The Apc 1322T mouse develops severe polyposis associated with submaximal nuclear beta-catenin expression.

BACKGROUND & AIMS: We previously demonstrated that the 2 APC mutations in human colorectal tumors are coselected, because tumorigenesis requires an optimal level of Wnt signaling. We and others subsequently showed that the truncated APC proteins in colorectal tumors usually retain a total of 1-2 beta-catenin binding/degradation repeats (20AARs); very few intestinal tumors have proteins with no 20AARs. The coselection of the "2 hits" at APC makes it difficult to undertake further mechanistic studies in this area in humans. In mice, however, second hits appear to vary with the strain or genetic background used. This suggested the possibility of creating suboptimal Apc genotypes in the mouse. METHODS: We have constructed a mouse, Apc(1322T), with a mutant protein retaining one 20AAR. After repeated backcrossing to the C57BL/6J background, we compared the 1322T animals with the widely used Min mouse in which the mutant Apc protein has zero 20AARs. RESULTS: In both mice, intestinal adenomas showed copy-neutral loss of heterozygosity, making them homozygous for the mutant Apc allele. 1322T animals had markedly more severe polyposis, with earlier-onset, larger, more numerous, and more severely dysplastic adenomas. 1322T tumors also had more marked Paneth cell differentiation and higher frequencies of crypt fission. Somewhat surprisingly, nuclear beta-catenin expression was lower in 1322T than Min tumors. CONCLUSIONS: We propose that the Apc(1322T) mutation produces submaximal beta-catenin levels that promote early tumor growth more effectively than the Apc(Min) mutation.

Apc mice: models, modifiers and mutants.

The mouse provides an excellent in vivo system with which to model human diseases and to test therapies. Mutations in the Adenomatous polyposis coli (APC) gene are required to initiate familial adenomatous polyposis (FAP) and are also important in sporadic colorectal cancer tumorigenesis. The (multiple intestinal neoplasia Min) mouse contains a point mutation in the Apc gene, develops numerous adenomas and was the first model used to study the involvement of the Apc gene in intestinal tumorigenesis. The model has provided examples of modifying loci (called Modifiers of Min: Mom) in mice, demonstrating the principle of genetic modulation of disease severity. A spectrum of Apc mutant mice has since been developed, each with defining characteristics, some more able to accurately model human polyposis and colon cancer. We will focus our review on Apc mutant mouse models, the advent of models with concurrent or compound mutations and the importance of genetic background when modeling polyposis and cancer. Brief consideration will be given to the use of these models in drug testing.

Pregnancy does not influence colonic polyp multiplicity but may modulate upper gastrointestinal disease in patients with FAP.

BACKGROUND: Reproductive factors have been shown by epidemiology studies to alter colorectal cancer risk in women. Familial adenomatous polyposis (FAP) patients carry a germline adenomatous polyposis coli (APC) mutation predisposing to multiple adenoma formation in the intestine. The Min mouse provides a good model of FAP, and we recently reported a significant increase in intestinal tumour multiplicity in a recombinant line of mice following pregnancy. AIM: We considered whether reproduction modulates intestinal tract disease in a large cohort of female patients with FAP (n = 180). RESULTS: Multiple regression analysis showed that the number of colonic polyps observed was not related to the person's pregnancy status nor the position of their APC germline mutation. The proportion of women attaining a high Spigelman stage (3 or 4) was unrelated to having a pregnancy prior to attaining the maximum Spigelman stage (p = 0.6). On the other hand, having a pregnancy significantly increased the proportion of women that attained the highest Spigelman stage when their APC germline mutation occurred within the mutation cluster region or at or after codon 1020 (50%, 6/12, p = 0.005 and 42%, 13/31, p = 0.006, respectively; multivariable logistic regression). CONCLUSION: Our data suggest that reproduction may influence disease severity in the upper gastrointestinal tract in patients with FAP.

Activation of AKT and nuclear accumulation of wild type TP53 and MDM2 in anal squamous cell carcinoma.

Human papilloma virus (HPV) infection is considered as an important aetiological factor for anal squamous cell carcinoma (ASCC) but is not sufficient for tumour progression. This carcinoma is poorly understood at the molecular level. Using the largest cohort of cases to date we investigated the molecular mechanisms underlying ASCC development, in particular the roles of TP53, MDM2 and AKT. Viral infection in our cohort occurred at high frequency (73%, 94/128) with HPV16 accounting for the majority (86%, 81/94) of infected cases. Only 4% (5/119) of ASCCs showed TP53 (exons 5-8) mutations, but a high frequency (91%, 100/110) of nuclear protein expression of TP53 was observed. There was a significant association (p < 0.001) between nuclear accumulation of TP53 and MDM2 protein although no MDM2 mutations were found, and copy number was normal. Cellular accumulation of phosphorylated-AKT was observed in 66% (82/125) of ASCCs and an association demonstrated between nuclear accumulation of MDM2 and activated AKT (p < 0.001). We observed a high frequency of copy number gain at PIK3CA (47%), and some coding sequence mutations (4%). Amplification of PIK3CA was associated with presence of phosphorylated-AKT (p= 0.008). There was no association between virus infection and TP53 nuclear accumulation (p = 0.5). However, a significant association was found between infection and MDM2 nuclear staining, and between infection and activated AKT (p = 0.04, p = 0.01, respectively). We propose that activation of AKT, possibly through the PI3K-AKT pathway, is an important component of ASCC tumorigenesis that contributes to MDM2 and TP53 accumulation in the nucleus.

A novel exon duplication event leading to a truncating germ-line mutation of the APC gene in a familial adenomatous polyposis family.

Familial Adenomatous Polyposis (FAP) is an autosomal dominant condition predisposing to multiple adenomatous polyps of the colon. FAP patients frequently carry heterozygous mutations of the APC tumour suppressor gene. Affected individuals from a cohort of FAP families (n=22), where no germ-line APC mutation was detected by direct sequencing, were analysed by Multiplex Ligation-dependent Probe Amplification (MLPA). MLPA identified a previously unreported APC mutation involving duplication of exon 4. Subsequent analysis of cDNA from affected family members revealed expression of mutant mRNA species containing two copies of exon 4, resulting in a frameshift and premature stop codon. Bioinformatic analysis of the relevant APC genomic segment predicted a role for homologous recombination possibly involving Alu repeats in the generation of this genotype. Our results highlight the importance of MLPA as an adjunct to exon-by-exon sequencing in identifying infrequent mutational events in cancer predisposing genes.

The stem cell marker CD133 associates with enhanced colony formation and cell motility in colorectal cancer.

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133-expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause "off-target" effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133- populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133- populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133- population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133- population of SW480. Prolonged culture of a pure CD133- population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene.

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.