The Role of MDM2 In Neuroendocrine Tumors: A Novel Therapeutic Target

Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells which secrete hormones that cause distinct...


Introduction
Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells which secrete hormones that cause distinct clinical syndromes. They are considered rare tumors, but annual incidence has increased significantly with recent SEER analysis showing a 6.4-fold increased incidence from 1973 (1.09 per 100,000) to 2012 (6.98 per 100,000) [1]. They are highly diverse neoplasms that can be divided into several subgroups based on embryonic derivation, histology, functionality, and origin of primary tumor. One of the most important classifications, based on histologic differentiation and grade, divides NETs into well-differentiated and poorly differentiated neoplasms [2]. Well-differentiated NETs are indolent tumors with a slow progression profile, while poorly differentiated neuroendocrine carcinomas (NECs) are aggressive tumors with worse outcomes [3]. Well-differentiated and poorly differentiated tumors have vastly different responses to treatment and thus require different clinical approaches [2]. While there have been therapeutic advances in NETs during the last decade, treatment options are still limited, especially in those with higher grade tumors. The multiple types of NETs and the differences in their clinical behavior demonstrates the need for further research and the development of more targeted therapies. Given this, a more detailed understanding of the molecular pathogenesis and the development of novel therapeutics is needed.
While most poorly differentiated NECs harbor alterations in tumor protein p53 (TP53) and retinoblastoma (RB1) genes, there is limited data about their expression/alteration in other NET subgroups and how these features may contribute to the separation of well from poorly differentiated NETs with a proliferation index above 20% [4]. There is also little known about the relationship between the transcription factor p53 pathway and the E3 ubiquitinprotein ligase mouse double minute 2 homologue (MDM2; also called HDM2 for its human homologue). Given the paucity of genetic information regarding MDM2-TP53 pathway in NETs, we will summarize the role of MDM2 overexpression and its relation to TP53 as a potential targeted therapy in preclinical models and early phase clinical trials in these neoplasms.

TP53 and MDM2
Transcription factor p53 has been called "the guardian of the genome" because it plays a critical role in regulating genomic stability and in suppression of tumor development. It is a haploinsufficiency gene in both mice and humans that is located on chromosome 17p13.1 and is essential in cell division as it regulates cell cycle phases [5]. TP53 itself is closely regulated by a number of positive and negative regulators and any small change in activity can deregulate the cell cycle, cause uncontrolled cell division, and predispose to tumorigenesis [5]. As more than 50% of all tumor types have an impaired p53 signaling pathway, researchers have successfully identified many downstream transcription targets for p53 [6]. One of these targets is the E3 ubiquitin-protein ligase mouse double minute 2 homologue (MDM2). Since discovery by Dona George in 1991, many in vitro and in vivo studies have described the tangled relationship between MDM2 and p53 [7].
The human MDM2 gene is located on chromosome 12 (q14.3-q15), a region with many genes thought to be involved in control of cell growth [8]. Reports indicate that MDM2 binds with wild type p53, targeting it for proteolytic degradation and thus inhibiting p53mediated gene expression [9,10]. It has been found that by adding exogenous MDM2, p53-mediated G1 phase cell cycle arrest and induction of apoptosis can be suppressed [8]. Additionally, p53 has been found to transactivate MDM2 expression suggesting that the MDM2 and TP53 complex may autoregulate MDM2 itself as well as TP53 function [8,10].
MDM2 also plays a critical role in carcinogenesis in a p53independent manner [8]. In addition to a p53 binding domain, MDM2 contains other domains that suggest that it can bind to DNA and affect transcription. It has been reported that MDM2 binds to retinoblastoma (RB) tumor suppressor protein as well as other transcription factors [8]. Preclinical studies showed the formation of these complexes in vitro and in cultured cells deficient of p53, therefore suggesting that MDM2 might play a p53-independent role in promoting cell cycle progression from G1 to S phase [8].

Role of P53 and MDM2 in different groups of neuroendocrine tumors
While p53 and MDM2 have been studied in multiple tumor types, the data regarding their role in neuroendocrine tumors is limited [11]. There are several differences in the mutational frequencies between high grade well-differentiated NETs and poorly pancreatic NETs confirmed that TP53 mutations were rare in welldifferentiated pNETs (<3%) [5]. However, the results also detected that about 70% of pNETs had an altered p53 pathway through aberrant activation of its negative regulators including MDM2 (22%), MDM4 (30%), and WIP1 (51%). These results clearly demonstrated that although most patients with well-differentiated pancreatic NETs have wild TP53, they do exhibit negative regulation of TP53 function which could be an important mechanism for the pNETs progression. Therefore, reactivation of TP53 could be a potential therapeutic strategy for these patients.

Targeting MDM2
Given the previous data highlighting the potential role of MDM2-TP53 pathway in NETs' tumorigenesis, several preclinical studies targeted MDM2 in NETs in vitro. In one study, MDM2 inhibitor, NVP-CGM097, was evaluated in NETs from different origins include mutated p53 pancreas (BON1), and lung (NCI-H727), and wild p53 midgut (GOT1) cell lines [12]. Each tumor cell line was incubated with NVP-CGM097 at different concentrations that ranged from 4-2,500 Nm. The study showed that wild type p53 GOT1 tumor cells of the midgut were sensitive and more responsive to NVP-CGM097 at higher concentrations. Co-incubation with other chemotherapies and targeted agents including 5-fluorouracil, temozolomide, or everolimus revealed increased anti-proliferative tumor effects in GOT1 cells [12]. The mutated p53 NCI-H727 and BON1 were resistant to NVP-CGM097. While this study encourages further workup of MDM2 inhibition for wild type p53 NETs, it also reveals that mutated p53 NETs are resistant to MDM2 inhibitors.
Another study analyzed nutlin-3a, a molecule that inhibits the MDM2-p53 pathway [13]. The study demonstrated that MDM2 was overexpressed in intestinal NETs and was higher in metastatic than in localized disease. The results confirmed the previous findings of wild p53 small intestinal NET cell lines exhibiting cell cycle arrest and apoptosis through nutlins reactivating a p53 antiproliferative response [13]. The antitumor effects of nutlin-3a were also confirmed in vivo in a xenograft mouse model. Additionally, the data suggested that MDM2 also influences the expression of oncogene FOXM1 in a p53-independent manner. Overall, these results demonstrated MDM2 overexpression in NETs and that targeting MDM2 overexpression could become a broad and clinically viable therapeutic strategy for NETs with wild-type p53.

Conclusion
The underlying molecular mechanisms driving NETs resistance are largely unknown and remain a significant scientific barrier.