Identification and Somatic Characterization of the Germline PTEN Promoter Variant rs34149102 in a Family with Gastrointestinal and Breast Tumors

Genetic variants located in non-coding regions can affect processes that regulate protein expression, functionally contributing to human disease. Germline heterozygous mutations in the non-coding region of the PTEN gene have been previously identified in patients with PTEN hamartoma tumor syndrome (PHTS) diagnosed with breast, thyroid, and/or endometrial cancer. In this study, we report a PTEN promoter variant (rs34149102 A allele) that was identified by direct sequencing in an Italian family with a history of gastroesophageal junction (GEJ) adenocarcinoma and breast cancer. In order to investigate the putative functional role of the rs34149102 A allele variant, we evaluated the status of PTEN alterations at the somatic level. We found that PTEN protein expression was absent in the GEJ adenocarcinoma tissue of the index case. Moreover, we detected the occurrence of copy number loss involving the PTEN rs34149102 major C allele in tumor tissue, revealing that the second allele was somatically inactivated. This variant is located within an active regulatory region of the PTEN core promoter, and in silico analysis suggests that it may affect the binding of the nuclear transcription factor MAZ and hence PTEN expression. Overall, these results reveal the functional role of the PTEN promoter rs34149102 A allele variant in the modulation of PTEN protein expression and highlight its contribution to hereditary cancer risk

A STUDY ON CORRELATION BETWEEN ELECTROMYOGRAPHICAL AND MORPHOLOGICAL FINDINGS OF BACK MUSCLES IN SCOLIOSIS, ESPECIALLY CHANGES IN THE INTRAMUSCULAR NERVE ENDINGS.

3. In the cases of neurofibromatosis with scoliosis, intramuscular nerve endings showed almost normal morphological appearances but, comparative histograms of the diameters of the end-plates in each 5μ. group showed 2 peak formations.. One of these cases recorded fibrillation voltage at the concave side showed abnormal staining of terminal filamints of the end-plates in deep back muscles at the same side. The sensory nerve endings showed perfectly preserved histological appearances. 4. In cases of rachitogenic scoliosis, motor end-plates of the back muscles were observed using the histochemical method of cholinesterase staining and vital staining with methylene blue. Histograms of the diameters of the end-plates showed a noted reduction in size and in number of units, but no apparent degenerative changes in sensory and motor endings or in terminal axons were seen. 5. In a case of discogenic scoliosis, silver impregnation was applied on the back muscles at both sides of the curvature. In the convex side, collateral branching and swelling of the nerve fibers was observed and also the motor end-plates showed an abnormality of staining in terminal arborization, but, fibrillation voltage was not recorded. 6. In congenital scoliosis, electromyographic findings failed to trace fibrillation voltage, but, terminal axons in deep muscles, showed collateral branchig and an increased terminal innervation ratio in a highly affected case. In slightly curved cases, atrophic changes, and intramuscular fibers and endings appeared almost normal. 7. In the so-called idiopathic scoliosis , about 50% had not traced fibrillation voltage in the paravertebral back muscles. Degenerative findings.of the intramuscular nerve fibers and endings were not observed, but comparative histograms of the diameters of the end -plates showed 2 peak formations in the convex side of the back muscles. Innervation ratio was not changed and sensory nerve endings showed normal appearances.8. In idiopathic scoliosis, characterised by fibrillation voltage which was traced in the paravertebral back muscles, intramuscular motor nerve endings were degenerated in deep muscles of the convex side, and disseminated muscle atrophy in the concave side. In deep muscles of the convex side, terminal axons showed collateral branching and motor endplates, club-like swelling, and 2 peak formations of the histograms of the diameters. However, sensory nerve endings were normal in appearance. These findings lead to a conclusion that changes in the motor endings observed in paralytic scoliosis differed from histological changes in the cases of idiopathic scoliosis, in which fibrillation voltage had been traced. In paralytic scoliosis, histological changes of the back muscles showed various kinds of degenerative findings, but, in cases of idiopathic scoliosis with fibrillation voltage they showed collateral branching and degenerative changes of the end-plates, respectively, in deep back muscles especially in the convex side. In other kinds of scoliosis, neuromuscular changes were largely influenced by their own basic disorders such as, degeneration of discs, abnormality of vertebral bodies, nutritional deficiencies and metablic disorders. In the cases of idiopathic scoliosis without fibrillation voltage intramuscular nerve endings were preserved in good condition, but almost all of the cases of non-paralytic scoliosis showed an abnormality of the histograms of the endplates, or atrophic changes in deep muscles. Prophylactic treatment must be employed on the back muscles for the prevention of further deformity and progression of scoliosis.An electromyographical and histological study on 20 cases of various kinds of scoliosis was carried out. In particular, a biopsy of the back muscles at the apex of the primary curve was performed using the methods of gold chloride staining, silver impregnation modified by Seto, vital staining with methylene blue, and the histochemical demonstration of cholinesterase on the subneural apparatuses of the endplates. Specimens were taken from superficial (M. longissimus dorsi) and deep (M. multifidus) back muscles of scoliosis patients and in all cases, an electromyographic fibrillation voltage was picked up under deep general anesthesia with ether. This was done from the paravertebral back muscles and a comparison of histologic and electromyographic findings was performed. The results obtained are summarized as follows : 1. Motor end-plates of normal back muscles, dissected at autopsy from adults were found to be concentrated in band-like narrow zones and situated at the mid-point of the muscle fibers. Through comparative histograms of the diameters of subneural apparatuses and those of the end-plates of gold chloride staining showed a close correlation. 2. Intramuscular endings in the cases of paralytic scoliosis showed remarkable collateral axonic sprouting and various kinds of pathological changes of the end-plates, multiple innervation, large and and small end-plates, thickened terminal filaments, failure to stain, irregular swelling, and abnormal terminal expansion. The motor endings of the musclespindle were also remarkably degenerated but sensory nerve fibers and endings remained undisturbed. In neuromuscular endings, pathological changes of the back muscles at the apex of the primary curve, displayed a distinguishing feature in a lack of uniformity in the degree of alignment and curvature, but, compared with superficial muscles, the deep muscles were significantly involved. In all cases a giant spike and fibrillation voltage was recorded from wide-spread various parts of the back muscles

Characterization of a rare variant (c.2635-2A>G) of the MSH2 gene in a family with Lynch syndrome

Abstract Introduction: Lynch syndrome is caused by germline mutations in one of the mismatch repair genes (MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. Case Description: Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. Conclusions: We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2)

FOXO3a from the Nucleus to the Mitochondria: A Round Trip in Cellular Stress Response

Cellular stress response is a universal mechanism that ensures the survival or negative selection of cells in challenging conditions. The transcription factor Forkhead box protein O3 (FOXO3a) is a core regulator of cellular homeostasis, stress response, and longevity since it can modulate a variety of stress responses upon nutrient shortage, oxidative stress, hypoxia, heat shock, and DNA damage. FOXO3a activity is regulated by post-translational modifications that drive its shuttling between different cellular compartments, thereby determining its inactivation (cytoplasm) or activation (nucleus and mitochondria). Depending on the stress stimulus and subcellular context, activated FOXO3a can induce specific sets of nuclear genes, including cell cycle inhibitors, pro-apoptotic genes, reactive oxygen species (ROS) scavengers, autophagy effectors, gluconeogenic enzymes, and others. On the other hand, upon glucose restriction, 5'-AMP-activated protein kinase (AMPK) and mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -dependent FOXO3a mitochondrial translocation allows the transcription of oxidative phosphorylation (OXPHOS) genes, restoring cellular ATP levels, while in cancer cells, mitochondrial FOXO3a mediates survival upon genotoxic stress induced by chemotherapy. Interestingly, these target genes and their related pathways are diverse and sometimes antagonistic, suggesting that FOXO3a is an adaptable player in the dynamic homeostasis of normal and stressed cells. In this review, we describe the multiple roles of FOXO3a in cellular stress response, with a focus on both its nuclear and mitochondrial functions

APC Splicing Mutations Leading to In-Frame Exon 12 or Exon 13 Skipping Are Rare Events in FAP Pathogenesis and Define the Clinical Outcome

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease

Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

Background Activation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38 alpha feeds into the canonical Wnt/beta-catenin pathway by inhibiting GSK3 beta through phosphorylation. Recently, we identified p38 alpha as a new druggable member of beta-catenin chromatin-associated kinase complexes in CRC.Methods The functional relationship between p38 alpha and beta-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APC(Min/+) mice). The role of p38 alpha in beta-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib.Results We used the GSK3 beta inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38 alpha nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3 beta inhibition, nuclear p38 alpha phosphorylates beta-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38 alpha is pharmacologically inhibited in addition to GSK3 beta, beta-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis.Conclusions p38 alpha seems to play a dual role as a member of the beta-catenin destruction complex and as a beta-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment

The chromatin remodeling factors EP300 and TRRAP are novel SMYD3 interactors involved in the emerging ‘nonmutational epigenetic reprogramming’ cancer hallmark

SMDY3 is a histone-lysine N-methyltransferase involved in several oncogenic processes and is believed to play a major role in various cancer hallmarks. Recently, we identified ATM, BRCA2, CHK2, MTOR, BLM, MET, AMPK, and p130 as direct SMYD3 interactors by taking advantage of a library of rare tripeptides, which we first tested for their in vitro binding affinity to SMYD3 and then used as in silico probes to systematically search the human proteome. Here, we used this innovative approach to identify further SMYD3-interacting proteins involved in crucial cancer pathways and found that the chromatin remodeling factors EP300 and TRRAP interact directly with SMYD3, thus linking SMYD3 to the emerging ‘nonmutational epigenetic reprogramming’ cancer hallmark. Of note, we validated these interactions in gastrointestinal cancer cell lines, including HCT-116 cells, which harbor a C-terminal truncating mutation in EP300, suggesting that EP300 binds to SMYD3 via its N-terminal region. While additional studies are required to ascertain the functional mechanisms underlying these interactions and their significance, the identification of two novel SMYD3 interactors involved in epigenetic cancer hallmark pathways adds important pieces to the puzzle of how SMYD3 exerts its oncogenic role

Understanding the Genetic Landscape of Pancreatic Ductal Adenocarcinoma to Support Personalized Medicine: A Systematic Review

Simple Summary Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high mortality. Most patients present with an advanced stage of the disease, highlighting the urgent need for early detection. Recent studies of individuals at high risk of PDAC showed benefits from participating in clinical management and surveillance programs. PDAC clinical management and surveillance programs are suggested for individuals with a germline pathogenic variant in a cancer predisposition gene or a strong family history. In the present study, we performed a systematic literature review to investigate the mutational portrait of the main genes (ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, TP53) involved in PDAC susceptibility. Our findings may support the development of tailored management and follow-up strategies in PDAC patients with specific germline genetic variants.Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies worldwide. While population-wide screening recommendations for PDAC in asymptomatic individuals are not achievable due to its relatively low incidence, pancreatic cancer surveillance programs are recommended for patients with germline causative variants in PDAC susceptibility genes or a strong family history. In this study, we sought to determine the prevalence and significance of germline alterations in major genes (ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, TP53) involved in PDAC susceptibility. We performed a systematic review of PubMed publications reporting germline variants identified in these genes in PDAC patients. Overall, the retrieved articles included 1493 PDAC patients. A high proportion of these patients (n = 1225/1493, 82%) were found to harbor alterations in genes (ATM, BRCA1, BRCA2, PALB2) involved in the homologous recombination repair (HRR) pathway. Specifically, the remaining PDAC patients were reported to carry alterations in genes playing a role in other cancer pathways (CDKN2A, STK11, TP53; n = 181/1493, 12.1%) or in the mismatch repair (MMR) pathway (MLH1, MSH2, MSH6, PMS2; n = 87/1493, 5.8%). Our findings highlight the importance of germline genetic characterization in PDAC patients for better personalized targeted therapies, clinical management, and surveillance