Role And Mechanism Of Wnt Signalling In Serrated Pathway Of Colorectal Carcinogenesis

Background and aimsColorectal cancer (CRC) is one of the most commonly occurring malignancies. A well-defined pathogenic pathway, the adenoma-adenocarcinoma sequence, is the predominant pathogenic pathway in colorectal tumorigenesis,and accounts for upwards of two thirds of all CRCs. Recently, an alternative "serrated pathway"of colorectal carcinogenesis with a hyperplastic polyp (HP)-serrated adenoma (SA)-adenocarcinoma sequence has been proposed, which may explain at least 7.5% of all and up to 17.5% of proximal CRCs. The propose of the serrated pathway of colorectal carcinogenesis have overthrew the view that HPs are non-neoplastic lesions, and raised new issues for the prevention and treatment of CRCs, also provide new clues for the pathogenesis of CRCs.The serrated polyps including:HP(hyperplastic polyp),SSA(Sessile serrated adenoma),TSA(Traditional serrated adenoma). The concept of SSA was recently introduced, representing a morphological intermediate between HP and TSA. More and more evidences indicate that SSA might be the precursor lesion for some cases of microsatellite unstable colorectal carcinoma. The diagnosis and differential diagnosis of SSAs are still unclear, lacking uniform standards. It is worth noting that a considerable part of which diagnosed SSAs before may be HPs. It is still difficult to distinguish HPs from SSAs.SSAs are now treated as adenomas for their malignant potential. However, a thorough study and a clear understanding on SAs is still lacking in China.So a preliminary study on the diagnosis and differential diagnosis of SAs seems very important.Although the concept of SA has been generally accepted, there have been several reports on SAs, the result is still conflicting, Whether the canceration of SAs the same as TAs is still unclear. It has been widely accepted that activation of the Wnt signalling pathway is a key early event involved in the multi-step process of colorectal tumorigenesis, but the role in SAs is not clear. Recently, some studies have reported the action of Wnt signalling in SAs,but the result is opposite.Some found that APC mutation andβ-catenin nuclear expression is frequent in SAs,while others reported that Wnt signalling is inactivated in SAs So further study is needed to make clear on the role of Wnt signalling in SAs.Aberrant activation of the Wnt signalling pathway in CRC occurs almost invariably through mutation of the two key regulators:adenomatous polyposis coli (APC) andβ-catenin. Up to 80% of CRCs have APC mutations, whereas 10% of CRCs haveβ-catenin mutations. However, it is reported that infrequent APC mutation has been reported in SAs.,only 3.8%(1/26). It implys that gene mutation may not play a key role on Wnt signalling in SAs. CpG island methylator phenotype, which characterized by a wide range of promoter methylation,plays an important role in the "serrated pathway"of colorectal carcinogenesis. Many reaserchers thought DNA methylation may provide an alternative mechanism to gene mutation for silencing APC, and could bridge the mutational gap. We can conlude from it that high frequency of DNA methylation may be the main reason of the reduction of APC expression.There still have no reports on the APC methylation status in SAs. Make sure that the two processes of cell adhesion and nuclear signaling is tightly coordinated and interrelated important for cell proliferation and differentiation. The key regulator of Wnt signalling,β-catenin, plays both a critical structural role in cadherin-based adhesions and also an essential activator of gene transcription. The control of the dual functions ofβ-catenin in cellular adhesion and in transcriptional regulation is crucial for maintaining normal cellular function,either function of P-catenin de-regulated will result in activation of Wnt sigalling,then will lead to human malignancies. Recently, some reasearchers found that the cytoplasm expression ofβ-catenin is infrequent in SAs than in TAs, and no nuclear expression was found in SAs. It implies us thatβ-catenin assembles with a-catenin and E-cadherin into adhension complexes without acting in nuclear transcription in SAs. Whetherβ-catenin that favors adhesion function rather than the function in transcription in SAs still needs further study.The object and significance of this article is that:first, preliminary study on the diagnosis and differential diagnosis of SAs, to guide clinical decision-making and avoid misdiagnosis; second, to make sure whether Wnt signalling acts in SAs, in order to understand the molecular basis deeply; third, to reveal the molecular mechanism of Wnt signaling activation in serrated pathway; forth, to reveal the adhension and transcription function ofβ-catenin in serrated pathogenesis, which will provide theoretical basis for clinical prevention and treatment.Materials and Methods:1. Sample collection: according to the diagnostic criteria of Snover etal in 2005, retrospectively analysis 97 paraffin serrated polyps specimen(includig 32 HP samples, 28 SSA samples,37 TSA samples) archived in our pathology department, and collect 51 fresh TA samples and 10 fresh normal colorectal tissue from patients who did 111 endoscopy polypectomy and polypectomy in our endoscopy department from February 2007 to July 2009. Among the 28 SSA sections,3 samples are mixed SSA-HP, among the 37 TSA sections,5 samples are mixed TSA-SSA. Each specimen was divided into two parts:one was frozen immediately in liquid nitrogen and stored at -80℃until required, the other was fixed in 10% formalin and embedded in paraffin. Paraffin specimen are maintained at room temperature. Clinical pathological datas were got from Nanfang Hospital cases, and informed consent was received from all patients.2,HE staining and immunohistochemistry:Each fresh sample was divided into two parts, one part was fixed in 10% formalin and embedded in paraffin, then all formalin specimen sliced,HE staining,immunohistochemistry and observed under microscope. Immunohistochemistry using two-step staining, heated-repairing, citrate antigen retrieval solution, specimens without adding antigen were used as an negative control, and specimens which known antigen were used as a positive control, thus allowing direct comparison within the same tissue specimen, then detected the expression of APC,β-catenin,α-catenin,E-cadherin in HPs,SSAs,TSAs and control groups of TAs,NCs.3. DNA extraction and PCR:According to the fresh tissue genomic DNA Kit manual provided by TIANGEN company, we extracted DNA from 10 NCs and 51 TAs. According to EZNA FFPE DNA Kit of OMEGA company, we extracted DNA from 32 HPs,28 SSAs,37 TSAs paraffin specimen. All DNA samples were preserved in -20℃. According to exon 15 of APC MCR region and exon 3 ofβ-catenin mutation region design primers using Primer 5.0 software. PCR amplification DNA section, amplified products by 2.5% agarosegelelectrophoresis to ensure that the ob servation fragment was successfullyamplifed, after succeeded amplified, purify the PCR production(using TIANGEN quick Midi Purification Kit),then send to company for sequencing. Assessment genotype with chromas 2.0 and lastcontrasted the gene bank data to find abnormal sequence, if we find mutation, then re-amplify the fragment and sequencing from both directions, then statistical analysis each mutation sample. Table 2-1 The primers sequence of APC exon 15 (MCR region) and (3-catenin exon 3 Statistical analysis:Data are presented as mean and standard deviation for continuous variables and as proportions for categorical variables. Data were analysed using one-way ANOVA, followed by Bonferroni test for multiple comparisons. Differences in categorical variables were determined by the Chi-square or Fisher's exact tests, as appropriate. Differences were considered significant if P<0.05. All significance tests were two-tailed. All statistical tests were performed using SPSS software Version 13.0 (SPSS Inc, Chicago, IL, USA).Results:1.Clinical and pathological features:According to our practial diagnosis and combines with foreign literature, initially identified the differential diagnosis of all kinds of serrated polyps. SSAs differ from HPs in:1.mitotic activity increases in the upper and middle part of crypts; 2. most nucleus of the superficial parts of crypts is round or oval; 3.the serrated structure starts from the base of the crypts; 4.other features:have flat crypts, branch crypts, inverted crypts; 5.50% of the crypts are immature; 6. other feature of abnormal mature cell:goblet cells can be seen at the base of crypts, goblet cells which on superficial parts of the crypts are outstretched and full of mucus. TSAs differ from SSAs in:1. stained nuclear; 2. stretched nuclear; 3. layered nuclear; 4. prominent nucleolus; 5. strong eosinophilic cytoplasm.2. APC,β-catenin, a-catenin and E-cadherin expression in HPs,SSAs,TSAs,TAs and NCs.Expression of E-cadherin,α-,β-catenin and APC was clearly evident at the cell-cell boundaries of all 10 normal colonic tissues and normal mucosa adjacent to tumors. However, membranous expression of E-cadherin,a-catenin and APC was only seen in 33.3%(17/51),27.5%(14/51) and 31.4%(16/51) of all investigated TAs, respectively.Membranous expression of beta-catenin was demonstrated in 29.4%(15/51) of TAs. Cytoplasmic accumulation and widespread or focal nuclear expression of beta-catenin was observed in 60.8%(31/51) and 19.6%(10/51) of all TAs, respectively. Mesenchymal tissue surrounding the epithelial cells did not express E-cadherin or any of the catenins.The frequence of E-cadherin membranous expression in HPs,SSAs and TSAs was significantly higher than that of TAs (33.3%,17/51, P=0.000, x2=5.029).The frequence of a-catenin membranous expression in HPs, SSAs, and TSAs was significantly higher than that of TAs(27.5%,14/51,P=0.000,χ2=56.568).The rate of APC membranous expression in HPs,SSAs and TSAs was significantly higher than that of TAs,too (31.4%,16/51, P=0.000,χ2=69.240).The frequence ofβ-cadherin membranous expression in HPs, SSAs and TSAs was significantly higher than that of TAs(29.4%, P<0.001). The rate of cytoplasmic accumulation of P-catenin in HPs, SSAs, and TSAs was significantly lower than that in TAs (60.8%, P<0.001). Widespread or focal nuclear staining for beta-catenin was observed in 19.6%(10/51) of TAs, while none of the HPs, SSAs, or TSAs showed nuclear labeling for beta-catenin (P<0.001). 3. APC andβ-catenin mutationThe result of APC andβ-catenin mutation is that:APC mutation was detected in only one TSA. The frequence of APC mutation in HPs, SSAs, and TSAs was significantly lower than that in TAs(51.0%,26/51,P=0.000,χ2=51.003). None ofβ-catenin mutation was observed in HPs, SSAs, TSAs and TAs.Conclusion:1. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys, the tumorigenesis of the SAs is distinct from that of TAs.2. P-catenin participates in adhesion function in serrated pathway rather than the function in Wnt signaling activation.3. Gene mutation can not be the reason that the moderate activation of Wnt signaling in serrated pathway, maybe there is another alternative mechanism.