Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

Genetic and Epigenetic Alterations of CDH1 Regulatory Regions in Hereditary and Sporadic Gastric Cancer.

E-cadherin is a key player in gastric cancer (GC) and germline alterations of CDH1, its encoding gene, are responsible for Hereditary Diffuse Gastric Cancer (HDGC) syndrome. This study aimed at elucidating the role of genetic variants and DNA methylation of CDH1 promoter and enhancers in the regulation of gene expression. For this purpose, we analyzed genetic variants of the CDH1 gene through Next-Generation Sequencing (NGS) in a series of GC cell lines (NCI-N87, KATO-III, SNU-1, SNU-5, GK2, AKG, KKP) and the corresponding CDH1 expression levels. By bisulfite genomic sequencing, we analyzed the methylation status of CDH1 regulatory regions in 8 GC cell lines, in a series of 13 sporadic GC tissues and in a group of 20 HDGC CDH1-negative patients and 6 healthy controls. The NGS analysis on CDH1 coding and regulatory regions detected genetic alterations in 3 out of 5 GC cell lines lacking functional E-cadherin. CDH1 regulatory regions showed different methylation patterns in patients and controls, GC cell lines and GC tissues, expressing different E-cadherin levels. Our results showed that alterations in terms of genetic variants and DNA methylation patterns of both promoter and enhancers are associated with CDH1 expression levels and have a role in its regulation.

Definition and management of colorectal polyposis not associated with APC/MUTYH germline pathogenic variants: AIFEG consensus statement.

An expert consensus panel convened by the Italian Association for Inherited and Familial Gastrointestinal Tumors (Associazione Italiana per lo Studio della Familiarità ed Ereditarietà dei Tumori Gastrointestinali, AIFEG) reviewed the literature and agreed on a number of position statements regarding the definition and management of polyposis coli without an identified pathogenic mutation on the APC or MUTYH genes, defined in the document as NAMP (non-APC/MUTYH polyposis).

E-cadherin Downregulation and microRNAs in Sporadic Intestinal-Type Gastric Cancer.

CDH1 gene, encoding E-cadherin, is a tumor suppressor gene frequently altered in gastric cancers (GCs) of both diffuse (DGC) and intestinal (IGC) histotypes, albeit through different mechanisms. The study aimed to characterize CDH1 expression in sporadic IGC and to investigate whether microRNAs (miRs) are involved in its transcriptional control. We evaluated CDH1 expression by quantitative real-time PCR (RT-qPCR) in 33 IGC patients and found a significant downregulation in tumor tissues compared to normal counterparts (p-value = 0.025). Moreover, 14 miRs, predicted to be involved in CDH1 regulation in both a direct and indirect manner, were selected and analyzed by RT-qPCR in an independent case series of 17 IGCs and matched normal tissues. miR-101, miR-26b, and miR-200c emerged as significantly downregulated and were confirmed in the case series of 33 patients (p-value < 0.001). Finally, we evaluated EZH2 expression, a target of both miR-101 and miR-26b, which showed significant upregulation in IGCs (p-value = 0.005). A significant inverse correlation was observed between EZH2 overexpression and CDH1, miR-101, and miR-26b levels (p-value < 0.001). Our results reinforce the link between CDH1 and IGC, highlighting the role of miRs in its transcriptional control and improving our understanding of GC subtypes and biomarkers.

Multigene Panel Testing Increases the Number of Loci Associated with Gastric Cancer Predisposition.

The main gene involved in gastric cancer (GC) predisposition is CDH1, the pathogenic variants of which are associated with diffuse-type gastric cancer (DGC) and lobular breast cancer (LBC). CDH1 only explains a fraction (10-50%) of patients suspected of DGC/LBC genetic predisposition. To identify novel susceptibility genes, thus improving the management of families at risk, we performed a multigene panel testing on selected patients. We searched for germline pathogenic variants in 94 cancer-related genes in 96 GC or LBC Italian patients with early-onset and/or family history of GC. We found CDH1 pathogenic variants in 10.4% of patients. In 11.5% of cases, we identified loss-of-function variants in BRCA1, BRCA2, PALB2, and ATM breast/ovarian cancer susceptibility genes, as well as in MSH2, PMS2, BMPR1A, PRF1, and BLM genes. In 78.1% of patients, we did not find any variants with clear-cut clinical significance; however, 37.3% of these cases harbored rare missense variants predicted to be damaging by bioinformatics tools. Multigene panel testing decreased the number of patients that would have otherwise remained genetically unexplained. Besides CDH1, our results demonstrated that GC pathogenic variants are distributed across a number of susceptibility genes and reinforced the emerging link between gastric and breast cancer predisposition.

Gut Microbiota Analysis in Postoperative Lynch Syndrome Patients.

Lynch syndrome (LS) is a dominantly inherited condition with incomplete penetrance, characterized by high predisposition to colorectal cancer (CRC), endometrial and ovarian cancers, as well as to other tumors. LS is associated with constitutive DNA mismatch repair (MMR) gene defects, and carriers of the same pathogenic variants can show great phenotypic heterogeneity in terms of cancer spectrum. In the last years, human gut microbiota got a foothold among risk factors responsible for the onset and evolution of sporadic CRC, but its possible involvement in the modulation of LS patients' phenotype still needs to be investigated. In this pilot study, we performed 16S rRNA gene sequencing of bacterial DNA extracted from fecal samples of 10 postoperative LS female patients who had developed colonic lesions (L-CRC) or gynecological cancers (L-GC). Our preliminary data show no differences between microbial communities of L-CRC and L-GC patients, but they plant the seed of the possible existence of a fecal microbiota pattern associated with LS genetic background, with Faecalibacterium prausnitzii, Parabacteroides distasonis, Ruminococcus bromii, Bacteroides plebeius, Bacteroides fragilis and Bacteroides uniformis species being the most significantly over-represented in LS patients (comprising both L-CRC and L-GC groups) compared to healthy subjects.

Shifts of Faecal Microbiota During Sporadic Colorectal Carcinogenesis.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

A novel APC promoter 1B deletion shows a founder effect in Italian patients with classical familial adenomatous polyposis phenotype.

Familial adenomatous polyposis is a Mendelian syndrome in which germline loss-of-function mutations of APC are associated with multiple adenomatous polyps of the large bowel, a multiplicity of extracolonic features, and a high lifetime risk of colorectal cancer. Different APC germline mutations have been identified, including sequence changes, genomic rearrangements, and expression defects. Recently, very rare families have been associated with constitutive large deletions encompassing the APC-5' regulatory region, while leaving the remaining gene sequence intact; the regulatory region contains a proximal and a distal promoter, called 1A and 1B, respectively. We identified a novel deletion encompassing promoter 1B in a large Italian family that manifested polyposis in three of the six branches descending from a founding couple married in 1797. By combining different molecular approaches on both DNA and RNA, we precisely mapped this deletion (6858 bp in length) that proved to be associated with APC allele silencing. The finding of the same deletion in two additional polyposis families pointed to a founder mutation in Italy. Deletion carriers from the three families all showed a "classical" polyposis phenotype. To explore the molecular mechanisms underlying promoter deletions, we performed an in silico analysis of the breakpoints of 1A and 1B rearrangements so far reported in the literature; moreover, to decipher genotype-phenotype correlations, we critically reviewed current knowledge on deletions versus point mutations in the APC-5' regulatory region.

Hereditary diffuse gastric cancer in two families: A case report.

Hereditary diffuse gastric cancer (HDGC) is associated with E-cadherin 1 (CDH1) germline mutations. In the present study, two unusual HDGC cases are described. Case 1 was a female with no family history of gastric cancer who developed Hodgkin's lymphoma at 19 years of age, and DGC at 32 years of age. Due to her young age (32 years), the patient was examined for CDH1 abnormalities and a deleterious mutation was identified. Her father and younger sister were identified to be carriers of the mutation. Case 2 was a 36-year-old female diagnosed with lobular breast cancer (LBC); her mother had LBC, and her grandmother had LBC and DGC. The molecular test was wild-type for breast cancer susceptibility genes 1 and 2; however, a large deletion in CDH1 was identified. At prophylactic gastrectomy, early DGC was identified. Early onset of DGC and LBC justifies testing for CDH1. A better knowledge of tumor natural history in carrier subjects is important to aid genetic counseling, in order to assess the surveillance time required prior to carrying out prophylactic surgery.

Digital PCR identifies changes in CDH1 (E-cadherin) transcription pattern in intestinal-type gastric cancer.

E-cadherin is a cell-cell adhesion protein encoded by CDH1 tumor-suppressor gene. CDH1 inactivating mutations, leading to loss of protein expression, are common in gastric cancer of the diffuse histotype, while alternative mechanisms modulating E-cadherin expression characterize the more common intestinal histotype. These mechanisms are still poorly understood. CDH1 intron 2 has recently emerged as a cis-modulator of E-cadherin expression, encoding non-canonical transcripts. One in particular, CDH1a, proved to be expressed in gastric cancer cell lines, while being absent in the normal stomach. For the first time, we evaluated by digital PCR the expression of CDH1 and CDH1a transcripts in cancer and normal tissue samples from 32 patients with intestinal-type gastric cancer. We found a significant decrease in CDH1 expression in tumors compared to normal counterparts (P = 0.001), which was especially evident in 76% of cases. CDH1a was detected at extremely low levels in 47% of tumors, but not in normal mucosa. A trend was observed of having less CDH1 in tumors expressing CDH1atranscript. The majority of tumors with both a decrease in CDH1 and presence of CDH1a also showed a decrease in miR-101 expression levels. On the whole, the decrease of CDH1 transcript, corresponding to the canonical protein, and the presence of CDH1a, corresponding to an alternative isoform, are likely to perturb E-cadherin-mediated signaling and cell-cell adhesion, thus contributing to intestinal-type gastric carcinogenesis.

Colorectal Adenomatous Polyposis: Heterogeneity of Susceptibility Gene Mutations and Phenotypes in a Cohort of Italian Patients.

AIMS: Colorectal adenomatous polyposis entailing cancer predisposition is caused by constitutional mutations in different genes. APC is associated with the familial adenomatous polyposis (FAP/AFAP) and MUTYH with the MUTYH-associated polyposis (MAP), while POLE and POLD1 mutations cause the polymerase proofreading-associated polyposis (PPAP). METHODS: We screened for mutations in patients with multiple adenomas/FAP: 121 patients were analyzed for APC and MUTYH mutations, and 36 patients were also evaluated for POLE and POLD1 gene mutations. RESULTS: We found 20 FAP/AFAP, 15 MAP, and no PPAP subjects: pathogenic mutations proved to be heterogeneous, and included 5 APC and 1 MUTYH novel mutations. The mutation detection rate was significantly different between patients with 5-100 polyps and those with >100 polyps (p = 8.154 × 10-7), with APC mutations being associated with an aggressive phenotype (p = 1.279 × 10-9). Mean age at diagnosis was lower in FAP/AFAP compared to MAP (p = 3.055 × 10-4). Mutation-negative probands showed a mean age at diagnosis that was significantly higher than FAP/AFAP (p = 3.46986 × 10-7) and included 45.3% of patients with <30 polyps and 70.9% of patients with no family history. CONCLUSIONS: This study enlarges the APC and MUTYH mutational spectra, and also evaluated variants of uncertain significance, including the MUTYH p.Gln338His mutation. Moreover this study underscores the phenotypic heterogeneity and genotype-phenotype correlations in a cohort of Italian patients.

Complementary molecular approaches reveal heterogeneous CDH1 germline defects in Italian patients with hereditary diffuse gastric cancer (HDGC) syndrome.

Germline inactivation of the E-cadherin gene (CDH1) is associated with hereditary diffuse gastric cancer (HDGC), a rare autosomal dominant syndrome predisposing to both diffuse gastric cancer (DGC) and lobular breast cancer (LBC). We searched for CDH1 germline defects in 32 HDGC Italian probands selected according to international consensus criteria and in 5 selected relatives. We used a series of molecular methods, including: DNA sequencing, multiplex ligation-dependent probe amplification, single-nucleotide primer extension, bisulfite sequencing, reverse-transcription PCR, and bioinformatics tools. We identified pathogenic mutations in 6 out of 32 probands (19%): one truncating and two missense mutations, one large deletion, one allelic expression imbalance and one splicing defect. Three out of six CDH1 constitutive alterations were novel. Our data support the need for a multimethod approach for CDH1 genetic testing, demonstrating that both DNA and RNA analyses are required to increase the detection rate of pathogenic mutations, thus reducing the number of patients without a clear molecular diagnosis. On the whole, our results indicate that not only DGC patients, but also subjects with personal or family history of LBC might benefit from CDH1 genetic testing. Moreover, our findings support the notion that prophylactic gastrectomy should be offered to asymptomatic CDH1 mutation carriers; indeed, while endoscopic analysis with histological examination of random gastric biopsies can miss cancer foci, gastrectomy performed in these subjects always revealed foci of cancer cells.

Consequences of the 118A>G polymorphism in the OPRM1 gene: translation from bench to bedside?

The 118A>G single nucleotide polymorphism (SNP) in the µ-opioid receptor (OPRM1) gene has been the most described variant in pharmacogenetic studies regarding opioid drugs. Despite evidence for an altered biological function encoded by this variant, this knowledge is not yet utilized clinically. The aim of the present review was to collect and discuss the available information on the 118A>G SNP in the OPRM1 gene, at the molecular level and in its clinical manifestations. In vitro biochemical and molecular assays have shown that the variant receptor has higher binding affinity for ß-endorphins, that it has altered signal transduction cascade, and that it has a lower expression compared with wild-type OPRM1. Studies using animal models for 118A>G have revealed a double effect of the variant receptor, with an apparent gain of function with respect to the response to endogenous opioids but a loss of function with exogenous administered opioid drugs. Although patients with this variant have shown a lower pain threshold and a higher drug consumption in order to achieve the analgesic effect, clinical experiences have demonstrated that patients carrying the variant allele are not affected by the increased opioid consumption in terms of side effects.

Understanding the role of the Q338H MUTYH variant in oxidative damage repair.

The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh(-)(/)(-) mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.

Influence of COMT Val158Met polymorphism on Alzheimer's disease and mild cognitive impairment in Italian patients.

COMT (Catechol-O methyltransferase) gene is one of the key players in synaptic plasticity and in learning and memory mechanisms. A single nucleotide polymorphism (rs4680; G to A) in the COMT coding region causes Val158Met aminoacid substitution in the corresponding protein, with Val allele exhibiting a 3- to 4-fold increase in enzyme activity compared to Met. With the purpose of examining the influence of COMT as a genetic risk factor for cognitive impairment, we analyzed a sample of 248 healthy subjects, 276 patients affected by Alzheimer's disease (AD), and 70 subjects with mild cognitive impairment (MCI), the latter condition possibly representing a prodrome for dementia. All subjects were analyzed for COMT rs4680 polymorphism and APOE genotype. Our study strengthens data showing that APOE ε4 allele is an independent risk factor for AD and also a risk factor for MCI. Neither COMT alleles nor genotypes proved to be independently associated with the risk of AD or MCI in our sample. However, we found an association between COMT GG genotype (Val/Val) and APOE ε4 carrier status and the risk of AD and MCI. In particular, when GG genotype is included into the multinomial analysis, the risk of AD and MCI due to APOE ε4 allele is increased of about 2-3 fold; moreover, the risk conferred by the combination of G and ε4 alleles is more pronounced in male patients. To our knowledge, this synergistic effect is here shown for the first time on a population sample representative of Caucasian patients.

Morphine metabolism, transport and brain disposition.

The chemical structures of morphine and its metabolites are closely related to the clinical effects of drugs (analgesia and side-effects) and to their capability to cross the Blood Brain Barrier (BBB). Morphine-6-glucuronide (M6G) and Morphine-3-glucuronide (M3G) are both highly hydrophilic, but only M6G can penetrate the BBB; accordingly, M6G is considered a more attractive analgesic than the parent drug and the M3G. Several hypotheses have been made to explain these differences. In this review we will discuss recent advances in the field, considering brain disposition of M6G, UDP-glucoronosyltransferases (UGT) involved in morphine metabolism, UGT interindividual variability and transport proteins.

How and why to screen for CYP2D6 interindividual variability in patients under pharmacological treatments.

Cytochromes P450 are members of a superfamily of hemoproteins that catalyze a variety of oxidative reactions in the metabolism of endogenous and exogenous hydrophobic substrates. Fifty-eight cytochrome P450 (CYP) isoenzymes belonging to 18 families have been identified in human cells; the corresponding genes are highly polymorphic, and genetic variability underlies interindividual differences in drug response. The polymorphisms of CYP2D6 significantly affect the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The number of functional CYP2D6 alleles per genome determines the existence of four different phenotypes, i.e. poor, intermediate, extensive, and ultrarapid metabolizers. CYP2D6 genetic variants include copy number variations, single nucleotide substitutions, frameshift and insertion/deletion mutations. This review reports some of the different methodological approaches used to screen for CYP2D6 variants and focuses on methods that have improved variation detection, from conventional techniques to more recent microarray technology and high throughput DNA sequencing. In addition, this review reports some results on clinical relevance of CYP2D6 polymorphisms and provides examples of variability in drug response associated with interindividual phenotypic differences.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay.

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.