Sima Salahshor

The links between axin and carcinogenesis

The products of the two mammalian Axin genes (Axin1 and its homologue Axin2) are essential for the degradation of β catenin, a component of Wnt signalling that is frequently dysregulated in cancer cells. Axin is a multidomain scaffold protein that has many functions in biological signalling pathways. Overexpression of axin results in axis duplication in mouse embryos. Wnt signalling activity determines dorsal–ventral axis formation in vertebrates, implicating axin as a negative regulator of this signalling pathway. In addition, Wnts modulate pattern formation and the morphogenesis of most organs by influencing and controlling cell proliferation, motility, and fate. Defects in different components of the Wnt signalling pathway promote tumorigenesis and tumour progression. Recent biochemical studies of axins indicate that these molecules are the primary limiting components of this pathway. This review explores the intriguing connections between defects in axin function and human diseases.

Colorectal cancer with and without microsatellite instability involves different genes

There is evidence supporting a multistep genetic model for colorectal tumorigenesis. In familial adenomatosis polyposis (FAP), the inherited defect is a mutation in the APC gene. The vast majority of all sporadic colorectal cancers also show mutations in the APC gene, and the tumorigenesis in sporadic colorectal cancer and FAP is assumed to involve the same genes. Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes and, as a result of defective mismatch repair, microsatellite instability (MSI) is frequently seen. Tumorigenesis in HNPCC was first thought to involve mutations in the same genes as in FAP and sporadic colorectal cancer. Recently, however, an alternative pathway to development of colorectal cancer has been suggested in colorectal tumors with MSI, compared to those tumors without the MSI phenotype. We used a consecutive series of 191 sporadic colorectal cancers to find out if there were any differences between the two groups of tumors regarding the prevalence of mutations in the APC, KRAS, TP53, and TGFβR2 genes. As expected, 86% (19/22) of MSI‐positive tumors showed a mutation in TGFβR2, while only one of 164 (0.6%) MSI‐negative tumors did. A highly statistically significant negative association was found between MSI and alterations in APC and TP53. The MSI‐positive tumors were screened for mutations in exon 3 of β‐catenin, which has been suggested to substitute for the APC mutation in the genesis of colorectal cancer, without finding mutations in any of the 22 MSI‐positive tumors. The number of mutations found in KRAS was lower in MSI‐positive than in MSI‐negative tumors but the difference was not statistically significant. Our results strongly support the idea that carcinogenesis in MSI‐positive and MSI‐negative colorectal cancer develops through different pathways. Genes Chromosomes Cancer 26:247–252, 1999.

Microsatellite instability in sporadic colorectal cancer is not an independent prognostic factor

SummaryHereditary non-polyposis colorectal cancer (HNPCC) is linked to an inherited defect in the DNA mismatch repair system. DNA from HNPCC tumours shows microsatellite instability (MSI). It has been reported that HNPCC patients have a better prognosis than patients with sporadic colorectal cancer. We examined whether the presence of MSI in a series of unselected colorectal tumours carries prognostic information. In a series of 181 unselected colorectal tumours, 22 tumours (12%) showed MSI. Survival analysis at 5–10 years follow-up showed no statistically significant difference in prognosis between MSI-positive and -negative tumours. Our results suggest that the MSI phenotype as such is not an independent prognostic factor.

Microsatellite Instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer.

Immunohistochemical expression analysis of mismatch repair gene products has been suggested for the prediction of hereditary nonpolyposis colorectal cancer (HNPCC) carrier status in cancer families and the selection of microsatellite instability (MSI)-positive tumors in sporadic colorectal cancer. In this study, we aimed to evaluate hMSH2 and hMLH1 immunohistochemistry in familial and sporadic colorectal cancer. We found that immunohistochemistry allowed us to identify patients with germline mutations in hMSH2 and many cases with germline mutations in hMLH1. However, some missense and truncating mutations may be missed. In addition, hMLH1 promoter methylation, commonly occurring in familial and sporadic MSI-positive colorectal cancer, can complicate the interpretation of immunohistochemical expression analyses. Our results suggest that immunohistochemistry cannot replace testing for MSI to predict HNPCC carrier status or identify MSI-positive sporadic colorectal cancer.

CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk

Mutations and diminished expression of the E‐cadherin gene (CDH1) have been identified in a number of epithelial malignancies. Although somatic CDH1 mutations were detected in lobular breast cancer with a frequency ranging from 10–56%, CDH1 alterations in more frequent ductal tumors appear to be rare. Here we have analyzed the coding region of CDH1 for mutations using denaturing high performance liquid chromatography and found 4 mutations in 83 ductal carcinomas (5%) and 3 mutations in 25 lobular carcinomas (12%). The germline of 13 patients with familial lobular tumors was also analyzed for mutations, but none were detected. In a case‐control study, we also tested whether a variant adenine allele in the promoter polymorphism −161C→A with a putative influence on the transcriptional activity of CDH1 in vitro confers any detectable risk of breast cancer. No significant difference in the allelic frequency between patients with breast cancer (326/1,152, 28.3%) and controls (190/696, 27.3%, p > 0.05; relative risk 1.05, 95% confidence interval 0.85–1.30) was found. A novel promoter polymorphism was identified at position −152, but the frequency of the variant cytosine allele was also similar in patients with breast cancer and controls (0.71% vs. 0.21%, p = 0.23). Transient transfection experiments using reporter constructs containing the nucleotide substitutions −161C/−152C and −161A/−152T showed only a slight decrease in the transcription activity compared to the wild‐type construct. These results do not support CDH1 as a prominent low‐penetrance cancer susceptibility gene, but indicate that CDH1 mutations contribute to the progression of both lobular and ductal tumors.

Frequent accumulation of nuclear E-cadherin and alterations in the Wnt signaling pathway in esophageal squamous cell carcinomas

Esophageal squamous cell carcinoma is frequently associated with poor prognosis, as a result of high levels of lymph node metastasis. So far, very few genetic abnormalities have been associated with this disease, and its molecular etiology remains largely unknown. To assess whether the Wnt pathway contributes to esophageal squamous cell carcinoma, we characterized the expression and subcellular localization of the key Wnt signaling components in all 30 cases of esophageal squamous cell carcinomas analyzed. We found abnormal expression and/or localization in glycogen synthase kinase-3 α/β (34%), Axin2 (48%), α-catenin (31%), MYC (73%) and cyclin D1 in 46% of cases. Only 13% of tumors showed nuclear accumulation of β-catenin. By contrast, 60% showed nuclear expression of E-cadherin using an antibody that recognizes the cytoplasmic domain of E-cadherin. When the same tumors were stained with antibody raised against the extracellular domain of E-cadherin, the expression was lost. A direct correlation was found between nuclear E-cadherin and the increased nuclear cyclin D1, one of the AP-1 target genes in these tumors. By transfection experiments, the cytoplasmic portion of E-cadherin was found to activate the AP-1 transcription factor pathway and induced cyclin D1 promoter activity, but β-catenin/Tcf transcription activity was unaffected. Nuclear expression of E-cadherin was also detected in tumors other than squamous cell carcinoma, including pancreatic and colon cancers, albeit at lower frequency. Nuclear accumulation of a portion of E-cadherin in esophageal squamous cell carcinoma and the other types of tumors indicates that, in addition to the previously implicated tumor suppressor activity of E-cadherin, modified forms of this glycoprotein might also play a role in growth promotion.

Low frequency of E-cadherin alterations in familial breast cancer

In order to explore the possible role of E-cadherin in familial cancer, 19 familial breast cancer patients, whose tumours demonstrated loss of heterozygosity (LOH) at the E-cadherin locus, were screened for germline mutations. No pathogenic germline alterations were detected in these individuals. However, a somatic mutation was found (49-2A→C) in one of the tumours. This tumour showed a pattern of both ductal and lobular histology. Another 10 families with cases of breast, gastric and colon cancer were also screened for germline mutations, and no mutations were found. A missense mutation in exon 12 of E-cadherin (1774G→A; Ala592Thr) was previously found in one family with diffuse gastric cancer, and colon and breast cancer. An allelic association study was performed to determine whether the Ala592Thr alteration predisposes to breast cancer. In total, we studied 484 familial breast cancer patients, 614 sporadic breast cancer patients and 497 control individuals. The frequencies of this alteration were similar in these groups. However, a correlation between the Ala592Thr alteration and ductal comedo-type tumour was seen. These results, together with previously reported studies, indicate that germline mutations and, more commonly, somatic mutations in E-cadherin may have an influence on the behaviour of the tumours, rather than predispose to breast cancer.

COL11A1 in FAP polyps and in sporadic colorectal tumors

BackgroundWe previously reported that the α-1 chain of type 11 collagen (COL11A1), not normally expressed in the colon, was up-regulated in stromal fibroblasts in most sporadic colorectal carcinomas. Patients with germline mutations in the APC gene show, besides colonic polyposis, symptoms of stromal fibroblast involvement, which could be related to COL11A1 expression. Most colorectal carcinomas are suggested to be a result of an activated Wnt- pathway, most often involving an inactivation of the APC gene or activation of β-catenin.MethodsWe used normal and polyp tissue samples from one FAP patient and a set of 37 sporadic colorectal carcinomas to find out if the up-regulation of COL11A1 was associated with an active APC/β-catenin pathway.ResultsIn this study we found a statistically significant difference in COL11A1 expression between normal tissue and adenomas from one FAP patient, and all adenomas gave evidence for an active APC/β-catenin pathway. An active Wnt pathway has been suggested to involve stromal expression of WISP-1. We found a strong correlation between WISP-1 and COL11A1 expression in sporadic carcinomas.ConclusionsOur results suggest that expression of COL11A1 in colorectal tumors could be associated with the APC/β-catenin pathway in FAP and sporadic colorectal cancer.

Differential gene expression profile reveals deregulation of pregnancy specific β1 glycoprotein 9 early during colorectal carcinogenesis

BackgroundAPC (Adenomatous polyposis coli) plays an important role in the pathogenesis of both familial and sporadic colorectal cancer. Patients carrying germline APC mutations develop multiple colonic adenomas at younger age and higher frequency than non-carrier cases which indicates that silencing of one APC allele may be sufficient to initiate the transformation process.MethodsTo elucidate the biological dysregulation underlying adenoma formation we examined global gene expression profiles of adenomas and corresponding normal mucosa from an FAP patient. Differential expression of the most significant gene identified in this study was further validated by mRNA in situ hybridization, reverse transcriptase PCR and Northern blotting in different sets of adenomas, tumours and cancer cell lines.ResultsEighty four genes were differentially expressed between all adenomas and corresponding normal mucosa, while only seven genes showed differential expression within the adenomas. The first group included pregnancy specific β-1 glycoprotein 9 (PSG9) (p < 0.006). PSG9 is a member of the carcinoembryonic antigen (CEA)/PSG family and is produced at high levels during pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18) of FAP adenomas and 75% (45/60) of sporadic colorectal cancer cases tested.ConclusionDetection of PSG9 expression in adenomas, and at higher levels in FAP cases, indicates that germline APC mutations and defects in Wnt signalling modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult except in association with cancer, and it appears to be an early molecular event associated with colorectal cancer monitoring of its expression may be useful as a biomarker for the early detection of this disease.

Nuclear expression of E-cadherin.

To the Editor: We read the paper by El-Bahrawy and colleagues in the January 2008 issue of The American Journal of Surgical Pathology with great interest and wish to share our findings of E-cadherin nuclear expression in solid pseudopapillary tumors (SPTs) of the pancreas with your readers. It is heartening that an independent group has corroborated nuclear E-cadherin expression in SPT, following our demonstration of this finding published in early 2007. Our study consisted of 18 cases of SPT in which there was consistent loss of membrane staining but nuclear immunoexpression of E-cadherin. Parenthetically, we have expanded our series to 30 cases and have observed nuclear E-cadherin in all cases of SPT. Tang and colleagues have also observed complete loss of membrane staining for E-cadherin in 9 cases of SPT, although they did not observe and/or comment on nuclear decoration in their cases. We note that the E-cadherin antibody used in the study by ElBahrawy et al is from Transduction Laboratories. Although the clone of the antibody is not mentioned in the paper, we assume that it is clone 36, a mouse monoclonal antibody that recognizes the cytoplasmic domain of the E-cadherin molecule. In our study that demonstrated nuclear expression of E-cadherin in SPT of the pancreas, we used the aforementioned clone from Transduction Laboratories, Franklin Lake, New Jersey. In a subsequent study of 20 cases of SPT, we explored the expression of E-cadherin using 2 monoclonal antibodies to E-cadherin: clone 36 directed against the cytoplasmic domain of E-cadherin, and an antibody from Vector Laboratories, Burlingame, CA, clone 36B5 that recognizes the extracellular portion of the E-cadherin molecule. Interestingly, the antibody that detects the extracellular domain of E-cadherin (clone 36B5), does not stain any of the cases of SPT, in other words, there is no membrane or nuclear immunoexpression of E-cadherin. Thus, the immunohistochemical staining pattern of E-cadherin in SPT of the pancreas is dependent on the type of antibody used: nuclear staining is only obtained with the antibody that recognizes the cytoplasmic fragment of E-cadherin. We also note that only 1 of 15 neuroendocrine tumors of the pancreas stained by El-Bahrawy and colleagues yielded nuclear positivity. We have also conducted an analysis of pancreatic neuroendocrine tumors and E-cadherin. In our hands, 34 of 57 primary neuroendocrine cases showed loss of membrane expression of E-cadherin, and 18 of these cases also showed nuclear expression with the antibody recognizing the cytoplasmic fragment of the E-cadherin molecule. Again, we and others have also explored E-cadherin expression in other pancreatic tumors and nonpancreatic cancers. Nuclear E-cadherin staining was noted in other tumors, but at a considerably lower level than SPT of the pancreas. In an effort to explain why the cytoplasmic tail of E-cadherin might translocate to the nucleus, Salahshor and colleagues generated 2 truncated E-cadherin constructs, which were then transfected into HEK293 cell lines. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, this study demonstrated that the intact cytoplasmic domain of E-cadherin is transported into the nucleus. It is also intriguing to note that none of the cases examined by ElBahrawy et al showed mutations in the E-cadherin gene. The implication of this being that the aberrant E-cadherin protein expression is not related to abnormalities of the gene. We have also examined the role of p120, a molecule which is thought to be a regulator of E-cadherin. All 29 cases in that series showed aberrant localization of p120, thus suggesting that p120 may be the effector of Ecadherin abnormalities in SPT. It is clearly apparent that nuclear E-cadherin is not the exclusive preserve of SPT of the pancreas, but we do concur with El-Bahrawy and colleagues that 100% of cases demonstrate this unique staining property. However, nuclear E-cadherin is also seen in pancreatic neuroendocrine tumors, pancreatic ductal adenocarcinomas, renal cell, esophageal squamous, and colorectal cancers. As awareness regarding nuclear localization of E-cadherin increases, more cancers with this staining pattern will emerge. Although the exact mechanism(s) for nuclear translocation of E-cadherin is not well elucidated at this juncture, it is clear that it is the cytoplasmic domain of the molecule that has a nuclear locale. It is also quite likely that p120 abnormalities are pivotal in the causation of E-cadherin abnormalities.