Gastroenteropancreatic endocrine tumors (GEP ETs) represent a heterogeneous group of neoplasms deriving from the gastrointestinal (GI) tract and pancreas diffuse neuroendocrine system [1].

Although considered rare entities, their incidence seems to be increasing by up to five cases per 100,000 persons per year [2]. In addition, due to long-term survival, their prevalence is even higher than those of oesophageal, gastric, pancreatic and hepatobiliary cancers in the US [2].

GEP ETs are generally considered “indolent” tumors, but two thirds of them present with metastatic diseases, and are often not suitable for radical surgery [3,4]. Prognosis of GEP-ETs depends on a number of variables, including primary tumor site, disease staging at time of diagnosis, and tumor proliferative activity, which is mainly expressed by the Ki67 value on tumor cells [5].

As far as medical treatment is concerned, several options have been proposed in the past, including somatostatin analogues [6], peptide receptors radionuclide therapy (PRRT) [7], and systemic chemotherapy [8]. Despite these therapeutic tools, the majority of advanced GEP-ETs have a progressive behaviour, particularly in those cases with higher Ki67 value [9,10].

In the past few years, as more knowledge on molecular alterations favouring carcinogenesis and spreading of GEP ETs has become available, a number of targeted agents interfering with key growth and angiogenic pathways have been explored in preclinical and clinical studies as novel and promising tools for GEP ETs treatment [11–13]. Recently, the mTOR inhibitor, Everolimus, and the multi-target antiangiogenetic agent, Sunitinib, have been shown to be effective in prolonging progression-free survival (PFS) in advanced progressive pancreatic endocrine tumors (PETs), and thus have been approved by the FDA for treatment of this disease [14].

However, a number of other compounds are under investigation. The present review will summarize existing data on targeted therapies for GEP ETs, focusing on combined treatments and novel targeted agents.

The inhibition of neoangiogenesis is considered a valid treatment approach, achieving good results in a number of solid tumors [15]. Angiogenesis is a central and complex process in tumor growth and metastasis, and involves a number of receptor tyrosine kinases (RTKs) and their ligands. Vascular endothelial growth factor (VEGF) is a specific key driver of angiogenesis in pancreatic endocrine tumors (PETs) [11].

The investigation of the VEGF pathway is also of particular interest for the biology of GEP ETs, especially of PETs, as angiogenesis switch, coupled by progressive expression of VEGF are key mechanisms in the transgenic mouse model (Rip1-Tag2) developing PETs [16]. The role of vascularisation in PETs is somewhat controversial, with some studies reporting that expression of VEGF correlates with a more aggressive tumor behaviour[17,18], and others that malignant tumors show lower VEGF expression than benign ones [19].

As tissues from malignant PETs also shows widespread expression of VEGF receptors (VEGFR), platelet-derived growth factor receptors (PDGFRs) α and β, and other receptors such as the stem-cell factor receptor (c-kit) [20], inhibitors of these kinases, have been tested in GEP ETs patients.

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates cell cycle and metabolism in response to environmental factors. mTOR mediates signaling transduction downstream of receptor tyrosine kinases, thus playing a critical role in different proliferative signals mediated through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway. The signaling pathways upstream of mTOR include several tumor suppressor genes, such as Phosphatase and tensin homolog (PTEN), Neurofibromatosis1 (NF1), and the tuberous sclerosis complex (TSC1/TSC2), which negatively regulate mTOR.

A number of studies investigated the expression of genes belonging to the PI3K/AKT/mTOR pathway in GEP ETs, suggesting its activation, which seems mainly due to mutations or reduced expression of its negative regulators, such as PTEN and TSC2 [33,34]. The activation of the mTOR pathway in pancreatic endocrine tumors is also supported by immunohistochemical expression of p-mTOR [35], and its downstream effector eukaryotic translation initiation factor 4E binding protein 1 (p-4E-BP1) which has also been reported to be an independent factor of poor prognosis [36].

The non-receptor protein tyrosine, Src, is the archetypal member of a family of nine membrane associated-tyrosine kinases (SFKs), the other members being: Lck, Fyn, Yes, Hck, Blk, Fgr, Lyn and Yrk, all of which are characterized by significant homology [49].

SFK are central mediators of proliferation, differentiation, migration, adhesion, invasion, and angiogenesis, and upon stimulation by growth factors, hormones, integrins or other factors, are activated throughout phosphorylation of signaling proteins for which the Src SH2 domain has high affinity.

SFK substrates include multiple downstream signaling pathway regulating cell proliferation, survival, motility, migration, cell-matrix adhesion dynamics, and regulation of cytoskeleton. Thus, not surprisingly, SFK play a significant role in signaling pathways involved in oncogenesis and tumor progression [50].

c-Src was the first proto-oncogene to be identified and an aberrant SFK activity has been reported in different tumor types, including breast, lung, colorectal cancer and pancreatic adenocarcinoma [51]. In most cases the overactivation of Src does not seem related to its mutations, but rather with overexpression and mutation of growth factor receptors or of other factors whose signaling pathways pass through SFKs.

Therefore SFKs have become a potentially attractive therapeutic target for different tumor types, and a number of SFKs are under evaluation in clinical trials.

Dasatinib (Sprycel^®, Bristol-Myers Squibb), Saracatinib (Astra Zeneca), and Bosutinib (Bosulif, Pfizer) are all dual-specific inhibitors of SFKs and Abl and have been clinically tested mainly in trials of patients with haematological malignancies. Dasatinib and Bosutinib have both been approved by the FDA as second-line treatment for haematological malignancies.

These compounds have been tested in preclinical studies on xenograft mouse models of solid tumors with promising results [52]. However, the efficacy of SFKs as single agents has not yet been replicated in clinical trials of patients with solid cancers [53,54]. It therefore seems that SFK inhibitors may be more useful as part of combined treatments, also because SFKs are frequently activated in response to other treatments such as chemotherapy [55]. On the other hand, it is possible that the concomitant inhibition of c-Abl should be avoided in some cancer types [56], but there are few SFK inhibitors in preclinical development that are not also inhibitors of c-Abl.

As far as regards digestive endocrine tumors, the overexpression of Lck, a member of SFKs, has been demonstrated in metastatic progressive PETs, both at the RNA and protein levels [57]. The expression of LCK as detected by innunohistochemistry was more frequent in liver metastases than pancreatic primary lesions (66% vs. 43%), and was not limited to the plasma membrane, as in normal pancreatic islets, but often cytoplasmic, possibly due to overproduction of the protein, or to mutations that might impair palmitoylation or myristoylation which are critical for its localization.

The expression and activity of Src and Fyn and high levels of Src family activity in comparison to several cancer cell lines of different origins have been described in PET cell lines. Immunoreactivity for Src was also present in PET human samples. The inhibition of SFK activity in PET cell lines has been shown to interfere with adhesion, spreading and migration of such cells [58]. Known substrates of Src, such as p130Cas and FAK, were identified in this PET model.

More recently the possible link between the SFK and mTOR pathways in PETs has been investigated [36]. The findings suggest a novel role for SFKs in controlling mTOR activity during adhesion in PET cell lines. SFKs control mTOR activation at the periphery of the cells and regulate its translation of a subset of mRNA involved in cell cycle progression. Interestingly, we also found that concomitant inhibition of SFK and mTOR activities, strongly impaired PET cell line growth, compared to the effect exerted by the single agents.

Moreover, while the treatment of PET cells with mTOR inhibitor triggered a prosurvival response dependent on PI3K/AKT signaling in PET cells, as also shown in other types of cancer, the simultaneous inhibition of SFKs blocked this escape signal [33].

Thus, the link between SFK and mTOR in PET cells represents a potential target for combined treatment for PETs.

The relevance of SFKs in neuroendocrine tumors has also been underlined by the recent finding that neuroendocrine cancer stem cells (N-CSC), present strong activity of both the Src and mTOR pathways, and that targeting Src inhibits the growth both of N-CSCs cells in vitro, and of tumors derived from them in vivo[59].

Further studies employing SFK inhibitors, as single agents or in combination with mTOR inhibitors or other treatments, in animal models of GEP ETs could further support the relevance of SFKs in this tumor type, and eventually set the ground for clinical trials.

The hedgehog (Hh) pathway, initially discovered in Drosophila, is a major regulator for cell differentiation, tissue polarity and cell proliferation [60]. The seven transmembrane domain containing protein smoothened, frizzled family receptor (SMO), serves as the key player for the Hh pathway, whose function is inhibited by another transmembrane protein Patched (PTC) in the absence of Hh ligands [61]. Binding of Hh to its receptor PTC releases this inhibition, allowing SMO to signal downstream, eventually to glioma-associated oncogene family zinc finger (Gli) transcription factors, primarily Gli2 transcriptional factor. As transcription factors, Gli molecules can regulate target gene expression by direct association with a specific consensus sequence located in the promoter region of the target genes [62]. Previous studies had shown that the Hedgehog signalling pathway was aberrantly re-activated in several cancers arising from the gastrointestinal tract, including the majority of pancreatic cancers [63–67].

In a preclinical study, it was shown for the first time that the Hedgehog signaling pathway is expressed in the Rip1Tag2 mouse model of pancreatic islet carcinogenesis [68]. It was found that hedgehog blockade with cyclopamine, a natural chemical that belongs to the group of steroidal jerveratrum alkaloids, led to marked in vivo growth inhibition of islet cell tumors combined with a decrease in proliferating tumor cells and an augmentation of apoptosis. The downregulation of the Hedgehog target gene Gli1 was demonstrated in the tumor of cyclopamine treated mice, and a significantly prolonged survival of the cyclopamine treated Rip1tag2 mice in vivo was achieved. Cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to Hedgehog signaling by direct binding to Smoothened [69].

After proving the impact of cyclopamin on islet cell tumors in a transgenic mouse model of PETs the orally bioavailable smoothened antagonist LDE225 (Novartis Pharmaceuticals) was used [70]. Treatment with LDE225 reduces tumor volume by 95% in Rip1Tag2 mice. After treatment for several weeks, no invasive carcinoma was found in histopathological evaluation. Down regulation of the hedgehog target genes Gli1, Ptch1 and Hip was found in the tumor cells of LDE225 treated mice, and survival was prolonged in the LDE225 treated Rip1Tag2 mice. Thus, these results provided the first evidence that targeting the hedgehog pathway with the orally bioavailable Smo antagonist LDE225 may be a very attractive target for patients with PETs. Clinical evaluation of pharmacological Hedgehog blockade as a novel cancer treatment strategy has been hampered by the lack of suitable substances that might serve as future drugs for use in humans. The novel small molecule hedgehog pathway inhibitor LDE225 was designed with the intention to overcome this shortcoming. LDE225 is highly bound to human plasma proteins (>99%) and is readily water soluble [70].

In contrast to the negative influence of sonic hedgehog (Shh) on developing pancreatic growth, there does appear to be a role for Indian hedgehog (Ihh) in the adult pancreas. Ihh is expressed in islet and β-cells in characteristic, small, highly localized aggregates or punctuates [71–74]. Patched homolog protein (Ptc-1) and Smo have been shown to be expressed in islets, localized to β-cells by coexpression with insulin [71]. The insulin-secreting clonal cell line INS-1 expresses Ihh, Ptc-1, and Smo. Activation of the Hh signaling pathway by ectopic misexpression of Shh, increased activity of the rat insulin I promoter [72]. The administration of cyclopamine decreased insulin I promoter activity, decreased insulin secretion, and the insulin content of these cells in a concentration-dependent manner. It is well known, that the hedgehog pathway is activated in islet and β-cells [68,70]. Activation of the hedgehog signaling pathway by ectopic overexpression of Sonic hedgehog increased activity of the rat insulin I promoter. Therefore treatment with a hedgehog inhibitor might be of potential interest for patients with metastatic insulinoma. The effect of LDE225 was analysed to determine whether reduction of Hh signaling by LDE225 affects β-cell function in vivo. A quantitative real-time PCR was performed for treated and untreated Rip1Tag2 mice and it was found that inhibition of hedgehog signaling with LDE225 reduced endogenous insulin mRNA expression. Therefore, if LDE225 or other hedgehog inhibiting compounds are to be used in patients, attention should be given to changes in blood glucose levels [75].

In conclusion, targeting the hedgehog signaling pathway with LDE225 might be an interesting tool for future clinical testing in patients with PETs. Combination of LDE225 with already established drug regimens in PETs are likewise imaginable.

As the knowledge of molecular alterations in GEP ETs increases, a number of potentially “targetable” genes are being investigated. Among potential therapeutic strategies, the inhibition of angiogenesis and of the mTOR pathway, which are the better investigated targets, with approved drugs, still need investigation to elucidate the possible advantage of combined treatments in overcoming induced resistances, and in identifying biomarkers able to predict clinical efficacy, possibly employing biomarkers of response to treatment [76].

Other novel, interesting targets for which a strong biological rationale exists, and where specific inhibitors are available, are the Src Family Kinases and the Hedgehog Pathway.

A number of other specific critical issues which emerge should be carefully considered when designing clinical trials with targeted agents:

In conclusion, while there exists optimism, as novel therapeutic strategies are emerging for patients with GEP ETs, the increasing number of options also requires an increased need for accurate and rigorous studies to answer the clinically relevant questions.