Pancreatic Neuroendocrine Tumors: Signaling Pathways and Epigenetic Regulation

1. Introduction

Pancreatic neuroendocrine tumors (PNETs) are a rare and heterogeneous group of neoplasms arising from pancreatic islet cells. They account for approximately 2% of all pancreatic malignancies and are characterized by their slow-growing nature and potential for metastasis [1]. Current treatment options for PNETs are limited, and the prognosis for patients with advanced disease remains poor, underscoring the urgent need for innovative therapeutic strategies [2]. The pathogenesis of PNETs is intricate, characterized by complex interactions among numerous signaling pathways. Each pathway plays a role in different facets of tumor development, encompassing cell proliferation, survival, migration, and angiogenesis. Together, these pathways constitute a complex network that, when disrupted, can result in uncontrolled tumor growth and metastasis [3]. Germline and somatic whole-genome sequencing provided a comprehensive analysis of PNET-related genetic variations [4]. Most PNETs occur sporadically, with only around 10% of the cases being associated with germline mutations. Germline mutations such as multiple endocrine neoplasia type 1 (MEN1), von Hippel-Lindau syndrome (VHL), neurofibromatosis type 1 (NF1), and occasionally tuberous sclerosis complex (TSC) are the most identified PNET-associated mutations [5,6]. For sporadically occurring PNETs, MEN1, DAXX (death domain associated protein), ATRX (α-thalassemia/mental retardation syndrome X-linked), and genes related to the mammalian target of rapamycin (mTOR) pathway harbor commonly identified somatic alterations [6].

While somatic and germline mutations remain of great significance for diagnosis and therapeutic treatment, emerging evidence highlights the pivotal role of epigenetic modifications in shaping the intricate landscape of PNET-related signaling [7]. Epigenetic modifications encompass a range of reversible alterations that modulate gene expression patterns without affecting the underlying DNA sequence, such as DNA methylation, histone modifications, and non-coding RNAs (ncRNAs) which coordinate together to govern cellular processes crucial for normal development and homeostasis [8]. In the context of PNETs, epigenetic dysregulation is starting to gain recognition as a significant player in the initiation and evolution of the disease [1,9,10]. Epigenetic alterations can lead to aberrant gene expression patterns, contributing to uncontrolled cell growth, evasion of cell death, and increased metastatic potential – hallmark characteristics of cancer [11]. This is particularly relevant in the case of PNET because PNETs have a low tumor mutational burden and are considered an epigenetic disorder, due to the fact that multiple high-frequency mutations, including MEN1, DAXX, and ATX, are all found involved in epigenetic regulation [7].

Several recent reviews have addressed the complicated signaling network in PNET development [1,3,12,13,14]. However, to the best of our knowledge, there is a deficiency in comprehensive studies that systematically review the epigenetic status of PNET-related signaling pathways, considering both PNET and other frequently encountered cancers simultaneously. Given that epigenetic mechanisms are common across various cancers [15,16], conducting a thorough examination of the epigenetic events within signaling pathways in the broader context of cancer research is certain to offer valuable insights. This approach will undoubtedly guide the identification of future directions for the relatively understudied field of epigenetic research in PNETs.

Because of our strong interest in enhancing the care of patients diagnosed with PNETs, we will concentrate on the epigenetic mechanisms that hold the highest clinical relevance. Presently, there are a total of eight FDA-approved anti-tumor epigenetic drugs primarily targeting DNA methylation and histone modifications [17]. Beyond their therapeutic applications, epigenetic markers, particularly in the realm of non-coding RNAs such as microRNAs (miRNAs), play a crucial role in cancer diagnosis [18]. This review will specifically delve into the three aforementioned epigenetic regulatory mechanisms: DNA methylation, histone modifications, and non-coding RNAs. This is by no means all-compassing, as we recognized that we will not discuss other epigenetic modifications including RNA methylation, histone ubiquitylation, phosphorylation, SUMOylation, ADP ribosylation, citrullination, and biotinylation at specific amino acidic residue [19].

2. Major Signaling Pathways and Molecules in PNETs

Various molecular alterations have been identified as correlated with the development of PNETs. These events manifest through diverse mechanisms, encompassing both genetic and epigenetic modifications, resulting in a complex regulatory network. Genetic variations of PNET include both familial and predominantly sporadic mutations [20]. Each of the hereditary syndromes, namely MEN1, VHL, NF1, and TSC, is characterized by distinct sets of signaling pathways and molecules [20]. Moreover, numerous signaling pathways exhibit sporadic mutations in PNET samples [20]. It is worth noting familial and sporadic mutations are not exclusive of each other in PNET. One such example is MEN1 signaling. MEN1 mutations play a pivotal role in the initiation and progression of pNETs, as over 40% of sporadic pNETs and all MEN1 patients exhibit somatic mutations in the MEN1 gene [21,22]. More interestingly, the status of MEN1 mutations and menin protein expression don’t always correlate well with each other. One study, which included the mutational analysis and immunohistochemistry results (IHC) of 169 PNET patients, showed that 80% of sporadic cases showed a loss of menin nuclear localization, while only 25% of the patients carried a mutation in the MEN1 gene itself [23]. This study clearly suggests that other regulatory mechanisms, besides genetic mutations, are involved in the altered level of signaling pathway in PNET pathology. The complicated regulatory mechanisms in PNET pathology have been nicely reviewed previously [1,3,12,13,14]. Here we adopt a different approach by discussing the PNET-related signaling events based on the major epigenetic regulatory mechanisms that they fall into, including DNA methylation, histone modifications, and ncRNAs (Figure 1). Dynamic alterations in DNA methylation, histone modifications, and ncRNA activity are integral aspects of the pathogenesis of other extensively studied cancers. However, the contributions of these epigenetic modifications to PNET pathology have yet to be fully explored.

Abbreviations: Acyl-CoA synthetase isoform 1(ACSL1); Secreted frizzled related protein 1 (SFRD1); WNT Inhibitory Factor 1(WIF1); Phosphatase and tensin homolog (PTEN); Receptor tyrosine kinase (RTK); Insulin receptor substrate 1 (IRS1); DEP domain containing 5 (DEPDC5); Hypoxia inducible factor 1 α (HIF1 α); Cyclin dependent kinase inhibitor 2A (CDKN2A); Wild-type p53-induced phosphatase 1 (WIP1); Mouse double minute 2 (MDM2); Pleckstrin Homology Like Domain Family A Member 3 (PHLDA3); Rab-like protein 6 (RABL6); Cyclin-dependent kinase 4 and cyclin-dependent kinase 6 (CDK4/6).

3. Epigenetic Regulation of PNET-Related Signaling Pathways

3.1. DNA Methylation

Methylation status has long been recognized as one of the primary mechanisms by which epigenetic modification is carried out within cells to regulate gene expression. DNA methyl transferases (DNMTs) cause DNA methylation by adding a methyl group to the cytosine in CpG DNA sequences (CpG islands) of promoter or enhancer. In cancer cells, dysregulated CpG methylation leads to promoter hypermethylation and downregulated gene expression, resulting in gene silencing of tumor suppressor genes. In addition, DNA demethylating enzymes might cause genome-wide hypomethylation, causing DNA instability. A variety of tumor suppressors as well as proto-oncogenes have been shown to be modified via methylation in the tumorigenesis [25].

3.1.1. MEN1

MEN1 is one of the most extensively studied genes in PNET. Germline mutations in this gene result in Multiple Endocrine Neoplasia Syndrome Type 1, causing different types of cancer including PNET, pituitary adenoma, and parathyroid hyperplasia. The genetic mutation responsible for MEN1 spans 9.8 kb of chromosome 11q13. The protein product of MEN1, termed menin, is a tumor suppressor protein ubiquitously expressed in the cell nucleus [26]. Considering menin’s crucial role as an epigenetic regulator, it is unsurprising that the analysis of DNA methylation in MEN1-associated PNETs revealed the promoter hypermethylation of numerous potential tumor suppressor genes [27]. Epigenetic modifications of MEN1 are also identified in non-endocrine tumors. Notably, in advanced breast cancer, the overexpression of the MEN1 gene is associated with promoter hypomethylation, which differs from the scenario observed in PNETs. [28].

3.1.2. mTOR-TSC

The mechanistic target of rapamycin (mTOR) is a kinase that in humans is encoded by the mTOR gene. It functions as a serine/threonine protein kinase that regulates a variety of cellular functions including cell growth and proliferation, motility, survival, protein synthesis, autophagy, gene transcription, and thus carcinogenesis. [29]. Activation of mTOR signaling can affect serine one-carbon metabolism enough to modulate DNA methylation and promote tumorigenesis [30]. Aberrant hypermethylation of the promoter of tumor suppressor genes followed by their silencing is important for aberrant mTOR-PI3K pathway activation in gastric cancer [31]. Tumor suppressor TRPM4, a calcium-activated nonselective cation channel, inhibits PI3K/Akt/mTOR signaling pathway to impede tumor migration and invasion. This gene has been shown to be methylated at its promoter with silenced or reduced expression in colorectal cancers, though this effect can be reversed with the calpain inhibitor calpeptin, providing possible therapeutic strategies for aggressive disease [32]. ZDHHC22, as a well-known member of the palmitoyltransferase family, regulates mTOR stability and activation of the AKT signaling pathway. Hypermethylation of the promoter CpG island of the ZDHHC22 gene is associated with poorer prognosis in breast cancer. Overexpression of ZDHHC22 inhibits breast cancer cell growth both in vitro and in vivo [33]. The major signaling molecules of mTOR pathway, including PTEN, PI3K, AKT, TSC and c-Myc, have been well documented in PNET pathology previously [34,35,36,37,38,39,40]. Promoter of PTEN gene has been shown to be hypermethylated while the methylation status of TSC promoter remains unchanged in PNET samples [41].

3.1.3. Hypoxia-Induced Factor 1α (HIF1α)-VHL

Hypoxia inducible factor1⍺ (HIF1⍺) is pro-tumorigenic factor that has found to lead to poor prognosis, metastasis, and recurrence in multiple cancer subtypes including breast, renal cell, gastric, and ovarian cancer. Its involvement in B-cell lymphoma, Chronic lymphocytic leukemia, colon cancer, small cell lung cancer and PNET have also been established [42]. HIF1⍺ is regulated by hypoxia. Under normoxic conditions, HIF1α interacts with von Hippel Lindau (VHL) protein (pVHL) resulting in its polyubiquitination and proteasomal degradation [43]. During cellular hypoxia, HIF1⍺ translocates to the nucleus where it becomes stabilized activating genes involved in cellular proliferation, angiogenesis, glucose homeostasis and metastasis [44]. In patients with von Hippel Lindau syndrome, the tumor suppressor gene is mutated and pVHL is absent limiting HIF1⍺ degradation. Patients with VHL are at increased risk for developing hemangioblastomas, retinal angiomas, renal cell carcinomas, pheochromocytoma, and pancreatic lesions including PNETs [45]. Methylation status of the HIF binding region within the hypoxia response element correlates with increased expression of epidermal growth factor receptor (EGFR) under hypoxic conditions in breast cancer. These changes can be reversed by treatment with DNA methyltransferase inhibitors such as azacytidine or decitabine, suggesting that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors [46]. HIF1α driven transcriptional response in hypoxia in pediatric neuroblastoma is subject to epigenetic control via DNA methylation status of gene regulatory regions. Hypoxia exposure induces a global DNA hypermethylation in neuroblastoma cells and HIF1A itself might control DNA methylation [47]. In VHL disease, loss of VHL leads to activation of its binding partner HIF1α, causing multi-organ tumorigenesis including PNET [48,49]. The VHL gene promoter has been shown to be hypermethylated in a subset of familial PNET samples [50].

3.1.4. RAS-MAPK-NF1

The RAS-MAPK pathway mediates cellular responses to growth signals and is often dysregulated in cancer. The mitogen-activated protein kinase (MAPK) pathway involves extracellular signal regulated kinases 1&2 (ERK1/2) that mediates multiple cellular functions including proliferation, growth, and senescence [51]. The rat sarcoma (RAS) gene is an oncogene part of the large family of GTPases and acts as a control switch for the EKR1/2 pathway. In order to transmit signals to downstream effector proteins, RAS must properly bind to cellular membranes which requires posttranslational modifications such as DNA methylation and histone acetylation [52]. Global DNA hypermethylation correlated with the RAS and MAPK oncogenic pathways (among others) is significantly associated with high-grade tumors, platinum resistance, and poor prognosis in high-grade serous ovarian carcinoma (HGSOC). Treatment with demethylating agents shows significant growth retardation in ovarian cancer cells through differential inductions, such as cell apoptosis or G2/M cell cycle arrest [53]. There have been newly identified biomarkers for colorectal cancer metastasis that are related to MAPK, including Eye Absent Homolog 4 (EYA4) [54]. 5-methylcytosine (m5C) regulator status and expression is a robust predictor of prognosis and therapy response in pancreatic ductal adenocarcinoma (PDAC) [55]. Differentially expressed genes, both hypermethylated–low expression and hypomethylation–high expression, in the Ras/MAPK pathways are seen in neurofibromatosis type 2 vestibular schwannomas [56]. Genomes of acute myeloid leukemia (AML) cells resistant to the BCL-2–selective inhibitor venetoclax (VEN) show extensive differential methylation with activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein. VEB sensitivity can then be at least partially restored by silencing or pharmacologic inhibition of MCL-1 [57]. RASSF1A is a negative regulator of RAS-MAPK signaling pathway. In addition, RASSF1A can mediate DNA repair through nucleotide excision repair [58]. RASSF1A might be the most frequently inactivated tumor suppressor identified in human cancers and has been found to be inactivated in more than 40 types of human malignancies [59]. The expression of RASSF1A is lower in bladder and prostate cancer patients due to promoter hypermethylation. If this is reversed and RASSF1 is overexpressed, enhanced cytotoxicity to chemotherapeutic drugs is observed [60]. Urinary methylation assays can even be used to help predict prostate tumor stage and grade, and related tests utilizing RASSF1A methylation in various contexts have been investigated in endometrial, breast, and colorectal cancers [61,62,63,64]. Methylation of the RASSF1 gene promoter is also significantly higher in lung tumor tissues and is associated with lymph node metastasis and clinical stage. Interestingly, it appears that associations exist between methylation levels and cigarette smoking-related parameters [65,66]. RASSF1 promoter methylation, under the impact of the methyltransferase genes DNMT1 and MGMT, can also be a papillary thyroid cancer genetic marker [67]. In PNET, the promoter of RASSF1 gene was found to be hypermethylated in 75% of NF-PNET where its inactivation results in the degradation of beta-catenin [68]. Inhibition of RAS-MAPK attenuate PNET cell growth [69]. Methylation has been associated with RASSF1A downregulation, and, interestingly, up-regulation of RASSF1C in PNET samples [70]. Neurofibromatosis type 1 (NF1) encodes a RAS GTPase activating protein called Neurofibromin, which has been found to be mutated in both sporadic cancers and in familial syndrome. Mutations of NF1 result in excessive RAS signaling. NF1-associated sporadic cancers include glioblastoma, neuroblastoma, AML, lung cancer, ovarian cancer, and breast cancer [71]. In the NF1 familial syndrome, patients with neurofibromatosis will present with benign cutaneous neurofibromas, and are predisposed to breast, gastric, and pancreatic neuroendocrine tumors [10,72]. Despite the significant importance of NF1 in PNET regulation, there is currently no reported information concerning the DNA methylation of NF1 in the progression of PNETs.

3.1.5. ATRX/DAXX

ATRX and DAXX form a histone chaperone complex that deposits histone variant H3.3 into specific genomic regions [73]. Mutations in ATRX constitute the most prevalent genetic abnormalities in gliomas and, interestingly, ATRX alterations are associated with favorable outcomes. CRISPR/Cas9 knockout ATRX glioblastoma cells demonstrated compromised H3K9 trimethylation, which led to decreased DNA repair by the tumor cells. In addition, increased response to DNA damage-inducing agent temozolomide was confirmed in these cells, suggesting that these mutations might serve as a prognostic maker in predicting chemosensitivity [74]. In high-grade meningiomas, there is a significant increase of ATRX expression at both gene and protein levels, particularly in the nuclear compartment, and an increased DAXX protein expression level. In these tumors, ATRX/DAXX is suggested to have a role in telomere maintenance due to low variability of telomere length [75]. In histopathologic evaluation of human pituitary adenomas, ATRX or DAXX protein loss was observed, suggesting either homozygous loss of the gene or the presence of another silencing mechanism such as promoter methylation [76]. Mutations in ATRX and DAXX have been identified in PNET tumor samples [34,35,77]. The DAXX gene has been shown to be hypermethylated at promoter region in a fraction of PNET tumor samples [78].

3.1.6. CDKN2A-RB1

Abnormal DNA methylation is found in the early stages of kidney cancers. Genistein, a potent antioxidant and demethylating agent in soybean-enriched foods, induces cell apoptosis and inhibits the cell proliferation of multiple types of kidney cancer cells. It achieves this through the expression of CDKN2a via decreased CDKN2a methylation [79]. CDKN2A promoter methylation correlates with a poor prognosis and progression-free survival in both ovarian and colorectal cancers [80,81]. Peroxisome proliferator-activated receptor alpha (PPARα), the molecular target of fibrates commonly used to treat dyslipidemia and diabetes, inhibits DNMT1 activity and abolishes methylation-mediated CDKN2A repression in colorectal cancer cells. It also appears to inhibit tumor growth and DNMT1 activity in vivo, indicating that it may be an applicable agent for epigenetic therapy of colon cancer patients [82]. In addition, methylation of the CDKN2A gene is recorded as an early epigenetic event in the progression of cervical tumorigenesis. There is a strong and significant correlation between CDKN2A gene methylation and risk of cervical cancer as well as pre-cancerous lesions [83]. Furthermore, the level of CDKN2A gene methylation in human gastric cancer (GC) samples is increased compared to noncancerous tissues. Variations in methylation patterns and different loci are associated with changes in overall survival [84]. Inducing methylation in GC cells increased their sensitivity to palbociclib, an anti-CDK4/6 chemical for cancer treatment, both in vitro and in vivo [85]. As part of the CDKN2A-CDK4/6-RB1 axis, retinoblastoma tumor suppressor protein1 (RB1) plays critical roles in tumor suppression. RB1 inhibits cellular proliferation through repressing the transcription of genes essential for cell cycle progression, serving as a negative regulator of the cell cycle [86]. Aberrant DNA methylation at the RB1 gene promoter has been reported in multiple types of tumor [87]. Members of the CDKN2A-CDK4/6-RB1 pathway, including CDKN2A, P14^ARF, and RB1 have shown promoter hypermethylation in PNET tumor samples [41,88,89,90], suggesting that DNA methylation-mediated downregulation of CDKN2A-RB1 signaling might contribute to PNET development.

3.1.7. p53

p53, also known as tumor protein TP53, is a regulatory protein that is often mutated in human cancers. DNA hypermethylation of TP53 has been found associated with the pathogenesis of cervical cancer in Northeastern Indian patients [91]. Resveratrol, a chemical mostly found in red grapes and products made from these grapes, alters the methylation of histones in the TP53 promoter in human breast cancer cells and upregulates the expression of SET domain-containing lysine methyltransferase 7/9 (SET7/9) in colorectal cells to positively regulates p53 through its mono-methylation function [92,93]. Luteolin, a dietary flavone molecule, modulates various signaling pathways involved in carcinogenesis and has apoptotic effects mediated by DNA demethylation of the nuclear factor erythroid 2-related factor 2 (NRF2) promoter and the interaction of Nrf2 and p53 in human colon cancer cells [94]. DNMT3B has been shown to be elevated in nasopharyngeal carcinoma tissues and predicts a poor prognosis. In addition, ionizing radiation can induce DNMT3B, which might be one of the reasons for radio-resistance; silencing DNMT3B restores p53 via DNA demethylation, which leads to cell cycle arrest and apoptosis both in vitro and in vivo [95]. In gastric cancer, PBX/Knotted Homeobox 2 (PKNOX2) is a candidate tumor suppressor, which exerts its tumor suppressive effect by promoting the upregulation of p53. PKNOX2 mRNA expression is largely silenced in gastric cancer cell lines and primary gastric cancer via promoter methylation, which is associated with poor outcomes [96]. Genetic profiling of BON-1 and QGP-1 PNET cell lines showed homozygous TP53 mutations which suggests loss-of-function of p53 contributes to PNET pathology [97], which is consistent with the observation that advanced metastatic PNET patients often display mutations in TP53 and RB1 locus [77]. In addition, both P53 and its downstream effector PHLDA3 have been found to be hypermethylated at their gene promoter regions, respectively [41,98], suggesting that p53 signaling regulates PNET development at both genetic and epigenetic levels.

3.1.8. Notch Signaling

The Notch signaling pathway is a highly conserved cell signaling system present in most animals, whose proteins span the cell membrane [99]. Notch signaling is responsible for differentiation and tissue homeostasis, and its dysregulation has been attributed to development of multiple cancers including leukemias such as T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL) [100]. In patients with breast cancer, elevated expression of histone methyltransferase NSD3 was associated with recurrence, distant metastasis, and poor survival. In mice, NSD3 promoted malignant transformation of mammary epithelial cells [101]. NSD3-induced methylation of H3K36 is crucial for Notch-dependent tumor initiation and metastasis, suggesting that Notch-related methylation factors may be an actionable therapeutic target in localized and metastatic breast cancer [102]. In colorectal cancer, the upregulation of oncogenic histone cluster 2 H2B family member F (HIST2H2BF) enhances malignancy aggressiveness in humans and increases liver metastasis in mice through the activation of Notch signaling. Reactivation of this cluster is found related to promoter CpG hypomethylation [103]. High expression of SETD1A, a histone methyltransferase that specifically methylates H3K4, acts as a key oncogene in ovarian cancer and is associated with a poor prognosis. Accordingly, overexpression of SETD1A augments cell proliferation, migration, and invasion in vitro via initiation of Notch signaling, and downregulation of SETD1A reduces tumorigenesis in vivo [104]. Among North Indian patients with cervical cancer, the methylation rate of Notch1 and Notch3 promoters is notably elevated compared to healthy tissues, accompanied by a downregulation in protein expression. In addition, Notch1 promoter methylation increases with age, severity of the disease, and HPV infection in patients with cervical cancer [105]. Low expression of the methyltransferase DNMT3A, a key regulator of DNA methylation, is associated with more aggressive types of chronic lymphocytic leukemia (CLL). Dnmt3a knockout mice consistently developed CLL and showed a general upregulation of Notch signaling genes with sensitivity to Notch inhibition using daptomycin [106]. In PNETs, the expression of Notch1 signaling was highly related to the cancer progression [107]. Activation of Notch 1 inhibits tumor growth, suggesting that Notch 1 serves as a tumor suppressor [108,109,110]. Accordingly, Notch1 activation has been shown to be induce stable disease in a fraction of PNET patients in a Phase II trial [111]. As of now, there is no documentation of DNA methylation occurring in the Notch1 signaling pathway in PNET tumor samples.

3.1.9. Wnt/β-catenin

Wnt/β-catenin signaling has been widely known to be closely related to tumor progression, metastasis, migration, and invasion [112]. The Wnt pathway includes both canonical (β-catenin dependent) and non-canonical signaling. The canonical Wnt signaling has been known to play a large role in the pathogenesis of colon cancer with greater than 90% of colorectal cancers carrying a mutation that activates Wnt signaling. When Wnt signaling is activated, Wnt molecules will bind to the FZD-LRP5/6 co-receptor complex. LRP6 requires acetylation by p300 to become activated for Wnt signaling [113]. In epithelial ovarian cancer (EOC) cells, alternations of DNA methylation regulate members of the Wnt/β-catenin pathway [114]. Interestingly, the combination of curcumin, commonly recognized as turmeric, with decitabine hinders the formation and migration of ovarian cancer cell colonies. This effect might be achieved, at least partially, by downregulation of DNMT3a protein which in turn contributes to inhibition of the epithelial-to-mesenchymal transition (EMT) process. [115]. Adherens Junctions Associated Protein 1 (AJAP) forms a complex with E-cadherin and β-catenin in the cytomembrane, thereby diminishing the nuclear translocation of β-catenin and impeding the Wnt/β-catenin signaling pathway. In salivary adenoid cystic carcinoma (SACC) tumors, AJAP has been found downregulated, primarily due to promoter hypermethylation which causes AJAP gene silencing. Silencing AJAP1 in SACC cells markedly amplifies both in vitro and in vivo processes of proliferation, invasion, and metastasis. Furthermore, it independently contributes as a risk factor to the prognosis of SACC (38). Zinc-finger protein 471 (ZNF471) exerts its tumor-suppressive functions through suppressing EMT, tumor cell stemness and inhibition Wnt/β-catenin signaling. In both breast cell lines and tissues, there is notable downregulation of the promoter CpG methylation of ZNF471, as observed in comparison to normal mammary epithelial cells and their corresponding surgical-margin tissues [116]. In colorectal cancer, SET and MYND domain-containing protein 2 (SMYD2) suppresses the expression of adenomatous polyposis coli 2 (APC2), an inhibitor of the Wnt/β-catenin pathway. The decreased APC2 expression in colorectal cancer cells is due to SMYD2-mediated DNA methylation, which requires synergism with DNMT1 [117]. Lysine-specific histone demethylase 1A (KDM1A) regulates the stemness of thyroid cancer and promotes thyroid cancer progression via the Wnt/β-catenin pathway. Mechanistically, KDM1A downregulates APC2 and DKK1, two antagonists of the canonical Wnt pathway, by demethylation. Additionally, GSK-LSD1, a highly selective inhibitor of KDM1A, significantly inhibits thyroid cancer progression and enhances the sensitivity of thyroid cancer to chemotherapy [118]. In the context of PNET development, precise regulation of the Wnt/β-catenin signaling pathway is essential for normal pancreas development [119]. However, the dysregulation of Wnt/β-catenin leads to elevated expression of β-catenin and the decreased expression of Wnt/β-catenin inhibitors, a phenomenon frequently observed in higher-grade pNET tumors [120]. In addition, aberrant methylation of SFP1, an inhibitor of Wnt/β-catenin pathway, caused downregulation of SFP1 protein expression and enhanced tumor growth. Conversely, overexpression of SFP1 and WIF-1, another Wnt/β-catenin inhibitor, resulted in inhibition of tumor growth both in vitro and in vivo [121]. Furthermore, there is a strong correlation between promoter CpG hypermethylation and decreased expression of O6-methylguanine-methyltransferase (MGMT), a downstream target of the Wnt/β-catenin pathway, in PNETs [122,123].

3.1.10. NF-κB

NF-κB signaling plays critical roles in cancer development by mediating the inflammatory tumor microenvironment [124]. There have been numerous reports of epigenetic regulation of NF-κB signaling in cancers. In colorectal carcinoma, protein arginine methyltransferase 5 (PRMT5) catalyzes methylation of the multifunctional protein Y-box binding protein 1 (YBX1) and causes NF-κB activation which in turn leads to increased cell proliferation in vitro [125]. In addition, NF-κB signaling has been involved in the regulation of fatty acid-binding proteins (FABPs), dysregulation of which have been shown in many types of cancer. FABP5 promoter hypomethylation correlates with its increased expression, which forms a positive feed-back loop with NF-κB to promote tumor metastasis [126]. On the contrary, DRD2, involved in restricting NF-κB signaling and EMT in breast cancer (BC), undergoes downregulation due to promoter hypermethylation. DRD2 demonstrates the ability to suppress tumorigenesis in both in vitro and in vivo settings. Notably, there exists a positive correlation between DRD2 expression and extended survival times in BC patients [127]. In non-small cell lung cancer (NSCLC), genetic deletion and methylation contribute to decreased expression of circadian gene hepatic leukemia factor (HLF). HLF inhibits NF-κB/p65 signaling via increasing activity of PPAR in malignant tissues [128]. In PNET samples, upregulation of NF-kB is positively correlated with tumors with higher grade. In addition, down-regulation NF-kB and STAT3 inhibits proliferation, viability, and spheroids growth of PNET cell lines [129]. Presently, there is no documentation on the DNA methylation status of NF-κB in PNET.

3.1.11. Somatostatin Receptor 2 (SSTR2)

Somatostatins are a family of peptide hormones which are secreted by pancreatic islet δ cells and binds to somatostatin receptor (SSTR) signaling to inhibit the release of a variety of enzymes including insulin, glucagon, pancreatic amylase, along with other hormones secreted by pancreas. Upon binding of SST, SSTR exerts anti-proliferative functions, thereby serving as a tumor suppressor in PNET [69]. Since SSTR2 has been found highly-expressed in a majority of neuroendocrine neoplasms [130], the antitumor activity of SSTR2 has been explored in treatment of NE including PNET [131]. Methylation profiling of PNET samples showed hypermethylated promoter of SSTR2 gene [132] and, not surprisingly, DNMT inhibition recovered SSTRs expression in cell cultures and facilitated delivery of novel peptide receptor radiotherapy against PNET [133].

3.1.12. Smad3

SMAD transcriptional factors, which are part of transforming growth factor-β (TGF-β) signaling pathway, regulate cell differentiation and proliferation [134,135]. Depending on the cell status, activated SMAD can lead to either induction or inhibition of cell growth [135]. In PNETs, TGF-β/SMAD signaling mainly functions to inhibit cell growth via the SMAD downstream effectors, p21WAF1/CIP1 tumor suppressor [135,136]. Loss of homozygosity (LOH) of the SMAD3 gene has been observed in a fraction of PNET patients [137,275]. In addition, inhibition of TGFβ/ SMAD by inhibition of SMAD7 causes over-proliferation of islet β cell proliferation in adult mice, suggesting that TGF-β/Smad signaling might plays a role in PNET progression [138]. Smad3 has been shown to be undergone promoter CpG methylation in colorectal cancer [139,140]. Nevertheless, there is currently no information available regarding the DNA methylation of Smad3 in PNET. A complete list of DNA methylation profiling of PNET-related pathways was shown in Table 1.

3.2. Histone Modifications

Histone modifications include both acetylation and methylation of histones which control the conformation of genomic DNA. Acetylation is a post-translational histone modification process that modulates the opening of the chromatin structure allowing for gene transcription. Through the opposing activities of histone deacetylase (HDACs) and histone acetyl transferases (HATs), the process of acetylation is reversible through either the addition or removal of acetyl groups from the amino- terminal ɛ-group of lysines on histones. HDACs catalyze the removal of the acetyl moieties from acetylated histones and are generally associated with transcriptional repression [144]. Whereas histone acetylation is linked to transcriptional activation, histone methylation can either be repressive or activating. This depends on which lysine residue (K) on which histone (H3, H4) is modified, and the extent of methylation, i.e. di- or tri-methylation (me2, me3). For example, there are markers for inhibitory histone methylation, such as H3K9me2/3 and H3K27me2/3. In contrast, H3K4me2/3, H2K36me3, and H3K79me3 serve as markers for activating histone methylation. The mediation of the histone methylation process involves histone lysine methyltransferases (HMTs) and histone demethylases (HDMs) [145].

Histone modifications have been shown to regulate many important biological functions including cell cycle progression, metabolism, differentiation, and development. They are found involved in various tumors and are involved in vital chromosomal translocation-mediated oncogenic protein fusion and carcinogenic events [146,147]. Histone modifications play a crucial role in the different stages of cancer and have been linked to a variety of malignancies including solid and hematologic tumors. For example, increased levels of HDACs are correlated with advanced diseases, and poor outcomes. For example, increased levels of HDAC 5 have been correlated with metastatic PNETs. Similarly, elevated expression of HDACs 1, 2, and 3 is linked to poor outcomes in gastric and ovarian cancers [148,149]. Both histone acetylation and methylation have been found involved in regulation of multiple signaling pathways in cancer development [147].

3.2.1. MEN1

Despite the lack of evidence to support a direct role for histone acetylation or deacetylation in MEN1-associated endocrine cells, there is indirect evidence through examining the effects of HDAC inhibitors on MEN1-associated cell lines. Menin protein regulates the cyclin B2 promoter region by modifying histone H3 acetylation and H3K4me3 methylation levels in mouse embryonic fibroblast cells (MEF) [150]. HDAC inhibitors such as TSA, thailandepsin-A (TDP-A), sodium butyrate (NaB), valproic acid (VPA), and BET protein bromodomain inhibitors (BETi) have been well studied in neuroendocrine tumor cell lines including BON-1, NCI-H720, NCI-H727 and QGP-1 [151,152,153,154,155]. HDAC inhibitors have been found to induce dose dependent growth inhibition on NET cells. The anti-tumor effects of HDAC inhibitors have been further demonstrated in a BON-1 xenograft model [153] and a pancreatic beta cell-specific MEN1 knockout mouse model [155]. In addition, menin itself can serve as part of a mixed-lineage leukemia histone methyltransferase complex that functions to promotes histone methylation [156]. Polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1 in diffuse large B-cell lymphoma (DLBCL) cells. In cells with EZH2 gain-of-function mutations, combinatorial inhibition of MEN1 using MI-503 or VTP50469 with EZH2 inhibitor Tazemetostat (EPZ-6438) has been shown to decrease tumor burden and survivability in a xenograft model [157]. Aberrations of EZH2 gene, along with MEN1 and others, can cause an imbalance in the activities of histones via methylation, contributing to parathyroid tumorigenesis in parathyroid carcinoma as well [158]. MEN1 even appears to play a role as a tumor suppressor gene in colorectal cancer, with recent work finding a hotspot mutation in MEN1 that affects 4% of BRAF mutant cancers [159].

3.2.2. mTOR-TSC

PI3-AKT-mTOR signaling is the key mechanism that normal cells use to metabolize glucose. In glioblastoma, this pathway has been found to be acetylated resulting in the amplification of c-Myc and promotion of glutaminolysis. mTORC2 regulates c-Myc levels through Akt-independent phosphorylation of class II HDACs (4,5,7,9), which leads to the acetylation of FoxO1 and FoxO3 and release of c-Myc. Upregulation of mTORC2, c-Myc, and acetylated FoxO have been found to be attributed to worse prognosis in glioblastoma patients. Pharmacologic inhibition of PI3K and mTOR kinase have been found to suppress FoxO acetylation, decrease c-Myc levels, and result in tumor cell death [160]. mTORC1 and mTORC2 cooperatively regulate H3K27 causing hypermethylation which subsequently promotes glioblastoma tumor cell survival both in vitro and in vivo, suggesting that dynamic regulation of histone methylation by mTOR complexes could be exploitable as a novel therapeutic target against this deadly tumor [161]. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that dephosphorylates phosphatidylinositol-3,4,5 trisphosphate (PIP3) to negatively regulate the PI3K-AKT signaling pathway. The PI3/AKT signaling pathway is responsible for upregulating protein synthesis, cell migration, and tumor induced angiogenesis [162]. Mutations of PTEN are responsible for many cancers including brain, breast, prostate, endometrial carcinoma, head and neck cancers, melanoma, and PNET [163]. PTEN is acetylated at K125 and K128 by the p300/CBP associated factor (PCAF), resulting in its inactivation and upregulation of the PI3K-AKT signaling pathway [164]. TSA and SAHA have been found to up-regulate PTEN by inducing PTEN membrane translocation through acetylation at K163 in 293T cells. Further studies have determined that HDAC6 inhibition, using the HDAC6 specific inhibitor tubastatin A, caused PTEN acetylation and activation [165]. TSC2 forms a complex with TSC1, which negatively regulates mTOR. Dissolution of the TSC2/TSC1 complex allows for the activation of mTOR. [166]. Mutations in either TSC1 or TSC2 lead to the onset of tuberous sclerosis complex, characterized by the formation of hamartomas, also called benign tumors, in the skin, brain, lungs, heart, and kidneys [167]. Patients with TSC are also at greater risk for developing malignancies, such as renal cell carcinoma, breast cancer, thyroid cancer, and PNETs [168]. There is currently no report of histone modifications of mTOR pathway regarding PNET.

3.2.3. HIF1α-VHL

Epigenetic regulation of HIF1⍺ signaling has been well established in a variety of cancers. P300 is a transcriptional component of HIF1⍺ which acetylates HIF1⍺ at Lys-709 increasing its stability as shown in both osteosarcoma and renal cell carcinoma cell lines [169]. HDAC inhibitor (HDACi) SAHA has been found to decrease HIF1⍺ levels in tumor cell lines via direct acetylation of heat shock protein 90 (Hip90), a HIF⍺ chaperone protein, using HDAC6 [170]. In addition, HIF1α R282 has been shown methylated by protein arginine methyltransferases 3 (PRMT3) in CRC, which is necessary for its stabilization and oncogene function. PRMT3 inhibition with a novel therapeutic molecule (MPG-peptide) decreases tumor growth and angiogenesis in vivo, potentially offering a future therapeutic strategy [171]. Presently, there is no available information on the histone modifications of the mTOR pathway regarding the HIF1α-VHL signaling in PNET.

3.2.4. RAS-MAPK-NF1

RAS has been found acetylated in human cancer lines on lysine 104 resulting in destabilization of its Switch II domain, which modulates the interaction between RAS and GEF as well as RAS and PI3K for GAP-induced GTP hydrolysis. Essentially, acetylation stands as a negative regulator of RAS function exerting anti-oncogenic effects [172]. Other studies have established a link between RAS signaling and H3K9ac modification, which is one of the most widely studied acetylation site of histone H3 tails and has been commonly observed in ovarian cancer, hepatocellular carcinoma, oral cancer, and cervical cancer [173]. Through binding to xeroderma pigmentosum A protein (XPA), RASSF1A regulates the acetylation and deacetylation cycle of XPA [174]. Not only does RASSF1A acts as a tumor suppressor through DNA repair, but RASSF1A has also been found to increase microtubule stability through HDAC6 which prevents tumor cell migration, invasion, and metastasis [59]. The effect of RASSF1A was also demonstrated in NSCLC xenograft model and in human bronchial cells [175]. Patients with NF1 familial syndrome sometimes develop benign plexiform neurofibromas, which can develop to form peripheral nerve sarcomas known as malignant peripheral sheath tumors (MPNSTs). Bromodomain containing protein 4 (BRD4), a histone acetyltransferase, binds to acetylated histone H3K27Ac in MPNSTs, and has been found to be one of the most highly unregulated genes in MPNSTs. The Bromodomain-containing inhibitor, JQ-1 has been demonstrated to decrease MPNST tumor growth in vitro and in vivo [176]. A phase II trial was conducted in patients with MPNST using JQ-1 (NCT02986919); however, the trial was discontinued due to lack of participants. Currently, there is a new Phase 1/2 Trial that is evaluating the efficacy of Bromodomain-containing inhibitor AZD5153 in combination with PL1 therapy and MEK inhibitor therapy for the treatment of neurofibromatosis [177]. In PNET, the only report of histone modifications in RAS-MAPK signaling comes from one of the receptor tyrosine kinases, IGF2, which shown aberrant H3K4 methylation in the pancreatic islets of MEN1-deficient mice [178].

3.2.5. ATRX/DAXX

The chromatin remodeler ATRX and the histone chaperone DAXX are mutated in a variety of cancers including glioblastoma multiforme, pediatric adrenocotical carcinoma, osteosarcoma, neuroblastoma, myelodysplastic syndrome, acute myeloid leukemia, and PNETs [179]. As mentioned in Section 3.1, ATRX and DAXX form a histone chaperone complex that deposits histone variant H3.3 into specific genomes. The first discovery of ATRX/DAXX mutations came from PNETs where they were found to be mutated in 23% of the tumors. These mutations are inactivating mutations and exhibit alternative lengthening of telomeres (ALT) phenotype. Recently, DAXX has been found to exist in two distinct H3.3. complexes, which include DAXX-ATRX complex and DAXX-SETB1-KAP1-HDAC1 complex [180]. The DAXX-SETB1-KAP1-HDAC1 represses endogenous retroviral (ERV) sequences. ERVs lead to genomic instability through insertion into protein coding regions or by promoting transcription of neighboring genes leading to tumorigenesis [181].

3.2.6. CDKN2A/RB1

RB1 inhibits G1 to S transition through the repression of E2F target genes which are involved in DNA synthesis and cell cycle progression. RB1 antagonizes E2F activity through binding to E2F transcription factors as well as multiple corepressor molecules including HDACs (HDAC1, 2, 3) [182,183]. HDAC inhibitors such as SAHA have been shown to cause RB1-mediated silencing of cell cycle oncogenes [184]. More recently, HDAC5 has been found to act as a corepressor for RB1 but can be inhibited by CDK 4/6. CDK 4/6 inhibition was found to specifically enhance HDAC5 binding to RB1. Resistance to CKD 4/6 inhibitor therapy such as palbociclib was found to be related to low HDAC levels in cancer cells, which means HDAC5 could act as a useful biomarker in guiding therapy [185]. HDAC inhibition has been shown to have anti proliferative effects by inducing cell cycle arrest in G1 via cycling-dependent kinase inhibitors or downregulation of cyclins and CDKs [186]. Inhibition of HDACs induced elevated expression of CDK inhibitors, resulting in the blockade of the cell cycle at the G1 phase and disruption of the G1/S transition. This occurred through the reactivation of RB1 function, leading to the inhibition of E2F, halt of G1 progression, and cell apoptosis [187,188]. HDAC inhibitors such as TSA, SAHA, and VPA have been shown to result in cell cycle arrest at the G2/M checkpoint [189]. HDACs not only play a transcriptional role in cell cycle progression, but also can act as a cell cycle regulator through modulating Aurora B kinase in mitotic progression [190]. Maggi et al. reported that histone demethylase retinoblastoma binding protein 2 (Rbp2) was found overexpressed in PNET tumors. In addition, aberrant expression of Rbp2 altered histone demethylation and contributed to PNET pathogenesis [191].

3.2.7. P53

One of the first recognized non-histone proteins affected by acetylation is p53 [192,193]. P53 is a tumor suppressor gene that is negatively regulated through MDM2 and transiently stabilized and activated by various intracellular processes. While the phosphorylation of serine residues was previously recognized as the primary stabilizer of p53, it has been discovered that acetylation also plays a role in stabilizing p53 during cellular stress and contributes to enhancing transcriptional activity of p53 [193]. MDM2 has been reported to negatively regulate p53 acetylation, but can be reversed by tumor suppressor p14 ^ARF [193]. In H1299 carcinoma cells, acetylation status regulated degradation of p53 through directly interacting with MDM2 E3 ligase [194]. HDAC inhibitors such as TSA and SIRT1 were found to reverse inhibitory effects of cAMP on DNA damage-induced p53 stabilization and apoptosis [195]. TSA has also been found to upregulate PUMA promoter of p53 in gastric cancer through inhibition of HDAC3 [196]. HDAC inhibitors may lead to p53 activation and pro-apoptotic events, but they do not need p53 for exerting anticancer effects. Rather, studies have shown HDAC inhibitors can independently act to upregulate p21 and BID as a backup mechanism for tumor suppression [197]. Recent evidence has found that p53 itself can be a driver of HDAC inhibition, which in turn leads to autophagy and programmed cell death [198]. HDAC inhibitors VPA and TSA were found to induce apoptosis in pancreatic cancer derived cell lines, Panc1 and PaCa44, through upregulation of p21 and Puma resulting in mutant p53 degradation [199]. Currently, there is no available information on histone modifications of p53 in PNET research.

3.2.8. Notch Signaling

In Notch signaling pathway, ligand and notch receptor binding releases Notch Intracellular Domain (NICD), which translocates to the nucleus to activate gene expression through assembling a coactivator complex which includes RBJP and histone acetyltransferase p300. During this process, HDAC3 increases NICD stability through reducing p300 mediated acetylation of NICD protein. HDAC3 expression was found to be significantly higher in T-ALL patients and in CLL B cell samples [100]. VPA has been found to activate Notch signaling in tumor cells, resulting in suppressed growth of carcinoid tumors in a mouse tumor xenograft [108,200]. These finds were further confirmed in a phase II pilot study that examined the effects of VPA on eight patients with low grade neuroendocrine tumors (carcinoid and pancreatic). Pretreatment tumor biopsies revealed low Notch1 levels, and a 10-fold increase in Notch1 activity on post VPA treatment tumor biopsy [111].

3.2.9. Wnt/β-catenin

Histone modifications have been shown to be involved in regulation of Wnt signaling via complicated mechanisms. Upon activation, Wnt molecules bind to the FZD-LRP5/6 co-receptor complex. LRP6 requires acetylation by p300 to become activated for Wnt signaling. During Wnt signaling, β-catenin is released from the destruction complex and can become acetylated by CBP, p300, and PCAF, which allows for upregulation of β-catenin activity. β-catenin will then undergo nuclear translocation to activate gene transcription [113]. When Wnt signaling is in its inactive state, beta-catenin becomes phosphorylated by the protein destruction complex and targeted for proteasomal degradation. Once degraded, a repressive complex containing T cell factor (TCF)/ lymphoid enhancer factor (LEF) and transducing like enhancer protein (TLE) utilizes HDACs to repress target genes [201]. HDACis such as TSA have been found to inhibit the Wnt/β-catenin pathway by increasing β-catenin phosphorylation, reducing β-catenin nuclear translocation, and inhibiting β-catenin/TCF complex formation in pituitary corticotrope tumor cells (AtT20) [202]. TSA and SAHA have demonstrated anti-tumor effects in colon cancer cell lines with β-catenin mutations by downregulating the Wnt transcription factor TCF7L2 [203]. Curcumin, as discussed in Section 3.1, serves as an inhibitor of acetyltransferase, specifically targeting p300 in the Wnt pathway. Currently, curcumin is undergoing Phase I clinical trials for the treatment of breast, colon, and pancreatic cancers [204]. Currently, there is no available information on histone modifications of Wnt/β-catenin in PNET research.

3.2.10. NFκB

Post translational modifications, including acetylation and methylation, play a major role in the regulation of NF-KB signaling. These modifications occur in the cytoplasm and allow for the activation of IκB kinases (IKKs) resulting in the degradation of IκBalpha, which allows for translocation of NFκB into the nucleus [205]. Within the nucleus, Re1A, a subunit of NFκB, undergoes acetylation by p300/CBP at lysine 310 which promotes transcriptional activity of NFκB [206]. Stat 3 is an oncogenic transcription factor that is commonly activated in cancer. Stat 3 has been found to maintain tumor NFκB activity through acetylation of Re1A. This has been studied in human A2058 melanoma cell lines and in DU145 prostate cancer cells [207]. To date, there have been no documented instances of histone modifications affecting NFκB in PNET.

3.2.11. SSTR2

In the PNET cell lines BON-1 and QGP-1, histone 3 acetylation was observed on SSTR2 [208]. This finding was subsequently corroborated by Veenstra et al. [209], suggesting that the regulation of SSTR2 expression is likely influenced by both histone acetylation and DNA methylation [132]. In addition, using an in vivo BON-1 xenograft mouse model, Kidd et al. showed that the combination treatment of HDACi (VPA) and camptothecin-somatostatin conjugate significantly reduced growth comparing to monotherapies, respectively [210], suggesting that combinatorial therapy might hold promise in PNET patient treatment.

3.2.12. Smad3

Epigenetic regulation of Smad3 via histone modifications plays important roles in cancer progression and metastasis [211]. In lung cancer, the actin binding protein profilin-2 binds to and thereby prevents HDAC1’s access to the promoters of Smad2 and Smad3, which causes activation and promotes EMT and angiogenesis in lung cancer cells [212]. Besides acetylation, histone methylation has also been observed in lung cancer cells. Smad3 mediates recruitment of the histone methyltransferase SETDB1, which controls the expression of SNAIL1 and EMT in breast cancer [213]. Despite its crucial regulatory role in PNET, as outlined in Section 3.1, there is presently no information on histone modifications of Smad3 in the pathology of PNET. A complete list of histone modifications in PNET-related signaling pathways are listed in Table 2.

3.3. Non-Coding RNAs

Alterations in both coding and ncRNAs have been widely implicated in cancer pathophysiology [218]. There are a few recent reviews which described the limited literature concerning the role of ncRNA in pancreatic adenocarcinoma cancers and PNET [219,220,221]. However, comparing to other heavily studied cancers, there is still lack of study of both miRNAs and long non-coding RNAs (lncRNAs) due to the low incidence of PNET cases.

3.3.1. MEN1

The regulatory landscape of the MEN1 signaling pathway in cancer has recently become a focal point of research, with ncRNAs emerging as influential players in this intricate network. NcRNAs exert nuanced control over gene expression, contributing to the dynamic modulation of the MEN1 pathway in cancer progression. For instance, multiple miRNAs, including miR-142-3p, let-7i, miR-125a-5p, miR-199b-5p, and miR-1274b_v16.0, and miR-193b have been found differentially expressed in MEN1 parathyroid tumors comparing to normal parathyroid tissues [222]. Furthermore, lncRNA NEAT1 has been implicated in the dysregulation of MEN1 signaling in multiple endocrine neoplasia type 1 (MEN1) syndrome, acting as a competing endogenous RNA to sequester miR-34a and alleviate its inhibitory effect on MEN1 [26,223,224]. Additionally, miR-29a has been identified as a negative regulator of MEN1 in parathyroid adenomas, influencing cell proliferation and apoptosis [225]. These examples underscore the intricate interplay between ncRNAs and the MEN1 pathway, highlighting the diverse regulatory roles of ncRNAs in shaping the landscape of cancer progression. Investigating these molecular interactions offers promising avenues for understanding the underlying mechanisms and identifying novel therapeutic targets in MEN1-associated cancers. Human maternally expressed gene 3 (MEG3) encodes an RNA transcript that exhibits tumor suppressive ncRNA functions (lncRNA MEG3) with an unknown contribution to cellular processes in its limited translated form [226,227,228]. MEG3 levels are downregulated in PNET through a variety of mechanisms. In murine embryonic stem cells undergoing differentiation into pancreatic islet-like endocrine cells (PILECs), a biallelic MEN1 mutation resulted in reduced menin protein levels, subsequently leading to decreased transcription of MEG3 [178]. In both human PNET samples and MIN6 murine insulinoma cells, hypermethylation of the MEG3 promoter are associated with downregulation of MEG3 compared to normal tissue [229]. Luzi et al. used BON-1 luciferase reporter cell line to show that menin acted as a negative regulator of miR-24-1, which in turn downregulated menin in a negative feedback loop [230]. In a subsequent investigation, it was demonstrated that reduced menin expression resulting from mutations in neuroendocrine cancers disrupted the feedback loop between menin and miR-24-1. This disruption further contributed to a decrease in menin levels due to unregulated expression of miR-24-1 [231]. These studies unequivocally demonstrate that diminished menin levels, with miR-24-1 as a driving factor, play a role in the pathogenesis and progression of neuroendocrine neoplasms [232].

3.3.2. mTOR

The intricate regulation of the mTOR signaling pathway in cancers has garnered substantial attention, with ncRNAs standing out as prominent players in modulating the mTOR pathway, influencing various facets of cancer progression. For example, miR-99a has been identified as a critical regulator by directly targeting mTOR, suppressing its expression and impeding cell proliferation in hepatocellular carcinoma [233]. In breast cancer, the lncRNA HOTAIR acts as a molecular scaffold to facilitate the assembly of the mTOR signaling complex, promoting tumor growth [234]. Additionally, the lncRNA TUG1 has been shown to modulate mTOR signaling in renal cell carcinoma, influencing cellular processes such as proliferation and apoptosis [235]. In MIN6 luciferase reporter cell line, overexpression of miR-144 downregulated PTEN which in turn caused upregulation of p-AKT pathway and a subsequent increase of beta cell proliferation [236]. This piece of evidence is in line with the expression pattern of p-AKT and miR-144 in a differential expression analysis of 29 human insulinoma samples in comparison with normal tissues [236]. In addition, overexpression of a lncRNA H19 plasmid activated VGF and PI3K/AKT pathway in QGP-1 cell line, suggesting a link between lncRNA H19 and an increase in proliferation and metastasis of neuroendocrine tumors [237].

3.3.3. HIF1α

The regulatory influence of ncRNAs has been recognized as pivotal players in modulating the dynamic HIF-1α signaling cascade, influencing critical aspects of cancer progression. Notably, miR-210 has been identified as a key regulator by directly targeting HIF-1α, promoting angiogenesis and metastasis in various cancers [235,238]. In renal cell carcinoma, the lncRNA H19 acts as a molecular sponge for miR-29a, derepressing HIF-1α expression and fostering tumor growth [239]. Additionally, miR-20a has been implicated in the regulation of HIF-1α in colorectal cancer, influencing cellular responses to hypoxia [240]. The lncRNA MALAT1 has also been associated with HIF-1α modulation in breast cancer, affecting tumor invasion and metastasis [241]. These examples underscore the intricate roles of ncRNAs in shaping the dynamics of HIF-1α signaling in cancers. There are a few reports regarding miRNA-mediated regulation of HIF1α signaling in PNET. Thorns et al. compared the differential expression of miRNAs in 37 PNET tissue samples and 9 non-neoplastic controls, from which they were able to identify two miRNAs. While levels of miR-642 were positively correlated with Ki-67 scores, levels of miR-210 expression were positively correlated with metastasis [242]. The connection between miR-210 and its targets has not been elucidated in PNET, although in liver metastasis samples from colorectal adenocarcinoma patients, miR-210 was shown to affect disease progression possibly through interaction with the HIFa pathway [243,244].

3.3.4. Ras-MAPK

The Ras-MAPK signaling pathway plays a pivotal role in regulating various cellular processes, and the influence of ncRNAs on this pathway has emerged as a significant area of exploration in cancer research. For instance, miR-21 has been implicated in the activation of Ras-MAPK signaling in pancreatic cancer, promoting cell proliferation and invasion [245]. Conversely, miR-143 functions as a tumor suppressor by targeting KRAS, a key component of the Ras pathway, and inhibiting its downstream signaling in colorectal cancer [246]. In melanoma, the lncRNA SPRY4-IT1 has been shown to activate the Ras-MAPK pathway by regulating SPRY4 expression [247]. Additionally, the lncRNA MALAT1 has been associated with Ras-MAPK signaling in lung cancer, influencing cell proliferation and migration [248]. Using both QGP-1 GFP reporter cell line and a murine xenograft model, Zhang et al. showed that miR-431 promoted EMT, invasion, and metastasis by silencing DAB2IP, a tumor suppressor protein whose downregulation resulted in the direct activation of the Ras pathway [249].

3.3.5. DAXX/ATRX

Recently, ncRNAs have emerged as key regulators of the DAXX-ATRX signaling pathway, contributing to the complexity of cancer progression. For example, miR-1269a has been identified as a regulator that targets ATRX, promoting glioma cell proliferation and invasion [250]. In addition, miR-21 is found upregulated in gastric cancer. Accordingly, overexpression of miR-21 promotes tumor growth by targeting DAXX in gastric cancer cell lines [251]. Furthermore, the lncRNA XIST has been implicated in the regulation of DAXX-ATRX in glioblastoma, influencing both chromatin remodeling and tumor progression [252]. In the context of PNET research, Gill et al. performed microarray differential expression profiling of miRNA in 37 PNET tissue samples including local and distant metastases and found significant upregulation of miR-3653 in the metastatic group. Following bioinformatic analysis of three miRNA databases, ATRX was revealed to be a negatively regulated target for miR-3653, suggesting a possible link between miR-3653 and PNET pathogenesis through dysregulation of the DAXX/ATRX pathway [253].

3.3.6. RB1

Recent investigations reveal that ncRNAs play intricate roles in modulating RB1 signaling, contributing to the complex landscape of cancer progression. For instance, miR-106b-5p has been identified as an oncogenic miRNA that target RB1 signaling, promoting cell cycle progression and proliferation in laryngeal carcinoma [254]. Conversely, miR-34a and miR-15a/16 act synergistically to induce cell cycle arrest and apoptosis in NSCLC cell lines in a RB1-dependent manner [255]. In addition, the H19 ncRNA controls cell proliferation and metastasis by regulating essential RB1-E2F signaling in colorectal cancer [256]. Furthermore, the lncRNA PVT1 has been shown to regulate RB1 in gastric cancer, contributing to tumor growth [257]. Despite its critical regulatory role in PNET as described in Section 3.1, there have been no documented instances of ncRNAs affecting RB1 in PNET.

3.3.7. p53

The p53 signaling pathway, a crucial guardian of genomic stability and a key player in tumor suppression, is intricately regulated by ncRNAs in the complex landscape of cancer biology. For instance, the miRNA miR-125b has been implicated as an oncomiR that targets p53, promotes cell proliferation, and inhibits apoptosis in leukemia. On the other hand, the miRNA miR-34a, a well-established tumor suppressor, directly targets p53, inducing cell cycle arrest and apoptosis in multiple cancer types, including breast and lung cancers. The lncRNA TP53TG1 also acts as a positive regulator of p53 by enhancing its stability and transcriptional activity in gastric cancer [258]. Additionally, the NEAT1 functions as tumor suppressor in hepatocellular carcinoma in a p53-dependent manner [259]. Furthermore, Zhou et al. showed that MEG3 caused downregulation of MDM2 expression and stabilize p53, suggesting that lncRNA MEG3 stimulates p53 transcription and is linked to p53 longevity and activity [260]. As of now, there is no available information on the regulation of p53 signaling in PNET through ncRNA mechanisms.

3.3.8. Notch

Both miRNAs and lncRNAs play crucial roles in modulating Notch signaling in cancer development. For instance, the expressions of Notch-1 and miR-21 were found to have a positive correlation with the development of colorectal cancer, suggesting that miR-21 may function to promote cancer progression [261]. Conversely, miR-34a has been identified as a key player in negatively regulating Notch signaling by targeting Notch1 and Jagged1, leading to suppressed proliferation and enhanced apoptosis in colorectal cancer [262]. In addition, miR-34a has been demonstrated to directly inhibit Notch-1/2 by binding directly to their 3’ UTR in glioma cells [263]. Moreover, the lncRNA HOTAIR regulates the expression of notch3 by competitively binding with miR-613, making the notch3-HOTAIR-miR-613 complex a potential drug target in pancreatic cancer [264]. In the context of PNET, by combining RNASeq and IHC expression analysis, He et al. showed that upregulation of lncRNA XLOC_221242 correlates with overexpression of DNER protein and activation of a variety of precursors in the Notch and Wnt signaling pathways, suggesting that lncRNA XLOC_221242 might interact with Notch signaling in the regulation of PNET progression [265].

3.3.9. Wnt/β-catenin

The Wnt/β-catenin signaling pathway is subject to intricate modulation by ncRNAs in the context of cancer progression. For instance, miR-21 has been identified as an oncomiRNA that promotes Wnt/β-catenin signaling activation by targeting negative regulators, contributing to enhanced cell proliferation and invasion in colorectal cancer [266]. Conversely, the tumor suppressor miRNA miR-34a directly targets Wnt/β-catenin pathway components, inhibiting cell growth and inducing apoptosis in hepatocellular carcinoma [267]. The lncRNA HOTAIR has been associated with Wnt/β-catenin pathway activation in breast cancer, fostering tumor progression [268]. Additionally, the lncRNA CCAT2 forms a feedback loop with Wnt/β-catenin signaling in colon cancer [269]. In the context of PNET, decrease of MEG3 caused upregulation of miR-183 in BON-1 cells. MiR-183 functions as a downstream target for MEG3 and is negatively regulated by MEG3. When miR-183 activity was increased, it upregulates BRI3, which positively relationship correlates with p38/ERK/AKT and Wnt/β-catenin pathways, suggesting a complicated tumor promoting mechanism which includes interaction among MEG3, miR-183, and BRI3 via p38/ERK/AKT and Wnt/β-catenin signaling [228,229]. In addition, in BON-1 and LCC-18 colonic neuroendocrine cell lines, overexpression of lncNEN885 caused a decrease of invasion and EMT by downregulating Wnt/β-catenin signaling. siRNA silencing of lncNEN885 restored markers of EMT and component of the Wnt/β-catenin pathway, suggesting that lncNEN885 might contribute to the regulation of neuroendocrine tumor metastasis through the Wnt/β-catenin pathway [270].

3.3.10. NFκB

The NFκB signaling pathway, a central regulator of inflammation, immunity, and cell survival, is intricately modulated by ncRNAs in the context of cancer. For example, miR-1892b has been identified as a key negative regulator of NFκB, thereby inhibiting inflammatory responses and tumor growth in breast cancer [271]. On the other hand, the oncogenic miRNA miR-21 promotes NFκB activation by targeting negative regulators in colon adenocarcinomas, as shown by evidence of diverse cell lines, xenograft mouse models, and gene expression profiling of patient tumor samples [272]. Additionally, the lncRNA MALAT1 has been associated with NFκB signaling in oral squamous cell carcinoma [273]. Furthermore, the lncRNA NEAT1 inhibits miRNA-216b and promotes colorectal cancer progression [274]. In the context of PNET, Huang et al. studied differential expression of miRNAs in 37 PNET samples and compared the differences between localized and metastatic tumors. Their findings indicated that MiR-196a is involved in NFκB signaling and exhibits a notable correlation with elevated tumor grade, advanced stage, and tissue invasion in PNET [275].

3.3.11. SSTR2

To date, there have been limited reports on the interplay between ncRNAs and SSTR2 signaling in cancers, with the existing literature mainly focusing on neuroendocrine neoplasms. For instance, the combination of SSTR2 with miR-7 and miR-148a caused inhibition of the growth of both lung and intestinal carcinoid cell lines [276]. In PNET, it has been reported that the combination of overexpression of miR-16-5p and the somatostatin analog octreotide resulted in the upregulation of SSTR2 expression in the INS-1 neuroendocrine cell line. This suggests that miRNA may play a role in advancing the development of combinatorial therapeutic approaches, particularly for patients who do not respond adequately to somatostatin analog treatment targeting SSTR2 alone [277].

3.3.12. Smad3

NcRNAs interact with TGF-β/Smad3 signaling to regulate important facets of cancer cell development, including EMT, invasion, migration, cancer cell stemness, and metastasis [278]. For example, miR-21 targets Smad7, an inhibitor of TGF-β/Smad3 signaling, which leads to enhanced cell proliferation and invasion in various cancers [279]. Conversely, the tumor-suppressive miRNA miR-145 directly targets Smad3, inhibiting TGF-β-induced EMT and metastasis in colorectal cancer [110]. In addition, miR-15a correlates with Smad3 expression NSCLC tissues. Not surprisingly, overexpression of miR-15a caused significantly downregulation of Smad3 expression and inhibited proliferation of A549 lung cancer cell line [280]. Furthermore, lncRNA H19 has been found associated with the regulation of TGF-β/Smad3 in hepatocellular carcinoma, impacting tumor growth [281]. Despite its important regulatory role in PNET pathology as described in Section 3.1, as of now, there are no reports on the miRNA-dependent regulation of Smad3 in research studies focusing on PNETs. A complete list of ncRNAs in PNET-related signaling pathways are listed in Table 3.

4. Future Directions for Epigenetic Research and Clinical Applications in PNET Patient Care

Up to this point, we have explored the epigenetic regulation of signaling pathways in the context of both PNET and other extensively studied cancers. It’s important to note that our intention is not to present exhaustive reviews that include detailed introductions to all the implicated signaling pathways. We acknowledge that providing such an in-depth review of the epigenetic regulation of even a single signaling pathway, like MEN1 signaling, would necessitate a comprehensive review in its own right [232,287,288]. Therefore, rather than aiming to cover all signaling pathways extensively, our focus is on discerning the disparities or gaps in our understanding of the epigenetic regulation of signaling pathways between PNET and other extensively studied cancers. The overarching goal is to utilize the understanding derived from existing knowledge in various cancers and translate this knowledge into advancements in research and clinical applications specific to PNET. This is particularly pertinent in the pursuit of more effective epigenetic drugs, akin to the successful cases observed in extensively studied cancers.

There has been notable advancement in comprehending the epigenetic control of cancers, although the available FDA-approved epigenetic therapies remain relatively scarce, with the majority targeting blood-borne cancers. Importantly, none of these approved therapies are directed at PNET. Although there have been endeavors to explore the use of FDA-approved drugs in clinical trials for PNET treatment, no successful reports have emerged thus far, as detailed in Table 4.

It is worth noting that currently approved FDA epigenetic drugs, such as DMTi and HDACi (Table 4), are lack of pathway specificity, thereby causing severe off-target effects and hindering their broad applications in the ever-growing field of precision medicine. In response to this limitation, ongoing efforts involve the utilization of CRISPR-guided systems to selectively activate or silence the promoter regions of either tumor suppressor or oncogenes, respectively [294]. However, this approach necessitates a more comprehensive understanding of the epigenetic regulation governing signaling pathways in PNETs.

As outlined in Section 3, it is evident that the epigenetic regulation of PNET remains an insufficiently explored domain in comparison to other extensively researched cancers, barring a few exceptions like the MEN1 and SSTR2 signaling pathways. This discrepancy is noticeable across various facets of epigenetic research, spanning from comprehensive genome profiling of PNET patient samples to laboratory-based animal studies and gene manipulation of cell lines (refer to Table 1, Table 2 and Table 3). For instance, in the field of DNA methylation research, there are lack of large-scale epigenetic profiling of PNET patient samples. As of 2022, there have been only nine studies reporting global-DNA methylation in human PNETs, which includes a total of 739 samples [295]. The lack of research samples could be, at least partially, due to the lack of overall PNET patient cases [1]. As discussed in section 1, PNETs make up around 2% of all pancreatic malignancies, characterized by their slow-growth tendencies and metastatic potential. These special characteristics resulted in a limited availability of patient tissue samples, particularly in the case of high-grade PNETs obtained from tumor autopsies [1]. Larger-scale studies and more advanced technologies are desperately needed to validate the existing results because identification of molecularly different NET subtypes will have a significant impact on clinical practice, given the high heterogeneity of PNET tumors. For example, in the research of some commonly occurring cancers, there have been reports of using advanced single-cell whole genome methylation profiling to obtain much more complicated information about of cancer epigenome [296].

The unique slowing growth characteristic of PNET also presents a practical challenge for research work in laboratories. For instance, STC-1, one of the most used mouse PNET cell lines, has a doubling time of 54 hours, which not only makes it hard to perform gene manipulation, but also creates a practical hurdle for creating syngenetic xenograft mouse models [297], which have been proven particularly suitable for studies of tumor immunity and immunotherapy response because of the presence of fully functional murine immune system [298]. Another challenge lies in the scarcity of PNET modeling systems. Currently, only a handful of human PNET cell lines, including the most widely used BON-1 and QGP-1, are accessible for research purposes. Consequently, there is a pressing need for further efforts to enhance and diversify the available tools for verifying both gain-of-function and loss-of-function in the continuously expanding list of genes associated with epigenetics research of PNET [299].

Despite the inherent challenges, some encouraging advancements have been achieved. Notably, recognizing the limited effectiveness of epigenetic cancer drugs has prompted investigations into combining these therapies with existing or emerging targeted treatments for a synergistic and combinatorial approach. This innovative strategy holds promise for improving clinical outcomes. For example, combination therapies that concurrently enhance SSTR expression using HDAC inhibitors and target SSTRs may demonstrate enhanced efficacy compared to individual therapies [10] (also refer to Section 3.2). Additionally, our laboratory has previously demonstrated that a combination of 5-azacytidine and chemotherapy can effectively reduce cell proliferation, activate silenced tumor suppressor genes, and diminish PNET tumors in vivo [300] .

Moreover, within the spectrum of diverse biomarkers, miRNAs display characteristics such as stability, relative abundance, and accessibility in blood samples. These qualities make miRNAs highly suitable as biomarkers for the diagnosis and monitoring of cancer [301,302]. Consequently, there is a call for more extensive global miRNA sequencing of PNET samples to establish a more dependable diagnostic platform for the optimal care of patients with PNETs.

5. Conclusion

In summary, the epigenetic regulation of PNETs exhibits similarities with broader cancer scenarios, providing valuable insights and therapeutic possibilities. Ongoing exploration of the specific mechanisms governing epigenetic changes in PNETs, alongside innovative strategies for drug development, holds considerable promise for enhancing the prognosis and quality of life for individuals grappling with this challenging malignancy. Notably, epigenetic changes play pivotal roles in immune surveillance and the development of drug resistance. As a result, the use of epigenetic drugs, including inhibitors targeting various enzymes like DNMTs, HMTs, HDMs, HATs, and HDACs, has the potential to effectively complement other treatments such as standard chemotherapy or immunotherapy in the care of PNET patients.