that the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway might be involved in NET tumorigenesis and progression, given its critical role in cell proliferation and angiogenesis [10–19]. In this context, it has been demonstrated that PI3K signaling pathway is constitutively activated by several aberrant receptor tyrosine kinases (RTKs), leading to high cell proliferation and vascularity [20–22]. NETs can also harbor different alterations of PI3K signaling, such as mutations in phosphatase and tensin homolog (PTEN), tuberous sclerosis complex 2 (TSC2) or PIK3CA genes, and other pathway abnormalities that lead to mTOR overexpression or Akt activation [20, 23–25]. For all these reasons, the inhibitors of PI3K/Akt/mTOR pathway are emerging as a new targeted therapeutic strategy for NETs [16, 21, 26–28].

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Growth factors

FGF-3 GDNF VEGF PDGF

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3 mTORC1 inhibitors -Rapamycin -Everolimus -Temsirolimus

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Proliferation Cell cycle progression Differentiation Angiogenesis Inhibition of apoptosis Migration Protein synthesis

Fig. 1 Overview of the PI3K/Akt/mTOR signaling pathway in cancer cells. This pathway is commonly up-regulated in tumor cells through: 1 direct upstream stimulation (receptor tyrosine kinases: RTKs); 2 intrinsically via activating genetic alterations in PI3K, Akt or mTOR, or via loss of function in PTEN; 3 indirect activation via

cross-talk with Ras pathway. Specific inhibitors of PI3K, Akt, and mTOR could represent a potential therapeutic innovative strategy in tumors showing an overactivation of this pathway, such as medullary thyroid cancer

Indeed, in May 2011, the mTOR inhibitor everolimus was approved by FDA for the treatment of patients with progressive unresectable pancreatic NETs. It has recently been observed that MTC cell proliferation also depends on the activation of the PI3K/Akt/mTOR signaling cascade [29–32]. Therefore, inhibition of this pathway may be a novel therapeutic approach for MTC [33, 34]. The aim of this review is to examine the role of the PI3K/Akt/mTOR pathway in the development and treatment of MTC.

factors (vascular endothelial growth factor, insulin-like growth factor-1/2, fibroblast growth factor-3, plateletderived growth factor, glial cell line-derived neurotrophic factor, etc.), amino acids, and hypoxia [20, 35]. The main pathway that controls mTOR functions is the PI3K/Akt signaling. Activation of PI3K/Akt is driven by several RTKs, stimulated by specific growth factors (Fig. 1). When these receptors are activated, PI3K is recruited and catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) in phosphatidylinositol3,4,5-trisphosphate (PIP3) and, thus, activates Akt. The latter, through the inactivation of mTOR’s negative regulators ‘‘TSC2’’ and ‘‘proline-rich Akt substrate 40 (PRAS40),’’ enables mTOR to carry out its multiple functions [26]. On the other hand, the main mTOR negative regulator is PTEN, a tumor suppressor which inhibits Akt, converting PIP3 back to PIP2 [36]. mTOR exists in two multiprotein complexes: mTOR complexes 1 and 2 (mTORC1 and mTORC2) (Fig. 1). The first consists of mTOR, mammalian LST8 (mLST8),

The PI3K/Akt/mTOR pathway and its role in carcinogenesis mTOR is a serine/threonine kinase involved in the regulation of cell proliferation, apoptosis, cell cycle, angiogenesis, metabolism, and protein synthesis [26]. mTOR signaling is regulated by numerous stimuli, such as growth

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PRAS40, and raptor [10]. Its activation results in the phosphorylation of 4E-binding protein (4EBP1) and ribosomal protein S6 kinases (S6K1), both of which regulate transcription and translation of critical growth genes [12, 37, 38]. 4EBP1 prevents the initiation of mRNA translation by binding to eukaryotic initiation factor 4E (eiF4E). When phosphorylated by mTORC1, 4EBP1 reduces its affinity for eiF4E, allowing eiF4E to stimulate translation initiation. Similarly, the phosphorylation of S6K1 promotes mRNA translation by phosphorylating or binding multiple proteins, which collectively affects translation initiation and elongation [39]. Interestingly, the activation of mTORC1 and then the phosphorylation of these downstream targets (4EBP1 and S6K1) preferentially lead to the translation of mRNAs for pro-tumorigenic genes implicated in cell cycle progression, cell differentiation, cell death, angiogenesis, and migration [12, 39]. mTORC2 consists of mTOR, mLST8, mammalian stress-activated protein kinase-interacting protein (mSIN1), protor, and rictor [40, 41]. mTORC2 phosphorylates Akt at Ser473 leading to full Akt activation and then to mTORC1 stimulation, and regulates the phosphorylation of PKCa. It has also been supposed that it controls cytoskeleton organization, cell cycle progression, and cell survival [40–44]. As highlighted before, given that the PI3K/Akt/mTOR signaling controls cell proliferation, cell death, and angiogenesis, an inappropriate activation of this pathway can result in cancer [13–16, 45]. Several genetic alterations are known to induce an overactivation of the PI3K/Akt/mTOR signaling, but the most common mechanism observed in human cancers is the aberrant activation of RTKs [46–49].

Relevance of the PI3K/Akt/mTOR pathway in MTC Activating mutations of the RET proto-oncogene are implicated in the pathogenesis of several forms of MTC [50]. Germline mutations of this gene have been detected in about 88–98 % of familial forms [5, 50], while somatic mutations of RET gene have been reported in about 23–70 % of sporadic MTCs [31]. The RET proto-oncogene encodes a RTK expressed in cells derived from the neural crest, including C cells [2]. The ligands of the RET receptor are growth factors belonging to the glial cell line-derived neurotrophic factor (GDNF) family [2, 3]. The activation of this receptor regulates growth, survival, differentiation, and migration of cells through the stimulation of several intracellular pathways, including RAS/RAF/MEK/ERK and PI3K/Akt/ mTOR (Fig. 1) [8, 51, 52]. Segouffin-Cariou [53] reported that rat fibroblasts expressing a MEN 2A mutant form of RET showed a constitutive activation of PI3K and Akt. Interestingly, the

overactivation of PI3K/Akt/mTOR pathway results to be a limiting factor for RET-MEN 2A oncogenic potential. Indeed, Drosten et al. [54] constructed adenoviral vectors expressing a truncated form of RET, which eliminates its oncogenic functions, and studied its effects on TT cells, in terms of cell cycle, proliferation, apoptosis, viability, and RET downstream signaling. The expression of the truncated RET was associated to a suppression of Akt activity and to a reduction of cell proliferation and viability. Moreover, they detected elevated levels of cyclin D1 mRNA, a cell cycle regulator which is known to be controlled by PI3K/Akt pathway. In summary, these results suggest that oncogenic RET activity in MTC is partially modulated by the overactivation of the PI3K/Akt/mTOR pathway [54]. Tamburrino et al. [31] reported that mTOR pathway has a role in the primary phases of MTC tumorigenesis. In fact, mTOR activation appeared to be an early event in C-cell transformation. They also showed that the activation of mTOR was higher in lymph-node metastasis than in primary MTC, suggesting a role of this pathway in tumor progression as well as initiation. In support of this hypothesis, Kouvaraki et al. [55] reported a strong correlation between eiF4E expression and tumor stage in thyroid cancer. Interestingly, a strong expression of the most critical downstream targets of mTOR signaling (4EBP1 and eiF4E) was commonly observed in MTC [55]. In order to deepen the possible role of mTOR in the development of familial MTC, Rapa et al. [30] screened 65 cases of MTC (11 of which occurring as familial tumors in MEN 2 syndrome) for PI3K mutations. Although they did not find any mutations, PI3K/Akt/mTOR pathway was highly activated in MTC and correlated to the germline RET mutation status. In fact, the vast majority of cases with RET germline mutations were highly positive for phospho-mTOR and phospho-S6K1. On the other hand, MTC with sporadic RET mutations or wild-type RET showed heterogeneous activation of mTOR pathway. Therefore, the association between RET status and mTOR activity appears to be independent on the type of mutation, but dependent on its hereditary nature. Although, it is difficult to elucidate the molecular mechanism at the basis of such phenomenon, it has been recently demonstrated that mTOR activity was exclusively RET-dependent in MTC cancer cell lines with endogenous mutations of the proto-oncogene [56]. In fact, the treatment of two different RET mutant MTC cell lines (TT and MZ-CRC-1) with AST487, a RET kinase inhibitor, inhibited cell proliferation and showed a profound suppression of the mTORC1 signaling in a dose-dependent manner. On the other hand, mTOR effectors were not suppressed in the FTC133 cell line, which harbors a homozygous PTEN loss of function, but it is wild type for RET [56].

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However, the aberrant activation of RET seems to be not the only factor implicated in the overactivation of PI3K/ Akt/mTOR pathway. In fact, while activation of mTOR signaling has been reported in 96–100 % of MTC [31, 55], the rate of activating RET mutations, particularly for sporadic MTC, is lower [57]. Therefore, several other factors, that need to be explored, may be involved in the overactivation of mTOR signaling in MTC.

Utility of PI3K/Akt/mTOR inhibitors in MTC: preclinical studies RAD001 (everolimus), a synthetic derivative of rapamycin, is a potent selective mTORC1 inhibitor (Fig. 1), which exerts its activity binding with an intracellular protein, FKBP12. This complex interacts with mTORC1 and inhibits signaling downstream [16]. In 2010, GrozinskyGlasberg et al. [58] investigated the in vitro effects of everolimus in a human MTC cell line (TT) and in two primary cultures from human MTC. Everolimus significantly inhibited cell viability and decreased cell number in a dose-dependent manner and increased the number of TT cells in the G0/G1 phase of the cell cycle, but no effect of this drug on the modulation of apoptosis has been reported. Calcitonin and CEA levels appeared not to be affected by RAD001 in MTC cells, suggesting that this drug does not modify hormone secretion. Most of these data were confirmed in other MTC models. We recently showed a potent anti-proliferative effect of everolimus in both TT and MZ-CRC-1 cell lines. This antitumor activity was mediated by the induction of cell cycle arrest in G0/G1 phase, but not apoptosis. Interestingly, this study revealed that everolimus can also induce cellular senescence; indeed, the percentage of TT senescent cells, positive at the staining for b-galactosidase activity, doubled after the treatment with everolimus. In addition, incubation of TT and MZ-CRC-1 cell lines with everolimus did not influence calcitonin secretion [59]. The cell cycle arrest and the induction of senescence obtained specifically with mTOR inhibition could be mediated by cyclin D downregulation and/or p21 upregulation, which are known cell cycle regulators and participate in the launch of the senescence program [55, 60, 61]. Although everolimus has demonstrated significant growth-inhibitory effects on a variety of human malignancies, it is increasingly recognized that its mechanism of action may not be sufficient for achieving a potent anticancer effect due to its inability to inhibit mTORC2 activity. Indeed, the specific inhibition of mTORC1 by everolimus seems to be associated with a paradoxical Akt activation mediated by mTORC2, and by the inhibition of a negative feedback loop via S6K1 leading to the attenuation

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of its therapeutic effects [26]. Therefore, an upstream inhibition or targeting the PI3K/AKT/mTOR pathway at multiple levels may represent innovative therapeutic strategies to maximize blockade of the mTOR pathway and related antitumor activity. In fact, an upstream block of the PI3K/Akt/mTOR pathway through PI3K inhibitors seems to have additional effects on apoptosis (Fig. 1) [62]. Treatment of TT cells with the PI3K inhibitor LY294002 significantly suppressed cell proliferation through the inhibition of the phosphorylation of Akt and the induction of apoptosis. Moreover, LY294002 induced a progressive decrease in the expression of Chromogranin A and ASCL1, both proteins critical for the neuroendocrine cell differentiation and hormone production [62]. Unfortunately, LY294002 is too insoluble, making it ineffective in cancer clinical trials [63]. However, MK-2206, a novel orally bioavailable Akt-specific inhibitor, showed in vitro antitumor effects comparable to that observed after LY294002: 1) suppression of TT cell proliferation, 2) induction of apoptosis, and 3) decrease in expression of chromogranin A and ASCL1 [57]. Interestingly, the antitumor effect of MK-2206 resulted to be independent of RET inhibition, as RET activity was not blocked by MK-2206 incubation [57]. Further studies analyzed the possible effects of combined targeted therapies, which act at different sites of the PI3K/Akt/mTOR pathway (Fig. 1). The efficacy of BEZ235, a dual PI3K/mTOR inhibitor, which reduces PI3K, mTORC1, and mTORC2 kinase activity by competitive binding to the ATP-binding cleft of these enzymes, has been evaluated in vitro treating several thyroid cancer cell lines of four major pathological types [64]. BEZ235 was found to effectively inhibit cell proliferation in thyroid cancer cell lines. Anaplastic thyroid cancer cells were the most sensitive, followed by follicular undifferentiated, medullary, and well-differentiated thyroid cancer cell lines. Cancer cells harboring a PI3K gain-offunction mutation or a PTEN deletion demonstrated higher PI3K/mTOR pathway activity and greater sensitivity to BEZ235. In addition, the expression of p-S6 ribosomal protein and p27 correlated with the sensitivity of BEZ235 in thyroid cancer [64]. The concomitant targeting of RET and mTOR may represent another innovative therapeutic strategy in MTC. In fact at least in vitro, combined treatment with AST487 (a RET kinase inhibitor) and INK128 (an ATP-competitor inhibitor of mTORC1 and mTORC2) at low concentrations synergistically suppressed proliferation of RET mutant MTC cell lines through the induction of apoptosis [65]. In this regard, several natural compounds derived from plants have been tested for antitumor and cytotoxic properties. Withanolides and flavonoids are known to inhibit PI3K/ Akt/mTOR pathway. Several novel withanolides extracted

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from Physalis plant resulted to suppress activation of both RET and PI3K/Akt/mTOR signaling, leading to the inhibition of cell growth through cell cycle arrest and the induction of apoptosis in MTC cell lines [34]. The Ras/Raf/MEK/ERK signaling represents another crucial pathway involved in the pathogenesis and progression of MTC, particularly in RET-negative MTC [65]. Interestingly, Ras is capable of signaling through both Ras/ Raf/MEK/ERK and PI3K/Akt/mTOR pathways. In fact, both pathways are known to interact at multiple sites, and this can explain the occurrence of resistance to anticancer agents that target a single signaling [66]. Jin et al. [67] evaluated the antitumor activity of RAF265/CHIR-265, an ATP-competitive pan-RAF inhibitor, in combination with the dual PI3K/mTOR inhibitor BEZ235 in MTC preclinical models. This treatment strongly suppressed cell proliferation, both in vitro and in vivo, by inhibiting ERK and PI3K signaling and inducing cell cycle arrest in G0/G1 phase.

Everolimus as a possible therapy for MTC: clinical studies Few preliminary clinical studies suggest that mTOR inhibitors such as everolimus could be a possible treatment option for patients affected by MTC. Druce et al. [68] reported a case of a 57-year-old lady with MEN 2A and progressive metastatic MTC treated with everolimus (10 mg daily). The treatment was well tolerated and the disease, that was previously progressive, initially stabilized. Interestingly, the calcitonin doublingtime increased from 5.5–62 months during the treatment over the first 10 months. Subsequently, over a further 12 months, a decrease in the calcitonin doubling-time (27.9 months) and some minor size increase in the mediastinal lymph nodes and a new liver lesion were observed. Faggiano et al. [59] recently treated two patients affected by progressive metastatic MTC with everolimus in combination with somatostatin analog (octreotide). Patient # 1 was treated with everolimus initially at the dose of 10 mg a day, and then reduced to 5 mg a day because of hematological toxicity (grade 3 thrombocytopenia and grade 2 anemia). Serum calcitonin rapidly decreased one month after the start of everolimus, but it moderately rose again after the change of the dosage. Serum carcinoembryonic antigen (CEA) variations paralleled calcitonin variations, though being lower. Neurological symptoms secondary to vertebral metastasis, such as paraplegia, pain, and bladder dysfunction, improved during the treatment. Patient # 2 was initially treated with everolimus at the dose of 5 mg a day, because of a preexisting mild anemia, then, after a two months treatment, it was increased to 10 mg daily because no change in the serum markers occurred. The higher

dosage determined a marked decrease of serum calcitonin and CEA. Bone metastases were substantially unchanged, while a dramatic decrease in tumor vascularization and size was observed in laterocervical lymph node metastases. A multicenter phase II trial [69] recently investigated the efficacy and tolerability of everolimus in patients with progressive locally advanced or metastatic thyroid cancer of all histological subtypes. Forty patients with progressive, locally advanced, or I131 refractory thyroid cancers (9/40 MTC) were enrolled in this study, and they self-administered everolimus 10 mg orally once a day, until severe toxicity or disease progression. They achieved a confirmed objective response in 2/40 (5 %) patients, and a stable disease was described in 29/40 (76 %) patients. Clinical benefits were reported in 50 % patients. They did not observe any association between efficacy and histology, but it is reported a high proportion of disease control (stable disease [ 12 months) in patients with differentiated thyroid cancer and MTC. The progression free survival according to histology was not reached in MTC, but all patients enrolled with this kind of neoplasm showed at least a stable disease (2/ 9 experienced tumor shrinkage) at the end of the treatment, despite the initial condition of progressive, locally advanced, or metastatic cancer. Serum calcitonin and CEA levels were measured for 9 MTC patients. Calcitonin and CEA levels were C 50 % lower than baseline in 3 (30 %) and 4 (44 %) patients with MTC, respectively. Adverse effects registered during the therapy were predominantly grade 1 or 2; the most common effects reported were mucositis (84 %), anorexia (44 %), liver enzymes elevation (26 %), and rash (21 %). Some ongoing clinical trials are currently evaluating the antitumor activity of everolimus, alone or in combination with other drugs, in MTCs. A phase II trial is studying the efficacy of everolimus in patients with progressive or recurrent, unresectable, or metastatic MTC (Identifier: NCT01118065) [70]. Two studies are evaluating the efficacy of everolimus associated with the ‘‘pan-receptor’’ somatostatin ligand pasireotide (SOM230) in patients with progressive and advanced MTC (NCT01625520 and NCT01270321) [70]. Another trial is studying the effects of everolimus associated with cixutumumab, a monoclonal antibody against IGF-I receptor, in advanced neuroendocrine tumors, including MTC (Identifier: NCT01204476) [70]. Moreover, the effects of temsirolimus, another mTOR inhibitor, in combination with chemotherapy (vinorelbine ditartrate) are currently being studied in several neoplasms, including MTC (Identifier: NCT01155258) [70].

Conclusions Recent findings show that the overactivation of the PI3K/ Akt/mTOR pathway plays a crucial role in the

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pathogenesis of MTC. Indeed, most of the tumorigenic effects of RET mutations seem to be mediated by the activation of PI3K/Akt/mTOR cascade. Few preliminary clinical studies show the potential beneficial effects of everolimus in the treatment of MTC, but further studies are needed to confirm this hypothesis. In addition, the development of novel therapeutic strategies targeting RET/ PI3K/Akt/mTOR pathway at multiple sites and other survival pathways for cancer cells, such as Ras/Raf/MEK/ ERK signaling, may open a new scenario in the treatment of MTC. Acknowledgments This work was partially supported by the Italian Ministry of Education, Research and University (FIRB RBAP11884 M). Conflict of interest No potential conflict of interest relevant to this article was reported.

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