CD44 is the major ubiquitously expressed cell surface receptor for hyaluronate. The CD44 gene encodes several protein isoforms due to extensive alternative splicing and post-translational modifications. Some of these CD44 variable isoforms have been foreseen as key players in malignant transformation and their expression is highly restricted and highly specific, unlike the canonical CD44 standard isoform. In this study, we aimed at dissecting the mRNA splicing pattern of CD44 in normal stomach and gastric cancer (GC) cell lines (n ¼ 9) using cloning and quantitative mRNA amplification assays. Moreover, we assessed the RNA levels and protein expression pattern of relevant splicing forms in distinct premalignant and malignant gastric lesions (sporadic (n ¼ 43) and hereditary (n ¼ 3) forms) using real-time RT-PCR and immunohistochemistry. We also explored the association of CD44 and E-cadherin expression by immunohistochemistry, as E-cadherin has a pivotal functional role in GC. We established the pattern of CD44 variant forms in normal stomach and gastric malignancy. We observed that although exon v6-containing isoforms were rarely expressed in normal gastric mucosa, they became increasingly expressed both in gastric premalignant (hyperplastic polyps, complete and incomplete intestinal metaplasia, low-and high-grade dysplasia) and malignant lesions (cell lines derived from GCs, primary sporadic GCs and hereditary diffuse GCs (HDGCs)). Moreover, we verified that whenever E-cadherin expression was absent, exon v6-containing CD44 isoforms were overexpressed. The lack of expression of CD44 isoforms containing exon v6 in the surface and foveolar epithelia of normal stomach and, its de novo expression in premalignant, as well as in sporadic and hereditary malignant lesions of the stomach, pinpoint CD44 v6-containg isoforms as potential biomarkers for early transformation of the gastric mucosa. Further, our results raise the hypothesis of using CD44v6 as a marker of early invasive intramucosal carcinoma in HDGC CDH1 mutation carriers that lack CDH1 expression in their tumors.

The role of E-cadherin in tumorigenesis has been attributed to its ability to suppress invasion and metastization. However, E-cadherin impairment may have a wider impact on tumour development. We have previously shown that overexpression of mutant human E-cadherin in Drosophila produces a phenotype characteristic of downregulated Notch. Hence, we hypothesized that Notch signalling may be influenced by E-cadherin and may mediate tumour development associated with E-cadherin deficiency. De novo expression of wild-type E-cadherin in two cellular models led to a significant decrease in the activity of the Notch pathway. In contrast, the ability to inhibit Notch-1 signalling was lost in cells transfected with mutant forms of E-cadherin. Increased Notch-1 activity in E-cadherin-deficient cells correlated with increased expression of Bcl-2, and increased resistance to apoptotic stimuli. After Notch-1 inhibition, E-cadherin-deficient cells were re-sensitized to apoptosis in a similar degree to wild-type E-cadherin cells. We also show that Notch-inhibiting drugs are able to significantly inhibit the growth of E-cadherin-deficient cells xenografted into nude mice. This effect was comparable with the one observed in animals treated with the chemotherapeutic agent taxol, a chemical inducer of cell death. In conclusion, our results show that aberrant Notch-1 activation, Bcl-2 overexpression and increased cell survival are likely to play a crucial role in neoplastic transformation associated with E-cadherin impairment. These findings highlight the possibility of new targeted therapeutical strategies for the treatment of tumours associated with E-cadherin inactivation.

Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family are fundamental for the control of differentiation and proliferation of many adult tissues. C/EBPa has a crucial role in inducing terminal differentiation and is an established tumor suppressor gene in several cancer models. The objective of this study was to analyze the putative role of C/EBPa in gastric carcinoma (GC). We analyzed the expression of C/EBPa in normal and neoplastic gastric tissues, and assessed the role of C/EBPa on proliferation and differentiation of GC cells. In normal gastric mucosa, C/EBPa is expressed in the foveolar epithelium and co-localizes with the gastric differentiation marker trefoil factor 1 (TFF1). The expression of C/EBPa was found to be lost in 30% of GC cases. To evaluate the role of C/EBPa in cell proliferation and differentiation, we transfected GC cells with a full-length C/EBPa protein. We observed a significant decrease in proliferation in C/EBPa-transfected cells. This was accompanied by a decrease in Cyclin D1, an increase in P27 expression, and an increased expression of TFF1. Finally, we showed that inhibition of the Ras/MAPK pathway leads to increased C/EBPa and TFF1 expression, and decreased cell proliferation and cyclin D1 expression in GC cells. Our results suggest that C/EBPa (together with other members of the C/EBP family) has an active role in the control of differentiation and proliferation in normal gastric mucosa. In GC, loss of C/EBPa may be associated with the switch from a cellular differentiation to a cellular proliferation program, presumably as a consequence of Ras/MAPK pathway activation.

No abstract

Retention of intracellular secreted APP (isAPP) can be provoked by the neurotoxic peptide Aβ. The latter decreases in the cerebrospinal fluid of Alzheimer’s disease (AD) patients, as a consequence of its cerebral accumulation and deposition into senile plaques. Of similar relevance, secreted APP (sAPP) levels can be associated with AD. The studies here presented, reinforce the link between sAPP and Aβ and address putative therapeutic strategies. Laminin and gelsolin are potential candidates; both prevent Aβ fibril formation by complexing with Aβ, thus attenuating its neurotoxicity. We show that preincubation of Aβ with laminin and gelsolin has the effect of rendering it less potent to isAPP accumulation in cortical neurons. This appears to be related to a decrease in F-actin polymerization, whereas Aβ alone induces the polymerization. Further, Aβ decreases gelsolin levels, and the latter is involved in Aβ removal. Our data indicates that Aβ-laminin and Aβ-gelsolin complexes are less neurotoxic and also less potent than fibrillar Aβ at inducing isAPP retention. These results validate the potential of these proteins as therapeutic strategies that prevent the Aβ-induced effects. In hence, given that Aβ decreases the levels of proteins involved in its own clearance, this may contribute to the mechanisms underlying AD pathology.