From microbiota toward gastro-enteropancreatic neuroendocrine neoplasms: Are we on the highway to hell?

Receptor tyrosine kinases belong to a family of receptors that mediate cellular responses to extracellular signals such as growth factors, hormones and cytokines. These receptors play an important role in cell proliferation, survival and differentiation. Several receptor tyrosine kinases are frequently upregulated in GEP NENs, such as the receptor families for vascular endothelial growth factor, epidermal growth factor and tyrosine-protein kinase c-Met [74‐77]. Receptor tyrosine kinases activity results in the activation of several transduction systems, including the canonical Ras signalling pathway and PI3K–Akt–mTOR [78].

HP stimulated vascular endothelial growth factor receptor expression in human microvascular endothelial cells (HMEC-1) [79] and epidermal growth factor receptor expression in gastric epithelial cells [80]. HP strains carrying the type IV secretion system induce gastrin promoter activity via epidermal growth factor receptor [81]. Epidermal growth factor receptor is also activated by HP-integrin-β1 interaction [82]. HP infection may activate c-Met expression through cytotoxin-associated gene A protein in gastric epithelial cells, resulting in ERK1, 2 activation [83].

The RAS-RAF-MEK-ERK pathway, activated by several growth factors, is involved in cell growth and cell differentiation. Dysregulation of this crucial pathway occurs due to overexpression and/or overactivation of the RAS and RAF genes [84]. Mutations of RAS [85‐87] are very rare events in GEP NENs, with reported mutation frequencies [87] of HRAS 1% (2/150), KRAS 8% (10/125), NRAS 0.7% (2/274) or BRAF 1% (4/369). Although activating mutations of BRAF are rare in GEP NENs [88], wildtype BRAF and its activating small G protein, RAP-1, are highly prevalent in the majority of GEP NENs. Overexpression of RAP-1 is able to activate MAPK-signalling and the expression of mitogenic transcription factors of GEP NEN cells [89]. Some authors have reported that RAF-1 signalling cascade activation is associated with the modulation of neuroendocrine phenotype in BON-1 cells, a well-known neuroendocrine tumor cell line [90, 91].

Gastrin, via the cholecystokinin 2 receptor, is the principal regulator of ECL cell proliferation via a MAPK-activated signal transduction cascade [92] and induction of the activator protein-1 complex transcription factor [93]. The latter regulate several genes involved in cell cycle progression (eg, cyclin genes) [94]. ECL cell proliferation is associated with fos/jun transcription activation by the MAPK pathway after gastrin-mediated RAS activation [95].

HP rapidly activates MAPKs upon contact with gastric epithelial cells [96] and, indirectly, promotes gastrin-induced MAPK transduction pathways in ECL cells through histamine release [95]. Several bacterial factors are involved in MAPK activation, including the vacuolating cytotoxin A [97, 98] and cytotoxin-associated gene A protein [99]. However, the type IV secretion system appears to be crucial for complete phosphorylation of ERK and MAPK [96, 100].

The PI3K–AKT–mTOR pathway plays a relevant role in the pathogenesis and progression of GEP NENs [101]. AKT is the major kinase, which regulates cell survival and proliferation by inhibiting proapoptotic mitochondrial proteins and cell-cycle modulators. Dysregulation of this pathway is due to activation of PI3K or loss-of-function mutation of PTEN, TSC2 and GSK3. Studies in small bowel NEN cell lines revealed a stronger activation of PI3K/AKT/mTOR pathway compared to that observed in normal ECL cells [102].

In gastric epithelial cells HP infection induced PTEN phosphorylation, which activated AKT [103] and inhibited apoptosis [104]. Interestingly, the HP urease seems to have a relevant role in the activation of the PI3K-AKT-mTOR pathway in gastric cells [105].

Notch signalling pathway plays an important role in maintaining a dynamic balance between cell proliferation, differentiation and apoptosis and is an essential signalling in the regulation of inflammatory and immune responses. Notch signalling can have an oncogenic or tumour suppressor role. Histo-pathological studies have shown that Notch-1 is absent or poorly expressed in well-differentiated GEP NENs, suggesting a possible role as a tumour suppressor gene in these tumors [106].

After HP invasion, a significant reduction in the mRNA expression level of Notch-1 and Notch-2, together with low levels of active forms of Notch-1 and Notch-2, have been observed in GES-1, a human gastric epithelial cell line [107].

Wnt/β-catenin pathway is crucial to embryo development and adult tissue homeostasis. Aberrant activation of this pathway can cause uncontrolled cell growth and malignant transformation (Fig. 2). In NENs, cytoplasmic and nuclear beta-catenin accumulation, suggestive of Wnt/β-catenin signalling activation, has been reported in 1/12 gastro-intestinal NENs and 1/6 bronchial carcinoids [108]. Sun and colleagues found cytoplasmic accumulation and/or nuclear translocation of β-catenin in about 30% of gastro-intestinal NENs (27/80) [109]. In 72 cases of gastrointestinal NENs, accumulation of β-catenin in the cytoplasm and/or nucleus has been observed in 79% of cases (57/72) and mutations in exon 3 of β-catenin in 37% of tumors [110]. APC gene is a negative regulator that controls β-catenin concentrations and modulates cell adhesion. In ileal NENs, the APC gene was deleted in 15% (4/27) and somatic mutations of this gene were detected in 23% (7/30) of examined tumour samples, including 57% missense and 14% nonsense/frameshift mutations [111].

HP activates Wnt/β-catenin signalling through several mechanisms in gastric cells. Cytotoxin-associated gene A protein induces nuclear β-catenin accumulation in vivo and in vitro [112, 113] and activates the β-catenin through an independent phosphorylation manner in human gastric cancer epithelial cell lines or in rodent gastric cells [114]. The vacuolating cytotoxin A induces Wnt/β-catenin signalling through the activation of PI3K/AKT pathway [115]. HP can also activate Wnt/β-catenin pathway by recruiting tumor-associated macrophages [116]. Wnt/β-catenin activation in HP infection has been linked to angiogenesis in the gastric mucosa, which is an important process for tumorigenesis [117].

The TGF-β exists in at least three isoforms: TGF-β1, TGF-β2, and TGF-β3 [118]. TGF-β signalling is mediated by TGF-β receptors 1 and 2 and intracellular SMAD proteins. These factors are involved in cell cycle regulation, apoptosis, tumor angiogenesis and invasion [119‐121]. A high expression level of TGF-β receptor 1 (intensity scores 2 and 3) has been detected in almost 100% of GEP NENs [122]. The tumor suppressor SMAD4 has been demonstrated to be often mutated or deleted in small intestinal NENs in approximately 45% of cases (22/48) [123, 124].

TGF-β1 is a potent stimulator of ECL cell proliferation through downregulation of SMAD4 and activation of the ERK1/2 pathway [125]. In vivo studies have shown that HP infection induced upregulation of TGF-β1 in gastric mucosa. This effect is positively correlated with the vacuolating cytotoxin A genotype and the grade of chronic inflammation [126].

The TP53 gene encodes p53, an important tumour suppressor modulating a network of genes implicated in DNA repair, cell growth arrest or cell senescence, apoptosis and autophagy [127]. The main effectors of TP53 expression are WIP1, MDM2, MDMX, ATM and ATR genes [128, 129]. Mutations in the TP53 gene have been consistently detected in poorly differentiated GEP NENs, with a frequency ranging from 20% to 73% of cases [130] and correlate with poor survival [131]. Hu and co-workers [132] observed a high rate of copy number gains of MDM2 in 22%, MDM4 in 40% and WIP1 in 51% of pancreatic NENs. High ATM expression in pancreatic NENs was associated with higher tumour differentiation, lower tumour size, lower recurrence rate and better prognosis [133], while loss of ATM expression was common in metastasized disease and resulted to be associated with a worse prognosis [134]. Interestingly, in the African rodent mastomys Tp53 seems to have a relevant role during the development of hypergastrinemia-induced ECLoma [135].

HP is able to inhibit the tumor suppressor TP53 through AKT activation and subsequent degradation of p53 in gastric epithelial cells [136]. Inhibition of p53 may provide advantages to HP and allow it to alter cellular homeostasis without triggering cell cycle arrest or apoptosis [137].

The family of CDKs belongs to a superfamily of 20 members, which catalyse the phosphorylation of key proteins and transcription factors implicated in cell cycle transition [138‐140]. Cyclin C/CDK3, Cyclin D/CDK4 and Cyclin D/CDK6 are involved in G₀–G₁ transition and the early G₁ phase by phosphorylating the tumour suppressor retinoblastoma protein and thus activating E2F [141]. These pathways are commonly altered in tumors, including GEP NENs [142, 143]. In 92 tumour samples of human pancreatic NENs, overexpression of CDK4 and retinoblastoma protein was detected in 58% and 68%, respectively. Gene amplifications of CDK4 or CDK6 were found in 19% (5/26) of investigated pancreatic NENs [144]. p27 is CDK inhibitor encoded by the CDKN1B gene and regulates the transition from cell cycle phase G₀/G₁ to S and is implicated also in cellular motility and apoptosis. Frameshift mutations or deletions of CDKN1B were reported in about 8–23% of small intestinal NENs [145, 146]. Loss of p27 protein expression, which occurred in 21% of 327 GEP NENs, was a predictor of poor prognosis [147]. The inactivation of RB1 gene, which occurs mainly by somatic mutations, has been reported in 71% of poorly differentiated pancreatic neuroendocrine carcinomas [148]. Both message and protein levels of cyclin D1 increased in vitro during ECL cell tumorigenesis [149, 150].

HP and related inflammatory response are associated in vivo with alterations in expression of cyclin D1 and CDKN1B and abnormalities in epithelial cell proliferation, cell cycle progression and apoptosis. HP infection can stimulate proliferation of gastric mucosal epithelial cells [151], through activating the MAPK pathway and promoting the expression of cyclin D1 [152]. In addition, HP decreases p27 expression in gastric cells through epigenetic mechanisms [153, 154]. Interestingly, low gastric p27 may promote carcinogenesis associated with HP infection by inhibiting apoptotic pathways [155].

The interleukin-6/STAT3/CDX2 pathway represents a relevant factor for the tumor progression of gastrointestinal NENs [156‐158].

It has been reported that the proinflammatory cytokine interleukin-6 is upregulated during HP infection in the gastric mucosa, with a potential involvement in gastrointestinal tumor development [159]. Interleukin-6 binds to the α-subunit of its specific receptor and activates two main signalling pathways: SHP-2/ERK and JAK/STAT [99], able to promote mucosal inflammation and carcinogenesis [160‐163]. In addition, HP infection induces CDX2 expression in patients with chronic gastritis and intestinal metaplasia [163].

COX-2 is able to modulate cell apoptosis and adhesion and promote tumor cell metastasis [164]. COX-2 overexpression was observed in 54% (126 of 234) of GEP NENs and was found to be positively correlated with Ki-67 labelling index and associated with poor prognosis [165].

COX-2 is induced in HP–positive gastritis and present at high levels in gastric antrum, where bacterial density is elevated [166]. This suggested that expression of COX-2 was a direct response to HP infection [167]. In patients with HP-positive gastric mucosal lesions, positive detection rate of COX-2 resulted significantly higher than that in HP-negative gastric mucosal lesions [168].

IBD is a group of inflammatory conditions of the colon and small intestine, that includes Crohn’s disease (CD) and ulcerative colitis.

Data from population study and large pathological and disease registry suggest an association between IBD and intestinal NENs. Among a cohort of 20,917 patients affected by CD, 9 NENs were observed, resulting, in a 7-fold increased neoplastic risk, as compared to the general population [169]. In a prospective observational 7-year follow-up cohort study in 590 patients with mono-institutional IBD diagnosis, neuroendocrine carcinoma and rectal carcinoid occurrence was increased (RR = 13.1, 95%CI: 1.82–29.7 and RR = 8.94, 95%CI: 1.18–59.7 respectively) [170]. Similarly, in a large retrospective study from US-based population database of electronic medical records of 62,817,650 individuals from 26 major healthcare institutions, 4530 of them were reported to have a large colonic carcinoid diagnosis and, in several cases, a personal history of CD or ulcerative colitis. For these subjects an increased risk to develop large bowel carcinoid was observed: OR 6.93 (95% CI 5.55–8.64, p < 0.0001) and OR 6.45 (95% CI 5.24–7.95, p < 0.0001), respectively [171]. Sciola et al. reported a prevalence for IBD of 4.8% in a series of 83 GEP NENs. This value was higher than that reported in general population [172]. In contrast, a recent Dutch nationwide study reported in the entire cohort of IBD patients from national pathological database 51 patients with concomitant IBD and colonic NENs with an estimated prevalence rate ratios between 2.8 and 4.1. These values were lower than ones from colonic resection specimens for diverticulitis and ischemia adjusted for resection type, sex and age, suggesting an incidental finding because of frequent colonic resection [173].

Detailed clinical and pathological features data for NENs in IBD is however lacking in literature. Data arose from case reports and surgical or pathological series revision with different collection data method and analysis [172, 174]. Interestingly, only a minority of patients with both diseases developed an aggressive NEN. In fact, only 8.3% (3/36) of NENs with IBD, reported by Derikx et al., showed distant metastasis (stage IV). In a recent retrospective study, Wong reported detailed clinical and pathological data in 17 patients with IBD and neuroendocrine proliferation. Eight patients (47.1%) were classified as neuroendocrine cell micronests with subcentimetric lesions and no oncological strength, while in the remain 9 (52.9%) a 1–11 mm, low grade and stage I NEN was reported [175].

IBD is associated with alterations in intestinal microbiota. The pathogenesis of the disease involves complex interactions between immune system, the microbiome and environmental factors in genetically susceptible individuals [176]. Despite an impressive number of 163 genetic loci of IBD susceptibility, most of which associated with both CD and ulcerative colitis, other factors as environmental exposures seem to contribute to disease pathogenesis. Most evidences point to the interaction between the host mucosal immune system and microbes, both at the epithelial cell surface and within the gut lumen, as one of the most important factors [177]. In patients with IBD, a compositional change in microbiota and an expansion of potential pathogens have been reported. In particular, a depletion in specific commensal bacteria, as Lachnospiraceae (class of Clostridia and philum of Firmicutes) and Bacteroidetes phylum, and an enrichment in Proteobacteria was reported [178] in patients with IBD compared to healthy subjects. There are no reliable data if microbial composition changes in human have a causative role in inducing intestinal inflammation or if could be a side effect (following acute infection or host inflammatory responses) and no specific pathogenic microorganism was recognized as singular cause of chronic IBD [179, 180]. However, alterations in gut microbiota were found in CD and ulcerative colitis, with a relevant impact of aging and disease stage. In fact, a decreased amount of Roseburia hominis and Faecalibacterium prausnitzii (Fprau) has been observed using RT-PCR in ulcerative colitis fecal samples [181]. In CD a global decrease in the biodiversity of the fecal microbiota with markedly reduced diversity of Firmicutes and in particular of the Clostridium leptum phylogenetic group was reported using a metagenomic approach [182]. An elegant paper from Sokol reported lower proportion of Fprau in resected ileal mucosa from patients with CD associated with endoscopic recurrence at 6 months, suggesting a significant role for microbiota variation in recurrent disease [183]. In this study an anti-inflammatory property of Fprau has been demonstrated both in vitro and in vivo.

Although only few studies are available in literature on microbiota and NENs, there are common patterns of microbiome composition with IBD. In 2008 Dorffel showed a significant depletion of Fprau in 12/23 patients with NENs, as previously reported in CD [181, 183]. Another study evaluated microbial fecal composition using microscopic examination and fluorescence and in situ hybridization in 66 patients with NENs (25 from foregut, 30 from midgut and 11 from hindgut origin), 25 healthy subjects and 50 patients with CD. Depletion of Fprau was observed in 67% of patients with midgut NENs, 84% of untreated CD and 56% of treated CD, while only in 3% of patients with chronic idiopatic diarrhea and 0% of healthy controls. In the same study fecal Enterobacteriaceae were significantly increased in NENs and CD patients. The effect of NEN therapy on microbiota was also analyzed. Somatostatin analogues had no influence on the concentration of habitual or occasional bacterial groups, while interferon alpha-2b and chemotherapy induced a massively increased in Fprau. Similar data were reported in successfully treated CD patients despite different drugs were used [184].

Therefore, a depletion of Fprau has been reported both in patients with IBD and NENs. A possible protective effect of Fprau on IBD inflammation has been proposed. Fprau is known to have a role in producing a large amount of butyrate, able to modulate the immune system and to protect the gut barrier integrity. The gut microbiota-derived butyrate, not also supplies energy source for intestinal epithelial cells, but also inhibits inflammation through epigenetic mechanisms [185]. In addition, recent data suggested a protective effect of Fprau for several tumors, such as colon carcinoma [186], breast cancer [187] and melanoma [188].