Temsirolimus (CCI-779, Torisel®, Pfizer) was the first mTOR inhibitor developed and identified to have antitumor activity [39]. After years of development, it was recently approved for the treatment of advanced renal cell carcinomas and pancreatic neuroendocrine tumors. Temsirolimus forms a complex by binding to the intracellular protein peptidylprolyl cis-trans isomerase FKBP1A (FKBP-12) that inhibits the activity of mTOR. This subsequently results in a G1-phase growth arrest, blocking its ability to phosphorylate S6K1 and the ribosomal protein S6, a reduction of HIF-1α, and VEGF expression [40]. In patients with advanced NET, a phase II study was conducted to evaluate the safety, efficacy, and pharmacodynamics of temsirolimus. Thirty-six patients with advanced and progressive NETs (21 carcinoids and 15 pNET) received weekly doses of intravenous temsirolimus. There was no difference in the objective response rates between carcinoids (4.8 %) and pNET (6.7 %). The intent-to-treat response rate for the entire cohort was 5.6 % (95 % CI 0.6–18.7 %), median TTP was 6 months, and 1-year PFS was 40.1 %. Two patients achieved partial responses (one patient with pNET and one patient with carcinoid tumor). Overall, the treatment was well tolerated with fatigue (78 %), hyperglycemia (69 %), and rash/desquamation (64 %) being the most common drug-related adverse events of all grades after a median of four cycles delivered per patient [30].

Pharmacodynamic analysis demonstrated that temsirolimus effectively inhibited the PI3K/Akt/mTOR pathway. Phosphorylation of the ribosomal protein S6 was significantly depressed (p = 0.02). Additionally, patients with an increased expression of phosphorylated Akt (p = 0.041) and a decreased expression in phosphorylated mTOR after 2 weeks of treatment were both associated with an increase in time to progression (p = 0.04 and p = 0.05, respectively). Elevated baseline levels of phosphorylated mTOR predicted a better response (p = 0.01). Even though the results of this study revealed temsirolimus value in downregulating mTOR’s downstream signaling, the authors concluded that it has limited clinical efficacy and does not support its use as monotherapy in patients with advanced NETs [30].

The limited benefit but excellent tolerability of this agent lends it to be partnered with additional agents. Preclinical studies suggest enhanced antitumor effects with temsirolimus and VEGF-targeted therapy. Therefore, a phase II study of temsirolimus in combination with bevacizumab, an anti-VEGF-A monoclonal antibody, in advanced, recurrent, or progressive pNETs (NCT01010126), was completed. Of the 56 patients eligible for response assessment, partial responses were seen in 41 % (23 of 56) patients, and 79 % of the patients (44/56) had disease stability at 6 months. Median progression-free survival was 13.2 months and overall survival was 34 months. This combination was very well tolerated with minimal toxicity. A minority of patients developed grade 3 or 4 drug-related adverse events including hypertension (18 %), hyperglycemia (13 %), fatigue (11 %), leukopenia (9 %), headache (9 %), proteinuria (7 %), and hypokalemia (7 %). The ORR of 41 % exceeds that reported to date for monotherapy with any targeted agent in pNET and provides compelling evidence to pursue this combination further [41].

Everolimus, a second-generation mTOR inhibitor, was recently approved for use in pNETs after demonstrating significant improvements in outcomes [25, 42]. Everolimus (40-O-(2 hydroxyethyl) derivative of rapamycin, RAD001, Afinitor®, Novartis) is an oral mTOR inhibitor that selectively inhibits mTORC1 and is absorbed rapidly, achieving peak concentration after 1.8 h and reaching steady state after 7 days [16]. It binds to FKBP-12 in a similar mechanism as temsirolimus, by forming a complex that induces the inhibition of mTOR kinase activity. It reduces the activity of mTOR’s downstream proteins by blocking phosphorylation of 4E-BP1 and inactivating S6K1. It also inhibits expression of HIF-1α and decreases expression of VEGF. Everolimus has been shown to reduce cell proliferation, angiogenesis, and glucose uptake from several in vivo and in vitro studies. In addition, it has demonstrated a potent dose-dependent inhibition of cell growth comprising G0/G1 phase arrest as well as induction of apoptosis in human pancreatic BON cells, a human pancreatic NET cell line exhibiting constitutively activation of PI3K/Akt/mTOR pathway [43]. Everolimus treatment also significantly inhibited cell proliferation in the rat insulinoma NET cell line (INS1) and decreased phosphorylation of all downstream targets of Akt, TSC2, and mTOR [44].

In a phase I study evaluating patients with advanced solid tumors including NETs, everolimus induced a dose and schedule-dependent inhibition of the PI3K/Akt/mTOR pathway. At 10 mg/day and ≥50 mg/week, there was an almost complete inhibition of phosphorylated ribosomal protein S6 (p < 0.001), expression of eIF4G (p < 0.001), and reduction of phosphorylated 4E-BP1 (p = 0.058). Furthermore, there was an overall increase in Akt phosphorylation (p = 0.006) and in cellular proliferation (p = 0.014). A total of four out of the 55 patients reached a clinical benefit (a partial response was observed in one patient, and three had stable disease). The dose-limiting toxicities consisted of grade 3 stomatitis, neutropenia, and hyperglycemia which were seen in five patients [45].

A phase II trial evaluated the activity of everolimus in combination with octreotide. Thirty patients with carcinoid and 30 patients with pNETs were treated with everolimus at 5 or 10 mg/day in combination with octreotide LAR 30 mg every 4 weeks. The intent-to-treat response rate was 20 %. The analysis showed that 13 (22 %) patients achieved partial responses, 42 (70 %) patients had stable disease, and 5 (8 %) patients progressed. The median PFS was 60 weeks. Median overall survival had not been reached; however, 1-, 2-, and 3-year survival rates were 83 %, 81 %, and 78 %, respectively. At study entry, among 37 patients with high chromogranin A levels, 26 patients (70 %) attained normalization or a reduction of more than 50 %. In pre- and posttreatment tumor biopsies, mean tumor Ki-67 expression decreased significantly from 6.7 to 2.1 % (p = 0.04). Overall, compared to patients that received the 5 mg dose, patients that received the 10 mg dose obtained a higher response rate (30 vs. 13 %) and had a prolonged median PFS (72 vs. 50 weeks). The most common toxicity was mild aphthous oral ulceration. Significant toxicities were uncommon and only 11 % of patients developed grade 3/4 hypophosphatemia, fatigue, and diarrhea [46].

Three RADIANT (RAD001 in advanced neuroendocrine tumors) trials were then designed to study efficacy of everolimus in NETs of different origins. These confirmed the value of everolimus in patients with advanced NETs. RADIANT-1 is a second open-label phase II trial in 160 patients with progressive chemotherapy-refractory metastatic pNET. Patients were stratified according to octreotide therapy with the primary endpoint assessing response rates in patients in stratum 1. Stratum 1 comprised of 115 patients treated with everolimus 10 mg daily alone and stratum 2 comprised of 45 patients treated with everolimus 10 mg daily plus octreotide LAR ≤30 mg every 28 days. In stratum 1, 11 patients (9.6 %) showed a partial response, 78 patients (67.8 %) had stable disease, and 16 patients (13.9 %) progressed, resulting in a clinical benefit of 77 %. The mean PFS was 9.7 months and overall survival was 24.9 months. In stratum 2, two patients (4.4 %) achieved partial response, 36 patients (80 %) had stable disease, and no patients with progressive disease, resulting in a clinical benefit of 84 %. The mean PFS was 16.7 months and overall survival was not reached after a follow-up period of over 16 months. This study supports the safety and antitumor activity of everolimus alone or in combination with octreotide in patients with progressive pNETs after failure of prior systemic chemotherapy [47].

Following these encouraging results, two pivotal phase III randomized trials were developed. RADIANT-2 evaluated the combination of everolimus plus octreotide LAR compared to octreotide LAR alone in 429 patients with low- to intermediate-grade advanced NET. Although the study failed to reach its primary endpoint, it demonstrated that everolimus plus octreotide LAR significantly improved PFS by 5.1 months (hazard ratio = 0.77, 95 % CI 0.59–1.00, p = 0.026); the mean PFS was 16.4 months in the everolimus plus octreotide LAR group and 11.3 months in the placebo plus octreotide LAR group. After adjusting for differences in baseline characteristics, everolimus plus octreotide LAR also significantly reduced the risk of disease progression by 40 % (hazard ratio = 0.60, 95 % CI 0.44–0.84, p = 0.0014) when compared to octreotide LAR alone [48].

A subgroup analysis of RADIANT-2 trial has shown that early combination therapy with octreotide might be associated with a better outcome compared to patients on octreotide with everolimus added on later (25.2 vs. 13.6 months). COOPERATE-2 study is an ongoing prospective randomized open-label phase II trial in pNET that aims to address the superiority of combination therapy and evaluates the treatment effect of everolimus with a novel somatostatin analog, pasireotide LAR, in comparison to everolimus monotherapy on PFS in patients with advanced progressive pNET (NCT01374451).

RADIANT-3 is the largest phase III pNET trial to date. This was a landmark double-blinded and placebo-controlled study that evaluated 410 patients with advanced, low-grade, or intermediate-grade pNET who received everolimus 10 mg/day (n = 207) or placebo (n = 203). Everolimus more than doubled progression-free survival (11 months vs. 4.6 months (p < 0.0001)) and was associated with a 65 % reduction in progression or death. Although the objective response rate to everolimus was low at 5 % (2 % in the placebo arm), there was a benefit seen in patients with prolonged stable disease in the everolimus arm (73 % vs. 51 % for everolimus and placebo, respectively). The overall survival was not significantly different between the two groups as more than 70 % of patients randomly assigned to placebo crossed over to the treatment arm after disease progression [1, 42]. There was a twofold increase in adverse events; the most common side effects were hematological, diarrhea, stomatitis, or hyperglycemia, ranging from 3 to 7 %. These side effects were manageable with dose reduction, drug interruption, or both [42]. In subgroup analyses, these benefits extended to patients regardless of ethnicity of history of previous therapies [49].

The approval of everolimus has changed the landscape for patients with advanced well-differentiated pNETs. Although the timing of everolimus in the treatment of advanced pNETs is not yet established, everolimus was equally effective in patients regardless of treatment history. The European Neuroendocrine Tumor Society (ENETS) 2012 determined that everolimus represents a novel therapeutic option in patients with unresectable pNETs after progression following chemotherapy, and in selected cases, everolimus should be considered as first-line therapy [50]. Similarly, the National Comprehensive Cancer Network (NCCN) recommended the use of everolimus as a possible first-line treatment for advanced unresectable well-differentiated pNETs (the NCCN clinical practice guidelines in oncology for neuroendocrine tumors version 1.2012. 2012. www.​nccn.​org).

Although there is convincing data for everolimus in pNETs, its efficacy in other NET subtypes, such as bronchopulmonary or colonic NET, has not been determined. Preclinical studies suggest that susceptibility to everolimus may depend on site of origin for NETs [51]. The RADIANT-4 trial will investigate the benefit of everolimus versus placebo in patients with advanced nonfunctional neuroendocrine tumor of gastrointestinal or lung origin (NCT01524783).

The PI3K/Akt/mTOR pathway is complex and perturbations can occur at multiple sites. Therefore, there are several potential targets and combinations of therapies compelling for further investigation. The use of PI3K inhibitors, Akt inhibitors, or mTORC1 and mTORC2 inhibitors as single agents or in combination can avert PI3K/Akt/mTOR pathway activation and reactivation [55]. A host of novel inhibitors that target key nodes with the PI3K/Akt/mTOR pathway have shown encouraging results in preclinical studies and are currently in early phase clinical trials.

Inhibitors of Akt either compete with ATP at the active site or bind distally to the catalytic site, inducing a conformational change that prevents ligand binding. Akt inhibition may be expected to abrogate negative feedback loops perpetuated by mTORC2 following mTORC1 inhibition [58]. Agents that inhibit both mTOR complexes may also overcome this problem. Therefore, inhibitors of both mTORC1 and mTORC2 and Akt inhibitors are attractive drug candidates.

Potential PI3K/Akt/mTOR pathway target upstreams of mTOR are the PI3K proteins themselves. Three classes (I–III) of PI3K have been characterized that vary in structure and substrate preference. The class I enzymes are activated directly by cell-surface receptors, and it is the catalytic domain of the class IA PI3K p110 subunits that are the most widely implicated in cancer [56]. Pan-PI3K inhibitors target all four class I p110 isoforms; however, PI3K inhibitors specific for individual class I p110 isoforms may allow for anticancer activity with an improved safety profile. The majority of therapeutic interventions or drugs under investigation are pan-p110 inhibitors, although a number of PI3K-targeted agents with isoform specificity have now been reported [55, 57]. It is of potential clinical significance that dual inhibition of PI3K and mTORC1/2 may be mediated through the shared structural homology between the catalytic domains of the PI3K p110 subunits and mTORC1/2 [60]. Agents that target both PI3K and mTOR will likely lead to improved inhibition of this pathway.

CC-223 is currently an experimental dual mTOR kinase inhibitor, inhibiting both TORC1 and TORC2 complexes. In a recent phase I trial, 101 solid tumor subjects were treated with CC-223 dosed at 45 mg once daily in 28 day cycles until disease progression. From the non-pancreatic NET cohort (n = 23), patients with progression within 12 months and receiving ongoing treatment with somatostatin analogs were included in the study. Biomarkers confirmed inhibition of TORC1 and TORC2 by p4EBP1 and pAKT, respectively. In 7 out of 13 subjects (54 %), PET imaging demonstrated a response at day 15 (>25 % change in SUV). All evaluable patients were stable on CC-223, with treatment ongoing up to nine cycles (median 6; range 4–9) (n = 10). Although not prospectively collected, there were six subjects with refractory carcinoid syndrome that reported complete resolution of flushing [5] and improvement in diarrhea [1]. Symptom improvement generally occurred within the first week of dosing and persisted despite dose reduction in five subjects [61].

The most common related adverse events of all grades were stomatitis, diarrhea, fatigue, anorexia, nausea, and rash. In addition, related serious adverse events included one case of transient dehydration/renal insufficiency. CC-223 dose reduction to 30 or 15 mg was required in 57 % of subjects, usually during cycle 1 or 2, but thereafter treatment was well tolerated [61].

These results are from an ongoing phase I/II study to assess the safety and efficacy of CC-223 in patients with advanced tumors (other than pNETs) unresponsive to standard therapies and to determine the appropriate dose and tumor type for later-stage clinical trials (NCT01177397).

There have been numerous preclinical data supporting the role of novel PI3K/Akt/mTOR pathway inhibitors in NETs, either through direct inhibition of specific pathway proteins or through indirect inhibition of molecular chaperones. The molecular chaperone heat-shock protein 90 (HSP90) is an emerging target for anticancer therapy as it is overexpressed in a number of tumors. The HSP90 inhibitor, IPI-504, has been studied in pNET cells. The potential activity of IPI-504 has shown to inhibit the growth of human insulinoma and pancreatic carcinoid cells by almost 70 %. IPI-504 also has antiproliferative effects by downregulating IGF-1 and several downstream factors of the PI3K/Akt/mTOR. Combination of IPI-504 with mTOR or Akt inhibitors also resulted in increasing antiproliferative effects [62]. This is a promising agent for the treatment of NETs.