Aida Jawhari

Abnormal immunoreactivity of the E-cadherin-catenin complex in gastric carcinoma: Relationship with patient survival

BACKGROUND & AIMS The E-cadherin-catenin complex plays a critical role in the maintenance of normal tissue architecture. Mutation of any of its components is believed to result in loss of cell-cell adhesion and contribute to neoplasia. The aim of this study was to examine the expression of E-cadherin and alpha-, beta-, and gamma-catenin in gastric carcinoma and dysplasia and determine any relationship with tumor characteristics and survival. METHODS Immunoperoxidase staining of E-cadherin and alpha-, beta-, and gamma-catenin was performed using 89 gastric carcinomas, lymph node metastases, and gastric biopsy specimens from 14 patients with dysplasia and 10 healthy controls. RESULTS Membranous staining was observed in control biopsy specimens for all components of the complex. Up to 57% of gastric dysplasia and 90% of tumors stained abnormally for one or more components of the cadherin-catenin complex. Abnormal E-cadherin and gamma-catenin staining occurred more frequently in diffuse than intestinal tumors (P < 0.0005 and < 0.05, respectively). No association with tumor grade or stage was found. A survival advantage was noted in intestinal and diffuse tumors retaining membranous expression of beta-catenin, independent of tumor type, grade, or stage (P < 0.005). CONCLUSIONS Abnormal expression of the E-cadherin-catenin complex occurs frequently in gastric carcinoma. The close correlation with poor survival suggests that abnormal beta-catenin may be a useful prognostic marker.

Fascin, an Actin-Bundling Protein, Modulates Colonic Epithelial Cell Invasiveness and Differentiation in Vitro

In epithelial tissue, cell-matrix and cell-cell adhesive interactions have important roles in the normal organization and stabilization of the cell layer. The malignant conversion of epithelial cells involves alterations in the expression and function of these adhesion systems that enable a switch to a migratory phenotype in tumor invasion and metastasis. Fascin is an actin-crosslinking protein that is found in the core actin bundles of cell-surface spikes and projections that are implicated in cell motility. We demonstrate that fascin is not detectable in normal colonic epithelium, but is dramatically up-regulated in colorectal adenocarcinoma. To test the hypothesis that fascin could participate in tumor invasive behavior, we developed a cell culture model to examine the effect of fascin expression on the adhesive interactions, invasiveness, and differentiation of colonic epithelial cells. We report marked effects on the organization of cell-surface protrusions, actin cytoskeleton, and focal adhesions in the absence of alterations in the protein levels of the major components of these structures. These effects correlate with alterations in cell movements on two-dimensional matrix, and increased invasiveness in three-dimensional matrix. The cells also show increased proliferation and decreased capacity for normal glandular differentiation in collagen gels. We propose that up-regulation of fascin, by promoting the formation of protrusive, actin-based, cell-motility structures, could be a significant component in the acquisition of invasive phenotype in colonic carcinoma.

Abnormal expression and function of the E-cadherin–catenin complex in gastric carcinoma cell lines

SummaryDysfunction of the cadherin–catenin complex, a key component of adherens junctions, is thought to confer invasive potential to cells. The aim of this study is to examine the expression and function of the E-cadherin/catenin complex in gastric carcinoma cell lines. Expression of E-cadherin, α, β and γ-catenin and p120ctn, and of the adenomatous polyposis coli protein (APC), together with function of the cadherin–catenin complex was examined in a panel of gastric carcinoma cell lines, using immunocytochemistry, Western blotting and a cell–cell aggregation assay. Protein interactions were examined by sequential immunoprecipitation and immunoblotting with antibodies to E-cadherin, α, β and γ-catenin, p120ctn and APC. Abnormalities of E-cadherin, α- and β-catenin expression, were associated with disturbance of E-cadherin–catenin complex composition, loss of membranous localization and loss of calcium-dependent aggregation in six gastric carcinoma cell lines. APC protein expression and interaction with β-catenin was preserved in five cell lines. We demonstrate frequent abnormalities of expression and function of E-cadherin and catenins, and associated disturbance of E-cadherin-mediated intercellular adhesion in gastric carcinoma cell lines. These findings support the tumour suppressor role of the E-cadherin and its contribution to the development and progression of the neoplastic phenotype in gastric carcinoma.

The E-cadherin/epidermal growth factor receptor interaction: a hypothesis of reciprocal and reversible control of intercellular adhesion and cell proliferation

The E‐cadherin/catenin complex is a calcium‐dependent cell–cell adhesion molecule, whose function is critical to the integrity of the adherens junction and which plays a role in the establishment and maintenance of normal epithelial morphology and differentiation. Loss of E‐cadherin‐mediated adhesion appears to be a fundamental aspect of the neoplastic phenotype which in some cases appears to be mediated by post‐translational modifications (i.e. tyrosine phosphorylation) of its interacting proteins, the catenins which link E‐cadherin to the actin cytoskeleton. There is increasing experimental evidence to suggest that epidermal growth factor receptor tyrosine phosphorylation may lead to the inactivation of the E‐cadherin/catenin complex in cancer cells through its interaction with β‐ or γ‐ catenin in the cytoskeleton. Modulation of epidermal growth factor receptor activity by pharmacological agents has the potential to regulate a variety of cellular processes including adhesion, differentiation, and proliferation. Copyright

Up-regulated cytoplasmic expression, with reduced membranous distribution, of the src substrate p120ctn in gastric carcinoma

p120ctn is a substrate of the tyrosine kinase pp60 src. Tyrosine kinases such as src localize to the adherens junctions and phosphorylate junctional proteins in both normal and transformed cells.1 p120ctn forms a complex with E‐cadherin at the adherens junction and is phosphorylated by ligands such as epidermal growth factor receptor as well as pp60 src. Phosphorylation of p120ctn has been shown to correlate with cell transformation. The aim of this study was to investigate in vivo expression of p120ctn in gastric carcinoma and to examine any relationship to pathological characteristics and patient survival. Immunohistochemical staining for p120ctn was performed in 68 gastric carcinoma specimens (19 diffuse, 49 intestinal type), in 22 lymph node metastases, and in gastric mucosal biopsies from 16 patients with gastric dysplasia and ten healthy controls. Up‐regulation of p120ctn cytoplasmic staining was seen in six (37 per cent) of the gastric dysplasia cases and in 45 (66 per cent) tumours (89 per cent of diffuse and 57 per cent of intestinal tumours). Loss of membranous distribution of staining for p120ctn was seen in 22 (32 per cent) tumours (52 per cent of diffuse and 24 per cent of intestinal tumours). The staining pattern in the primary tumour showed no correlation with tumour type, grade, or stage, or patient survival. Of 22 lymph node metastases examined, 13 (60 per cent) showed loss of membranous staining. In conclusion, staining for p120ctn in gastric carcinoma and dysplasia revealed marked up‐regulation of cytoplasmic staining, sometimes associated with reduced membranous expression. Up‐regulation of expression of p120ctn has not previously been described in human epithelial malignancy. The significance of these findings is uncertain, but they may reflect a change in tyrosine kinase signal transduction pathways, and a role for p120ctn in ligand‐induced mitogenic signalling and cell transformation. Copyright

The importance of the E-cadherin-catenin complex in the maintenance of intestinal epithelial homoeostasis: more than intercellular glue?

The intestinal epithelium is characterised by rapid cell turnover, with pluripotential stem cells in the crypts of Lieberkuhn providing a continuous supply of cells which are directed into a variety of maturation pathways. Development and maintenance of normal intestinal epithelial morphology requires regulation of daughter cells’ proliferative status, lineage allocation, migration, differentiation, and apoptosis.1 It has become increasingly evident that co-ordination of these processes and the consequent generation of the characteristic intestinal epithelial architecture are highly dependent on intercellular and cell-matrix adhesive interactions.2These adhesive events are not random, but through selective interactions, organise cells into diverse and highly distinctive patterns. Connections of cell junctions such as desmosomes, hemidesomosomes, and adherens junctions to the actin cytoskeleton, thus allow maintenance of cell polarity and tissue architecture.3 Variation in the functional state of these adhesive mechanisms is critical to the dynamic processes necessary for tissue morphogenesis in the embryo,4 where many morphogenetic events are correlated with a unique spatiotemporal pattern of cadherin expression.5 Such variation is also critical to the maintenance of this highly complex architecture in various physiological and pathological states, such as migration of cells up the crypt-villus axis6 and repair of mucosal injuries. In view of these facts, it is not surprising that many cell adhesion molecules have been implicated in cell signalling pathways.7 8 E-cadherin is a member of the large cadherin superfamily and is the predominant intercellular adhesion molecule expressed by intestinal epithelial cells.3 It is a calcium dependent transmembrane protein which forms a key component of adherens junctions. E-cadherin molecules form dimers at the cell surface, which interdigitate with other E-cadherin molecules on adjacent epithelial cells resulting in the formation of cell adhesion “zippers” (fig 1).9 10E-cadherin has classically been thought to be exclusively involved …

Follow-on RifAximin for the Prevention of recurrence following standard treatment of Infection with Clostridium Difficile (RAPID): a randomised placebo controlled trial

Background Clostridium difficile infection (CDI) recurs after initial treatment in approximately one in four patients. A single-centre pilot study suggested that this could be reduced using ‘follow-on’ rifaximin treatment. We aimed to assess the efficacy of rifaximin treatment in preventing recurrence. Methods A multisite, parallel group, randomised, placebo controlled trial recruiting patients aged ≥18 years immediately after resolution of CDI through treatment with metronidazole or vancomycin. Participants received either rifaximin 400 mg three times a day for 2 weeks, reduced to 200 mg three times a day for a further 2 weeks or identical placebo. The primary endpoint was recurrence of CDI within 12 weeks of trial entry. Results Between December 2012 and March 2016, 151 participants were randomised to either rifaximin or placebo. Primary outcome data were available on 130. Mean age was 71.9 years (SD 15.3). Recurrence within 12 weeks was 29.5% (18/61) among participants allocated to placebo compared with 15.9% (11/69) among those allocated to rifaximin, a difference between groups of 13.7% (95% CI −28.1% to 0.7%, p=0.06). The risk ratio was 0.54 (95% CI 0.28 to 1.05, p=0.07). During 6-month safety follow-up, nine participants died in each group (12%). Adverse event rates were similar between groups. Conclusion While ‘follow-on’ rifaximin after CDI appeared to halve recurrence rate, we failed to reach our recruitment target in this group of frail elderly patients, so the estimated effect of rifaximin lacks precision. A meta-analysis including a previous trial suggests that rifaximin may be effective; however, further, larger confirmatory studies are needed.

PWE-050 Follow-on rifaximin for the prevention of recurrence in clostridium difficile associated diarrhoea: a randomised controlled trial

Introduction Clostridium difficile associated diarrhoea (CDAD) is a common nosocomial infection that recurs in more than 1 in 4 cases. Garey et al. found that a course of rifaximin after standard therapy reduced relapse rate though not significantly1. We aimed to confirm or refute this finding. Method Design A parallel group, randomised, placebo controlled trial in 23 English hospitals. Funding NIHR RfPB Grant PB-PG-0110–21041. Norgine supplied drug and placebo without charge. Population age ≥18 with resolution of CDAD after treatment with metronidazole or vancomycin. CDAD diagnosis required evidence of toxin production or pseudomembranes at endoscopy. Exclusion criteria: pregnancy or breast feeding; life expectancy <4 weeks; unable to take intervention (hypersensitivity or swallowing disorder);>5 days elapsed since treatment. Randomisation was stratified by hospital using a remote, internet-based system. Participants, clinicians and researchers were blind to allocation. Intervention Rifaximin 1200 mg daily for two weeks then 600 mg daily for two weeks, in three divided doses. Comparator: identical placebo. Primary Outcome: relapse ≤12 weeks after treatment initiation. Sample size The planned sample size was 180 to detect a difference in relapse of 20% (30% placebo, 10% rifaximin) with 80% power, allowing for loss to follow-up of 20%. EudraCT 2012-003205-10; www.clinicaltrials.gov NCT01670149 Results Recruitment occurred December 2012–March 2015. Of 2157 patients screened, 151 were eligible, willing and randomised before funding limits were reached (74 placebo, 77 rifaximin). Primary outcome data were available on 130. Mean age was 71.9 (SD 15.3). 36% were in-patients at start of intervention. 18/61 (29.5%) on placebo relapsed within 12 weeks compared to 11/69 (15.9%) on rifaximin, a difference between groups of −13.7% (95% CI −28.1% to 0.7%, p=0.06). The risk ratio was 0.54 (95% CI 0.28 to 1.05, p=0.07). During 6 month safety follow up 9 participants died in each group (12%). Adverse event rates were similar between groups. Conclusion CDAD relapse rate was 13.7% lower than on placebo. The confidence interval means that lack of effect remains possible but the estimated effect size is similar to Garey’s trial, and those reported for fidaxomicin, with longer follow-up2. Age and mortality rate were higher in our trial which may reflect greater similarity to the population at risk. Comparative trials of cost effectiveness should follow. References . Garey et al. J Antimicrob Chemother 2011;66:2850–2855. . Crook et al. CID 2012;55(S2):S93–103. Disclosure of Interest G. Major: None Declared, L Bradshaw: None Declared, N Boota: None Declared, K Sprange: None Declared, A Jawhari: None Declared, M Diggle: None Declared, A Montgomery: None Declared, R Spiller Conflict with: Norgine Pharmaceuticals Ltd supplied product and comparator free of cost

Transcatheter embolisation in non-variceal gastrointestinal haemorrhage: an institutional review

Introduction The aim of this study was to assess the role of pre-angiographic imaging and the technical and clinical success of angiographic embolisation in the management of patients with acute non-variceal upper gastrointestinal (UGI) and lower gastrointestinal (LGI) bleeding. Methods We retrospectively assessed patients who had transcatheter angiography for acute gastrointestinal bleeding in a tertiary referral centre between 2006 and 2009. 36 patients were identified (mean age 77 years), of which 19 had LGI bleeding and 17 UGI bleeding. Data of clinical, endoscopic, angiographic and surgical interventions were collected. Clinical outcomes were recorded including: technical success, clinical success (no re-bleeding within 30 days), complications and mortality. Results 11 of the 36 patients had CT evaluation prior to catheter angiography. 5 of the 6 patients shown to have an active GI haemorrhage on CT had bleeding seen on angiography. All of the patients not demonstrating bleeding on CT were angiographic negative. All patients with UGI bleeding had upper GI endoscopy prior to angiography. 9 patients underwent endoscopic treatment for UGI bleeding. 3 patients in this group were eventually embolised. 8 patients did not have any endoscopic treatment and of these, 6 were embolised. 45% of patients having angiography within 24 h of endoscopy had active bleeding on angiography; as opposed to 17% of those waiting more than 24 h. Embolisation was performed in 50% patients (18 of 36; 9 UGI, 9 LGI) with a technical success of 95%. 12 patients had bleeding on angiography and were embolised. 6 patients underwent empirical embolisation. 5 patients required repeat angiography (1 technical failure, 4 clinical failures). Clinical success was 78% (7/9) in those with UGI haemorrhage and 67% (6/9) in those with LGI haemorrhage. The 30 day mortality in non-embolised group was 39% and the embolised group was 21%. Ischaemic complications occurred in 16% (3/18) of patients, all of whom were treated operatively. Conclusion Contrast extravasation is more likely to be demonstrated via transcatheter angiography if already seen on CT angiography. In those with UGI bleeding, angiography is more likely to demonstrate the haemorrhage if the time between endoscopy and angiography is less than 24 h. Our experience supports the early use of angiographic embolisation when gastrointestinal bleeding is not controlled at endoscopy.

Intestinal trefoil factor controls the expression of the adenomatous polyposis coli/catenin and the e-cadherin/catenin complexes in human colon carcinoma cells

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10(-9) M and 10(-8) M) for 20-24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, alpha-catenin and beta-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.