Atomic force microscopy and graph analysis to study the P-cadherin/SFK mechanotransduction signalling in breast cancer cells.

Physical forces mediated by cell-cell adhesion molecules, as cadherins, play a crucial role in preserving normal tissue architecture. Accordingly, altered cadherins' expression has been documented as a common event during cancer progression. However, in most studies, no data exist linking pro-tumorigenic signaling and variations in the mechanical balance mediated by adhesive forces. In breast cancer, P-cadherin overexpression increases in vivo tumorigenic ability, as well as in vitro cell invasion, by activating Src family kinase (SFK) signalling. However, it is not known how P-cadherin and SFK activation impact cell-cell biomechanical properties. In the present work, using atomic force microscopy (AFM) images, cell stiffness and cell-cell adhesion measurements, and undirected graph analysis based on microscopic images, we have demonstrated that P-cadherin overexpression promotes significant alterations in cell's morphology, by decreasing cellular height and increasing its area. It also affects biomechanical properties, by decreasing cell-cell adhesion and cell stiffness. Furthermore, cellular network analysis showed alterations in intercellular organization, which is associated with cell-cell adhesion dysfunction, destabilization of an E-cadherin/p120ctn membrane complex and increased cell invasion. Remarkably, inhibition of SFK signaling, using dasatinib, reverted the pathogenic P-cadherin induced effects by increasing cell's height, cell-cell adhesion and cell stiffness, and generating more compact epithelial aggregates, as quantified by intercellular network analysis. In conclusion, P-cadherin/SFK signalling induces topological, morphological and biomechanical cell-cell alterations, which are associated with more invasive breast cancer cells. These effects could be further reverted by dasatinib treatment, demonstrating the applicability of AFM and cell network diagrams for measuring the epithelial biomechanical properties and structural organization.

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer.

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with ß1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

Extracellular cleavage and shedding of P-cadherin: a mechanism underlying the invasive behaviour of breast cancer cells.

Cell-cell adhesion is an elementary process in normal epithelial cellular architecture. Several studies have shown the role mediated by cadherins in this process, besides their role in the maintenance of cell polarity, differentiation and cell growth. However, during tumour progression, these molecules are frequently altered. In breast cancer, tumours that overexpress P-cadherin usually present a high histological grade, show decreased cell polarity and are associated with worse patient survival. However, little is known about how this protein dictates the very aggressive behaviour of these tumours. To achieve this goal, we set up two breast cancer cell models, where P-cadherin expression was differently modulated and analysed in terms of cell invasion, motility and migration. We show that P-cadherin overexpression, in breast cancer cells with wild-type E-cadherin, promotes cell invasion, motility and migration. Moreover, we found that the overexpression of P-cadherin induces the secretion of matrix metalloproteases, specifically MMP-1 and MMP-2, which then lead to P-cadherin ectodomain cleavage. Further, we showed that soluble P-cadherin fragment is able to induce in vitro invasion of breast cancer cells. Overall, our results contribute to elucidate the mechanism underlying the invasive behaviour of P-cadherin expressing breast tumours.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

The interferon gamma receptor 1 (IFNGR1) -56C/T gene polymorphism is associated with increased risk of early gastric carcinoma.

BACKGROUND AND AIMS: It has been demonstrated that polymorphisms within inflammation-related genes are associated with the risk of gastric carcinoma (GC) in people infected with Helicobacter pylori. Recently, polymorphisms in the gene encoding the interferon gamma receptor 1 (IFNGR1) were found to be associated with increased susceptibility to H pylori infection. We aimed to determine the association between polymorphisms in the IFNGR1 gene and development of chronic gastritis and GC. METHODS: In a case-control study including 733 controls, 213 patients with chronic gastritis and 393 patients with GC, the IFNGR1 -611*G/*A, -56*C/*T, +1004*A/*C and +1400*T/*C polymorphisms were genotyped. A second independent case-control study including 100 controls and 65 patients with GC was used for confirmation of the original results. The effect of the -56*C/*T promoter polymorphism in the level of expression of the IFNGR1 gene was evaluated by an IFNGR1 -56*C/*T allele specific luciferase reporter assay. RESULTS: In patients with early onset GC (defined as being less than 40 years of age at the time of diagnosis) we found a significant over-representation of the IFNGR1 -56*T/*T homozygous genotype with an odds ratio (OR) of 4.1 (95% confidence interval (CI) 1.6 to 10.6). This result was confirmed in a second independent case-control study. In the luciferase reporter assay we observed a 10-fold increase (p<0.001) in luciferase expression associated with the IFNGR1-56*T allele. CONCLUSIONS: Our results indicate that the IFNGR1 -56C/T polymorphism is a relevant host susceptibility factor for GC development. Our data also indicate that this genetic polymorphism is functionally relevant and may be related to the early development of GC.

The NMD mRNA surveillance pathway downregulates aberrant E-cadherin transcripts in gastric cancer cells and in CDH1 mutation carriers.

Germline mutations in the gene encoding the tumour suppressor E-cadherin (CDH1) are the underlying genetic defect responsible for hereditary diffuse gastric cancer (HDGC). A remarkably high percentage ( approximately 80%) of CDH1 mutations in HDGC patients and carriers generate premature termination codons (PTCs). Here, we examined whether CDH1 transcripts harbouring PTCs are downregulated by nonsense-mediated decay (NMD), an RNA surveillance pathway that degrades PTC-bearing transcripts. Using an allele-specific expression (ASE) assay to differentiate between mutated and wild-type CDH1 alleles, we found that PTC-bearing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several CDH1 mutation carriers. We show that NMD is responsible for this robust downregulation, as CDH1 transcripts harbouring PTCs in the KATO-III gastric tumour cell line were upregulated in response to protein synthesis inhibitors or depletion of the NMD factors UPF1 and eIF4AIII. Analysis of HDGC patients harbouring CDH1 alleles with PTCs at a wide variety of different positions indicates an association of their predicted ability to induce NMD and an earlier age of onset of gastric cancer. This suggests that NMD may be detrimental for HDGC patients and therefore NMD is a potentially useful therapeutic target for CDH1 mutation carriers.

BRAF provides proliferation and survival signals in MSI colorectal carcinoma cells displaying BRAF(V600E) but not KRAS mutations.

BRAF kinase is a downstream target of KRAS and activates the MAPK pathway. These two molecules are prone to mutations in sporadic microsatellite unstable (MSI) colorectal carcinomas (CRC) and BRAF V600E mutations are inversely associated with oncogenic KRAS mutations. The biological significance of BRAF V600E oncogenic activation is not well established in this type of tumour. We aimed to study proliferation and survival effects induced by BRAF inhibition in MSI CRC cell lines harbouring distinct genetic backgrounds (BRAF V600E or KRAS G13D). Suppression of BRAF in BRAF V600E MSI CRC cell lines by RNA interference significantly inhibited proliferation and induced apoptosis, as demonstrated by BrdU incorporation and TUNEL assay, respectively. No significant differences were seen in proliferation and apoptosis, in cell lines harbouring KRAS G13D, after BRAF inhibition. We further analysed proliferation-associated molecules (pERK1/2, cyclin D1, p27 Kip1) and apoptosis-associated molecules (Bcl-2, Bax, pAkt, pBad, XIAP) in all cell lines. After BRAF down-regulation, we found a more pronounced decrease in ERK1/2 phosphorylation and cyclin D1 expression levels in BRAF-mutated cell lines in comparison to KRAS mutated cells. Upon BRAF inhibition, we also found an increase in p27(Kip1) levels and a more pronounced decrease in the levels of anti-apoptotic protein Bcl-2, specifically in cell lines with BRAF V600E. In conclusion, we have shown that MSI KRAS and BRAF mutant CRC cell lines respond differently to BRAF knockdown. This report provides evidence supporting BRAF as a good target for therapeutic intervention in patients with sporadic MSI CRC harbouring activating mutations in BRAF but not in KRAS.

Molecular pathology of familial gastric cancer, with an emphasis on hereditary diffuse gastric cancer.

Gastric cancer is one of the major causes of cancer-related death worldwide. Familial clustering is observed in about 10% of cases; 1-3% of cases are hereditary. In the latter group, a syndrome which has been well characterised is hereditary diffuse gastric cancer; this is specifically associated with CDH1 (E-cadherin) germline mutations in about 30% of families. In this article, the state of the art of familial gastric cancer regarding the clinical, molecular and pathology features is reviewed, as well as the practical aspects for a correct diagnosis and clinical management.

Characterization of the P373L E-cadherin germline missense mutation and implication for clinical management.

AIM: Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin germline mutations. One-third of these mutations are of the missense type, representing a burden in genetic counselling. A new germline missense mutation (P373L) was recently identified in a HDGC Italian family. The present work aimed at addressing the disease-causative nature of the P373L mutant. METHODS: Assessment of the P373L mutation effect was based on cell aggregation and invasion assays. LOH analysis at the E-cadherin locus, search for somatic E-cadherin mutations and for promoter hypermethylation were performed to identify the mechanism of inactivation of the E-cadherin wild-type allele in the tumour. RESULTS: In vitro the P373L germline mutation impaired the E-cadherin functions. E-cadherin promoter hypermethylation was observed in the tumour of the P373L mutation carrier. CONCLUSION: We conclude that the combination of clinical, in vitro and molecular genetic data is helpful for establishing an accurate analysis of HDGC-associated CDH1 germline missense mutations and subsequently for appropriate clinical management of asymptomatic mutation carriers.

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene.

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.

KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression.

In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS-BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS-BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Hereditary diffuse gastric cancer and E-cadherin: description of the first germline mutation in an Italian family.

AIMS: Germline mutation of the E-cadherin gene (CDH1) accounts for the Hereditary Diffuse Gastric Cancer (HDGC) syndrome. Fourteen pedigrees with Diffuse Gastric Cancer that fulfilled the International Gastric Cancer Linkage Consortium (IGCLC) criteria were selected and screened for CDH1 germline mutations. METHODS: The entire coding region of the CDH1 gene and all intron-exon boundaries were analyzed by direct sequencing in the 14 families fulfilling the IGCLC criteria. E-cadherin immunohistochemical expression was evaluated on tumour as well as normal formalin-fixed paraffin embedded tissues. RESULTS: A novel germline missense mutation was found. It was a single C-->T substitution in exon 8, resulting in a transition of CCG-->CTG (C1118T; Pro373Leu) demonstrated in the proband and her brother. At immunohistochemical analysis, the staining intensity was reduced and considered weakly positive (15%). CONCLUSIONS: The first CDH1 germline mutation of an Italian family is herein reported. The present missense mutation has never been described so far.

C/EBPbeta is over-expressed in gastric carcinogenesis and is associated with COX-2 expression.

The CCAAT/enhancer-binding protein beta (C/EBPbeta) transcription factor has been associated with several cancer models. In this study, the expression of C/EBPbeta was analysed in a series of 90 gastric carcinomas (GCs). We also assessed the effect of C/EBPbeta on COX-2 expression. In normal gastric mucosa, C/EBPbeta expression was restricted to cells in the proliferative zone. In intestinal metaplasia, dysplasia, and GC of the intestinal and atypical subtypes, C/EBPbeta was over-expressed (p < 0.0001, for the association with histological type). C/EBPbeta and Ki67, a marker of cell proliferation, were also co-expressed in primary GC. We also observed an overlap between C/EBPbeta and COX-2 expression in GC. Using GC cell lines we show that C/EBPbeta can regulate the expression of endogenous COX-2 and transactivate the promoter of the COX-2 gene, depending on its methylation status. These results suggest that C/EBPbeta may be a marker of neoplastic transformation and also play an active role in gastric tumourigenesis by regulating COX-2 expression.

H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid.

Follicular thyroid tumors are often aneuploid. It was advanced that chromosomal instability is closely associated to RAS mutations, but such association remains unproven. H-RAS can be alternatively spliced in two different proteins, p21 and p19, the former being the active protein. In order to investigate the relationship between RAS mutational status and ploidy in thyroid tumors, we analysed RAS genes in a series of 99 follicular lesions (14 nodular goiters, 70 follicular adenomas and 15 follicular carcinomas), eight thyroid carcinoma cell lines and a control group of 102 blood donors, correlating the presence of RAS mutations with the ploidy of the tumors and evaluating the two spliced forms of H-RAS. Overall, 20% of the follicular tumors harbored RAS mutations and 62% of the patients with follicular tumors (and 51% of blood donors) harbored the H-RAS 81T --> C polymorphism. The presence of RAS mutations was not associated with aneuploidy. The H-RAS polymorphism did not seem to confer a higher propensity for neoplastic transformation as it was also found in hyperplastic lesions, but was strongly associated with aneuploidy (P<0.0001). The presence of the H-RAS 81T --> C polymorphism was associated with significantly higher amounts of total H-RAS mRNA expression, higher amounts of p21 isoform and a higher fraction of neoplastic cells in S phase. Our results suggest that the H-RAS 81T --> C polymorphism may induce aneuploidy through overexpression of the active p21 isoform of H-RAS.

Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing.

BACKGROUND: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified. METHODS: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed. RESULTS: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining. CONCLUSIONS: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria.

BACKGROUND: Mutations in the E-cadherin (CDH1) gene are a well documented cause of hereditary diffuse gastric cancer (HDGC). Development of evidence based guidelines for CDH1 screening for HDGC have been complicated by its rarity, variable penetrance, and lack of founder mutations. METHODS: Forty three new gastric cancer (GC) families were ascertained from multiple sources. In 42 of these families at least one gastric cancer was pathologically confirmed to be a diffuse gastric cancer (DGC); the other family had intestinal type gastric cancers. Screening of the entire coding region of the CDH1 gene and all intron/exon boundaries was performed by bi-directional sequencing. RESULTS: Novel mutations were found in 13 of the 42 DGC families (31% overall). Twelve of these mutations occur among the 25 families with multiple cases of gastric cancer and with pathologic confirmation of diffuse gastric cancer phenotype in at least one individual under the age of 50 years. The mutations found include small insertions and deletions, splice site mutations, and three non-conservative amino acid substitutions (A298T, W409R, and R732Q). All three missense mutations conferred loss of E-cadherin function in in vitro assays. Multiple cases of breast cancers including pathologically confirmed lobular breast cancers were observed both in mutation positive and negative families. CONCLUSION: Germline truncating CDH1 mutations are found in 48% of families with multiple cases of gastric cancer and at least one documented case of DGC in an individual under 50 years of age. We recommend that these criteria be used for selecting families for CDH1 mutational analysis.