Glenn Matthews

The Wnt Antagonist sFRP1 in Colorectal Tumorigenesis

Regions of the short arm of chromosome 8 are deleted frequently in a range of solid tumors, indicating that tumor suppressor genes reside at these loci. In this study, we have examined the properties of the Wnt signaling antagonist secreted frizzled-related protein (sFRP) 1 as a candidate for this role at c8p11.2. An initial survey of 10 colorectal tumors, selected by the presence of isolated short deletions of the 8p11.2 region, identified three chain-terminating mutations, all within the first exon, which encodes the cysteine-rich domain. None of these tumors exhibited microsatellite instability, indicating intact mismatch repair gene function. The preserved sFRP1 alleles in the remaining seven tumors each contained a polymorphic three-base insertion in the signal sequence, but in a broader study, no association was found between this and the development of colorectal cancer. Epigenetic inhibition of sFRP1 transcription was investigated, and increased methylation of the promotor region was demonstrated in an additional cohort of 51 locally advanced colorectal cancers. Hypermethylation was identified in 40 of 49 (82%) cancers and in only 11 of 36 (30%) matched normal mucosal samples (P < 0.001). Semiquantitative analysis, by real-time PCR, of mRNA expression in 37 of the same cohort of 51 cancers revealed that sFRP1 mRNA expression was down-regulated in 28 (76%) cases compared with matched normal large bowel mucosa. The 3′ end of the sFRP1 mRNA also was found to be alternatively spliced, compared with the prototype liver and lung forms, in the colon and a number of other tissues, yielding an extended COOH terminus, which may influence its activity in a tissue-specific manner. The inactivation and down-regulation of sFRP1 observed are consistent with it acting as a tumor suppressor gene in colorectal carcinogenesis. Because β-catenin is constitutively active in the majority of colorectal tumors, it is unlikely that sFRP1 can act in the canonical Wnt response pathway. Therefore, we propose that the reduced activity or absence of sFRP1 allows the transduction of noncanonical Wnt signals, which contribute to tumor progression.

A role for iron in Wnt signalling

There is an emerging body of evidence implicating iron in carcinogenesis and in particular colorectal cancer, but whether this involves Wnt signalling, a major oncogenic signalling pathway has not been studied. We aimed to determine the effect of iron loading on Wnt signalling using mutant APC (Caco-2 and SW480) and wild-type APC (HEK-293 and human primary fibroblasts) containing cell lines. Elevating cellular iron levels in Caco-2 and SW480 cells caused increased Wnt signalling as indicated by increased TOPFLASH reporter activity, increased mRNA expression of two known targets, c-myc and Nkd1, and increased cellular proliferation. In contrast wild-type APC and β-catenin-containing lines, HEK 293 and human primary fibroblasts were not responsive to iron loading. This was verified in SW480 cells that no longer induced iron-mediated Wnt signalling when transfected with wild-type APC. The cell line LS174T, wild type for APC but mutant for β-catenin, was also responsive suggesting that the role of iron is to regulate β-catenin. Furthermore, we show that E-cadherin status has no influence on iron-mediated Wnt signalling. We thus speculate that excess iron could exacerbate tumorigenesis in the background of APC loss, a common finding in cancers.

Aberrant P-cadherin expression is an early event in hyperplastic and dysplastic transformation in the colon

Background: Colorectal adenomatous and, probably, hyperplastic polyp development requires epithelial remodelling and stratification, with loss of E-cadherin expression implicated in adenoma formation. We have shown that P-cadherin, normally expressed in stratified epithelia and placenta, is aberrantly expressed in disturbed epithelial architecture associated with colitis. Aims: (i) To investigate the role of P-cadherin in colonic polyp formation. (ii) To ascertain whether expression of P-cadherin is independent of or correlated with expression of its associated proteins— E-cadherin, β-catenin, and γ-catenin. (iii) To determine if P-cadherin is functional regarding catenin binding in polyps. Methods: Expression and localisation of cadherins (E- and P-) and their associated catenins (β- and γ-) were determined in aberrant crypt foci (ACF), in polyps with hyperplastic morphology (hyperplastic polyps and serrated adenomas), and in adenomatous polyps by immunohistochemistry, western blotting, and mRNA in situ hybridisation. Assessment of cadherin-catenin binding was evaluated by co-immunoprecipitation. Adenomatous polyposis coli (APC) mutation was assessed in adenomatous polyps. Results: P-cadherin was expressed from ACF through to hyperplastic and adenomatous polyps. Alterations in E-cadherin and catenin expression occurred later, with variant patterns in (i) ACF, (ii) hyperplastic polyps and serrated adenomas, and (iii) adenomatous polyps. P-cadherin present in adenomas was functional with regard to catenin binding, and its expression was independent of APC mutational status. Conclusions: P-cadherin is aberrantly expressed from the earliest morphologically identifiable stage of colonocyte transformation, prior to changes in E-cadherin, catenin, and APC expression/mutation. P-cadherin expression alone does not predict tissue morphology, and such expression is independent of that of associated cadherins and catenins.

Methylation profiling of rectal cancer identifies novel markers of early‐stage disease

Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery with transanal endoscopic microsurgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. Biomarkers associated with disease progression, particularly mesorectal nodal metastasis, are urgently required. The aim was to compare patterns of gene‐specific hypermethylation in radically excised rectal cancers with histopathological stage.

Antisense oligodeoxyribonucleotide-directed cleavage of maternal mRNA in Xenopus oocytes and embryos ☆

We have investigated the effect of specific antisense oligodeoxynucleotides (oligos) on endogenous histone H4 mRNA in Xenopus oocytes, eggs and embryos. In unfertilised eggs and non-matured oocytes, one 20-mer oligo (H4-1) mediated the RNAse H-like cleavage of up to 95% of H4 mRNA (which included polysomal mRNA), and cleavage was still obtained when the size of the oligo was reduced to a 10-mer; no cleavage was observed with 6- and 8-mers. The residual uncleaved mRNA appeared to be completely inaccessible to H4-1 since a second injection caused no further cleavage. A second 20-mer (H4-2) directed against a different region of H4 mRNA was much less effective (less than 5% cleavage). In fertilised embryos, injections of H4-1 and an oligo directed against the localised Vg1 mRNA caused less cleavage than in oocytes and also showed signs of inducing localised, non-specific mRNA cleavage. However we have been able to prepare fertilised embryos devoid of Vg1 mRNA by maturing and fertilising oligo-injected oocytes in vitro.

The Wnt antagonist sFRP1 is downregulated in premalignant large bowel adenomas

Our previous studies have implicated the Wnt antagonist, sFRP1, as a tumour suppressor gene in advanced colorectal cancer. In this study, we set out to investigate the relationship between sFRP1 expression and large bowel adenomas, a precursor of colorectal cancer. The induction of β-catenin/TCF mediated transcription is both a frequent early event in colorectal neoplasia, and a key downstream effect of wnt growth factor signalling. Lithium treatment of a small bowel mucosal cell line (FHs 74 int) induced sFRP1 within 8 h, indicating that this gene is positively regulated by β-catenin, contrasting with the suppression of sFRP1 expression, we saw previously in advanced colorectal cancers. We therefore investigated a series of 12 adenomas and matched large bowel mucosa samples. Real-time RT–PCR analysis showed a reduction in sFRP1 expression in all 12 dysplastic lesions (median 485-fold, IQR 120- to 1500-fold), indicating factors other than β-catenin influence sFRP1 levels. In a second series of 11 adenomas, we identified methylation of the sFRP1 promotor region in all 11 samples, and this was increased compared with the surrounding normal mucosa in seven cases. Immunohistochemical analysis using a polyclonal antibody supported these findings, with sFRP1 expression reduced in many of the adenoma samples examined. sFRP1 staining in normal mucosa adjacent to the dysplastic tissue was also reduced compared with the normal controls, suggesting that sFRP1 expression may be suppressed in a field of mucosa rather than in individual cells. This study identifies sFRP1 inactivation at the premalignant stage of colorectal cancer development, indicating that these pathways may be useful targets for chemoprevention strategies in this common solid tumour.

Reorganisation of Wnt-response pathways in colorectal tumorigenesis

In most colorectal tumours, APC mutation stabilises β-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of β-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca2+ receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both β-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These β-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

Biomarker-based treatment selection in early-stage rectal cancer to promote organ preservation

Total mesorectal excision (TME) remains commonplace for T1–2 rectal cancer owing to fear of undertreating a small proportion of patients with node‐positive disease. Molecular stratification may predict cancer progression. It could be used to select patients for organ‐preserving surgery if specific biomarkers were validated.

Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1–4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson “two hit” hypothesis.

Wnt signalling in adenomas of familial adenomatous polyposis patients

Background:Epigenetic silencing of Wnt antagonists and expression changes in genes associated with Wnt response pathways occur in early sporadic colorectal tumourigenesis, indicating that tumour cells are more sensitive to Wnt growth factors and respond differently. In this study, we have investigated whether similar changes occur in key markers of the Wnt response pathways in the genetic form of the disease, familial adenomatous polyposis (FAP).Methods:We investigated epigenetic and expression changes using pyrosequencing and real-time RT-PCR in samples from seven patients without neoplasia, and matched normal and tumour tissues from 22 sporadic adenoma and 14 FAP patients.Results:We found that 17 out of 24 (71%) FAP adenomas were hypermethylated at sFRP1, compared with 20 out of 22 (91%) of sporadic cases. This was reflected at the level of sFRP1 transcription, where 73% of FAP and 100% of sporadic cases were down-regulated. Increased expression levels of c-myc and FZD3 were less common in FAP (35 and 46% respectively) than sporadic tumours (78 and 67% respectively).Conclusion:Overall, the changes in expression and methylation were comparable, although the degree of change was generally lower in the FAP adenomas. Molecular heterogeneity between multiple adenomas from individual FAP patients may reflect different developmental fates for these premalignant tumours.