Diversity of genetic events associated with MLH1 promoter methylation in Lynch syndrome families with heritable constitutional epimutation.

PURPOSE: Constitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far. METHODS: We designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations. RESULTS: This strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5'-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription. CONCLUSION: This is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.

Messenger RNA Life-Cycle in Cancer Cells: Emerging Role of Conventional and Non-Conventional RNA-Binding Proteins?

Functional specialization of cells and tissues in metazoans require specific gene expression patterns. Biological processes, thus, need precise temporal and spatial coordination of gene activity. Regulation of the fate of messenger RNA plays a crucial role in this context. In the present review, the current knowledge related to the role of RNA-binding proteins in the whole mRNA life-cycle is summarized. This field opens up a new angle for understanding the importance of the post-transcriptional control of gene expression in cancer cells. The emerging role of non-classic RNA-binding proteins is highlighted. The goal of this review is to encourage readers to view, through the mRNA life-cycle, novel aspects of the molecular basis of cancer and the potential to develop RNA-based therapies.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

MUC5AC hypomethylation is a predictor of microsatellite instability independently of clinical factors associated with colorectal cancer.

Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC.

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin, microsatellite instability and KRAS mutations.

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4-9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6% of the patients (28/47) including 20 (71.4%) missense mutations, 7 nonsense mutations (25%) and 1 (3.6%) frameshift mutation. 89.3% (25/28) of the detected mutations were in exons 5-8, whereas 10.7% (3/28) were in exon 4. Among the 27 non frameshift mutations, 89% (24/27) were transitions and 11% (3/27) were transversions. 64.3% (18/27) of the altered amino acids corresponded to arginine. 74% (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25% (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.

Creatine and guanidinoacetate reference values in a French population.

Creatine and guanidinoacetate are biomarkers of creatine metabolism. Their assays in body fluids may be used for detecting patients with primary creatine deficiency disorders (PCDD), a class of inherited diseases. Their laboratory values in blood and urine may vary with age, requiring that reference normal values are given within the age range. Despite the long known role of creatine for muscle physiology, muscle signs are not necessarily the major complaint expressed by PCDD patients. These disorders drastically affect brain function inducing, in patients, intellectual disability, autistic behavior and other neurological signs (delays in speech and language, epilepsy, ataxia, dystonia and choreoathetosis), being a common feature the drop in brain creatine content. For this reason, screening of PCDD patients has been repeatedly carried out in populations with neurological signs. This report is aimed at providing reference laboratory values and related age ranges found for a large scale population of patients with neurological signs (more than 6 thousand patients) previously serving as a background population for screening French patients with PCDD. These reference laboratory values and age ranges compare rather favorably with literature values for healthy populations. Some differences are also observed, and female participants are discriminated from male participants as regards to urine but not blood values including creatine on creatinine ratio and guanidinoacetate on creatinine ratio values. Such gender differences were previously observed in healthy populations; they might be explained by literature differential effects of testosterone and estrogen in adolescents and adults, and by estrogen effects in prepubertal age on SLC6A8 function. Finally, though they were acquired on a population with neurological signs, the present data might reasonably serve as reference laboratory values in any future medical study exploring abnormalities of creatine metabolism and transport.

KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability.

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.

EFTUD2 haploinsufficiency leads to syndromic oesophageal atresia.

BACKGROUND: Oesophageal atresia (OA) and mandibulofacial dysostosis (MFD) are two congenital malformations for which the molecular bases of syndromic forms are being identified at a rapid rate. In particular, the EFTUD2 gene encoding a protein of the spliceosome complex has been found mutated in patients with MFD and microcephaly (MIM610536). Until now, no syndrome featuring both MFD and OA has been clearly delineated. RESULTS: We report on 10 cases presenting with MFD, eight of whom had OA, either due to de novo 17q21.31 deletions encompassing EFTUD2 and neighbouring genes or de novo heterozygous EFTUD2 loss-of-function mutations. No EFTUD2 deletions or mutations were found in a series of patients with isolated OA or isolated oculoauriculovertebral spectrum (OAVS). CONCLUSIONS: These data exclude a contiguous gene syndrome for the association of MFD and OA, broaden the spectrum of clinical features ascribed to EFTUD2 haploinsufficiency, define a novel syndromic OA entity, and emphasise the necessity of mRNA maturation through the spliceosome complex for global growth and within specific regions of the embryo during development. Importantly, the majority of patients reported here with EFTUD2 lesions were previously diagnosed with Feingold or CHARGE syndromes or presented with OAVS plus OA, highlighting the variability of expression and the wide range of differential diagnoses.

Evidence of constitutional MLH1 epimutation associated to transgenerational inheritance of cancer susceptibility.

Constitutional epimutations of DNA mismatch repair (MMR) genes have been recently reported as a possible cause of Lynch syndrome. However, little is known about their prevalence, the risk of transmission through the germline and the risk for carriers to develop cancers. In this study, we evaluated the contribution of constitutional epimutations of MMR genes in Lynch syndrome. A cohort of 134 unrelated Lynch syndrome-suspected patients without MMR germline mutation was screened for constitutional epimutations of MLH1 and MSH2 by quantitative bisulfite pyrosequencing. Patients were also screened for the presence of EPCAM deletions, a possible cause of MSH2 methylation. Tumors from patients with constitutional epimutations were extensively analyzed. We identified a constitutional MLH1 epimutation in two proband patients. For one of them, we report for the first time evidence of transmission to two children who also developed early colonic tumors, indicating that constitutional MLH1 epimutations are associated to a real risk of transgenerational inheritance of cancer susceptibility. Moreover, a somatic BRAF mutation was detected in one affected child, indicating that tumors from patients carrying constitutional MLH1 epimutation can mimic MSI-high sporadic tumors. These findings may have important implications for future diagnostic strategies and genetic counseling.

The complex intratumoral heterogeneity of colon cancer highlighted by laser microdissection.

AIMS: To evaluate the utility of laser microdissection in the comparison of phenotypes and genetic alterations between colon cancer and corresponding liver metastasis in the context of intratumoral heterogeneity. METHODS: Immunohistochemistry was performed on a series of 11 patients surgically treated for colon adenocarcinoma with liver metastases, using antibodies directed against six mucins. Immunohistochemistry was completed by laser microdissection of tumor zones with particular phenotype, luminal zone and invasion front of colon tumors. Microdissected samples were compared on the basis of microsatellite instability and alterations of CTNNB1, KRAS, and TP53. RESULTS: Our study demonstrated varying mucin expression within tumors, suggesting the existence of phenotypic intratumoral heterogeneity. A common immunohistochemical profile was observed in individual tumors between tumoral subpopulations and corresponding metastases. Nevertheless, the phenotypic characteristics were distinct from one patient to another. Laser microdissection underlined that phenotypic heterogeneity could rely on genotypic heterogeneity, and that some genetic alterations were common to microdissected samples from primary colon tumors and liver metastases. CONCLUSION: We illustrated intratumoral heterogeneity of colon cancer using laser microdissection, in combination with immunohistochemical and genotypic tools. This intratumoral heterogeneity could represent a major issue in the search of prognostic biomarkers.

Lynch syndrome in Tunisia: first description of clinical features and germline mutations.

PURPOSE: High rates of early colorectal cancers (CRC) are observed in Tunisia suggesting genetic susceptibility. Nevertheless, up to now, no molecular study has been performed in the Tunisian population. In our research, we evaluated the clinical characteristics of Tunisian families suspected of Lynch syndrome and the contribution of DNA mismatch repair (MMR) genes. METHODS: Thirty-one unrelated families suspected of Lynch syndrome were studied. Probands were tested for the presence of germline mutations in the MMR genes MLH1, MSH2, MSH6 and in MUTYH. Available tumours were analysed for microsatellite instability and expression of MMR proteins. Detailed family and medical histories were collected. RESULTS: A total of 134 cancers were noted in the 31 families, the most frequent type of cancer corresponding to CRC (69%), followed by uterine cancer (7.5%). Germline mutations were identified in 11 (35.5%) families (six MSH2, five MLH1, including seven novel mutations), seven of which fulfilled the Amsterdam criteria (sensitivity, 63.6%; positive predictive value, 58.3%). Noteworthy, germline mutations were detected in 52.6% of male patients tested, but in only 8.3% of females (p = 0.02). Moreover, CRC were essentially left sided in families without detected mutation (p = 0.017). Ages of onset of cancers and tumour spectrum were very similar in families with or without MMR germline mutation, contrasting with previous studies performed in other populations. CONCLUSIONS: MMR genes contribute significantly to CRC susceptibility in the Tunisian population. However, the cause of early CRC susceptibility remains unknown in most cases, especially in women and in patients with early left colon or rectal cancer.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface.

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.

Holt-Oram syndrome: clinical and molecular description of 78 patients with TBX5 variants.

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterised by the association of congenital heart defect (CHD), with or without rhythm disturbances and radial defects, due to TBX5 variants. The diagnosis is challenged by the variability of expression and the large phenotypic overlap with other conditions, like Okihiro syndrome, TAR syndrome or Fanconi disease. We retrospectively reviewed 212 patients referred for suspicion of HOS between 2002 and 2014, who underwent TBX5 screening. A TBX5 variant has been identified in 78 patients, representing the largest molecular series ever described. In the cohort, 61 met the previously described diagnostic criteria and 17 have been considered with an uncertain HOS diagnosis. A CHD was present in 91% of the patients with a TBX5 variant, atrial septal defects being the most common (61.5%). The genotype-phenotype study highlights the importance of some critical features in HOS: the septal characteristic of the CHD, the bilateral and asymmetric characteristics of the radial defect and the presence of shoulder or elbow mobility defect. Besides, 21 patients presented with an overlapping condition. Among them, 13 had a typical HOS presentation. We discuss the strategies that could be adopted to improve the molecular delineation of the remaining typical patients.

Paraganglioma after maternal transmission of a succinate dehydrogenase gene mutation.

CONTEXT: Inactivating mutations of SDHD, which is mapped to 11q23 and encodes the cybS subunit of succinate dehydrogenase, predispose to hereditary paraganglioma (PGL) and/or pheochromocytoma. So far no disease was shown to occur in case of maternal transmission of a SDHD mutation, suggesting the existence of genomic imprinting. A hypothetic model, involving the loss of the maternal copy of a tumor suppressor gene mapped to 11p15 in the tumoral tissue, has been proposed to explain this mode of inheritance. OBJECTIVE: Our objective was to investigate the possibility of maternal transmission of SDHD-linked PGL. DESIGN: A three-generation family carrying the SDHD W43X mutation was studied at the clinical, pathological, and genetical levels. RESULTS: The germline's mutation was probably inherited from the grandfather. In the second generation, three carriers (two females and one male), who had the same at risk 11q13-q23 haplotype, developed multiple cervical PGLs. In the third generation, one boy received the mutation from his mother and developed a glomus tympanicum PGL at 11 yr. He shared only the 11q23 haplotype with the other affected members of the family. Methylation analysis of the differentially methylated region upstream of the maternally expressed H19 gene, mapped to 11p15, showed that the seventh CTCF binding site is hypermethylated in the germline of the affected boy suggesting a gain of imprinting. CONCLUSION: Our data show that maternal transmission of a SDHD-linked PGL, even if a rare event, can occur. Therefore, we propose that children who inherited a pathogenic mutation from their mother should be considered as at risk of PGL.

Frequent mutations of the CA simple sequence repeat in intron 1 of EGFR in mismatch repair-deficient colorectal cancers.

AIM: To investigate the polymorphic simple sequence repeat in intron 1 of the epidermal growth factor receptor gene (EGFR) (CA-SSR I), which is known to affect the efficiency of gene transcription as a putative target of the mismatch repair (MMR) machinery in colorectal tumors. METHODS: The CA-SSR I genotype was analyzed in a total of 86 primary colorectal tumors, selected upon their microsatellite instability (MSI) status [42 with high frequency MSI (MSI-H) and 44 microsatellite stable (MSS)] and their respective normal tissue. The effect of the CA-SSR I genotype on the expression of the EGFR gene was evaluated in 18 specimens using quantitative real-time reverse transcription PCR and immunohistochemistry. RESULTS: Mutations in CA-SSR I were detected in 86% (36 of 42) of MSI-H colorectal tumors and 0% (0 of 44) of MSS tumors, indicating the EGFR gene as a novel putative specific target of the defective MMR system (P < 0.001). Impaired expression of EGFR was detected in most of the colorectal tumors analyzed [6/12 (50%) at the mRNA level and 15/18 (83%) at the peptide level]. However, no association was apparent between EGFR expression and CA-SSR I status in tumors or normal tissues. CONCLUSION: Our results suggest that CA-SSR I sequence does not contribute to the regulation of EGFR transcription in colon, and should thus not be considered as a promising predictive marker for response to EGFR inhibitors in patients with colorectal cancer.

Alpha1 antitrypsin deficiency due to an homozygous PI* Null Q0Cairo mutation: Early onset of pulmonary manifestations and variability of clinical expression.

Alpha-1 antitrypsin deficiency is an autosomal, codominant disorder caused by mutations of the SERPINA1 gene. This genetic disorder is mainly associated with development of pulmonary emphysema and/or chronic liver disease and cirrhosis. Here we report a very rare alpha-1 antitrypsin Null Q0cairo homozygous mutation characterized by a complete absence of alpha-1 antitrypsin in the plasma, in a non-consanguineous Moroccan family. This mutation has been previously described in heterozygosis in only three cases worldwide: an Italian/Egyptian family and two Italian families (Zorzetto et al., 2005). The main clinical features in two members of this Moroccan family were the severity and precocity of bronchiectasis, quickly spreading and seriously limiting respiratory function and physical activity by the second decade of age. Moreover, the index case presented with many episodes of pulmonary infections concomitant with severe neutropenia. The third member of the family presented with ankylosing spondyloarthritis and developed panniculitis later but had no respiratory symptoms. The presence of this alpha-1-antitrypsin Q0cairo homozygous mutation could explain the severity of clinical manifestations. Moreover, our observations highlight a great variability of clinical expression for the same mutation: early severe bronchiectasis, panniculitis, rheumatologic manifestations. This study further underlines the importance of genotyping by whole SERPINA1 gene sequencing in addition to serum alpha-1 antitrypsin determination, to enable detection of alpha-1 antitrypsin deficiency due to rare genotypes.

Description of 22 new alpha-1 antitrypsin genetic variants.

Alpha-1 antitrypsin deficiency is an autosomal co-dominant disorder caused by mutations of the highly polymorphic SERPINA1 gene. This genetic disorder still remains largely under-recognized and can be associated with lung and/or liver injury. The laboratory testing for this deficiency typically comprises serum alpha-1 antitrypsin quantification, phenotyping according to the isoelectric focusing pattern and genotyping if necessary. To date, more than 100 SERPINA1 variants have been described and new genetic variants are frequently discovered. Over the past 10 years, 22 new genetic variants of the SERPINA1 gene were identified in the daily practice of the University Medical laboratories of Lille and Lyon (France). Among these 22 variants, seven were Null alleles and one with a M1 migration pattern (M1Cremeaux) was considered as deficient according to the clinical and biological data and to the American College of Medical Genetics and Genomics (ACMG) criteria. Three other variants were classified as likely pathogenic, three as variants of uncertain significance while the remaining ones were assumed to be neutral. Moreover, we also identified in this study two recently described SERPINA1 deficient variants: Trento (p.Glu99Val) and SDonosti (p.Ser38Phe). The current data, together with a recent published meta-analysis, represent the most up-to-date list of SERPINA1 variants available so far.

Transcription factor AP-2alpha represses both the mucin MUC4 expression and pancreatic cancer cell proliferation.

MUC4 is a transmembrane mucin expressed in pancreatic ductal adenocarcinoma (DAC) in contrast to normal pancreas, and is an independent predictor of poor prognosis in patients with invasive DAC. Our aim was therefore to investigate the mechanisms that control MUC4 expression in pancreatic cancer cells. We focused our study on activator protein (AP)-2alpha transcription factor that acts as a tumour suppressor gene in several cancers. In a series of 18 human DAC, using immunohistochemistry, we confirmed that MUC4 was exclusively expressed in cancerous or preneoplastic lesions in 83% of the samples. On the contrary, AP-2 was mainly expressed by non-tumoural ductal cells (61%) or endocrine cells (67%). Moreover, MUC4 and AP-2 were never found co-expressed suggesting an inhibitory role of AP-2alpha in normal ductal cells. In CAPAN-1 and CAPAN-2 cells, transient AP-2alpha over-expression decreased both MUC4 mRNA and apomucin levels by 20-40% by a mechanism involving inhibition of MUC4 promoter. By chromatin immunoprecipitation and gel-shift assays, we demonstrated that this inhibition involved two AP-2 cis-elements located in the -475/-238 region of the promoter. CAPAN-1 clones, which stably over-expressed AP-2alpha, displayed a strong MUC4 down-regulation (-38 to -100%), a significant decrease of both cell proliferation and invasion concomitant to the up-regulation of p27 cyclin-dependent kinase inhibitor. In conclusion, our data provide evidence that AP-2alpha is an important in vivo negative regulator of MUC4 expression in human pancreatic tissue and that AP-2alpha may play a tumour-suppressive role in pancreatic DAC.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3-/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Low frequency of AXIN2 mutations and high frequency of MUTYH mutations in patients with multiple polyposis.

Familial adenomatous polyposis has been linked to germline mutations in the APC tumor suppressor gene. However, a number of patients with familial adenomatous polyposis (with either classical or attenuated phenotype) have no APC mutation. Recently, germline mutations in the Wnt pathway component gene AXIN2 have been associated with tooth agenesis-colorectal cancer syndrome. Moreover, biallelic mutations in the base excision repair gene MUTYH have been associated with polyposis and early-onset colorectal cancer. The aim of this study was to further assess the contribution of AXIN2 and MUTYH to hereditary colorectal cancer susceptibility. AXIN2 and MUTYH genes were screened for germline mutations by PCR and direct sequencing in 39 unrelated patients with multiple adenomas or colorectal cancer without evidence of APC mutation nor mismatch repair defect. Two novel AXIN2 variants were detected in one patient with multiple adenomas, but no clearly pathogenic mutation. In contrast, nine different MUTYH mutations were detected in eight patients, including four novel mutations. Biallelic MUTYH mutations were only found in patients with multiple adenomatous polyposis (7 out of 22 (32%)). Interestingly, five MUTYH mutation carriers had a family history consistent with dominant inheritance. Moreover, one patient with biallelic MUTYH mutations presented with multiple adenomas and severe tooth agenesis. Therefore, germline mutations are rare in AXIN2 but frequent in MUTYH in patients with multiple adenomas. Our data suggest that genetic testing of MUTYH may be of interest in patients with pedigrees apparently compatible with autosomal recessive as well as dominant inheritance.