Activity Of Hedgehog Signaling Is Altered In Rectal Aberrant Crypt Foci And The Predictive Value For Colorectal Cancer

ObjectivesColorectal cancer (CRC) is one of the major causes of cancer related death in many countries including China. Among the multiple strategies for reducing the associated mortality, early detection and treatment under endoscope is currently considered most effective.Aberrant crypt foci (ACF) were described in 1987 by Bird and Good as putative preneoplastic lesions in the colon of carcinogen- treated rodents. ACF are defined by their characteristic morphology:the crypts are enlarged, they have thickened layer of epithelial cells, they have increased pericryptal space, they have irregular lumens, and they are microscopically elevated. ACF are identified in histological sections and under endoscopy after stain with methylene blue or indigo carmine. ACF have been used as a short-term bioassay to evaluate the role of nutritional components and,chemopreventive agents at an early stage of colon carcinogenesis. However, although they are thought to be the earliest identifiable neoplastic lesions in the colon carcinogenetic model, the development of these lesions is not clearly related to the early development of tumors. Therefore, there is a strong need to clarify the role of ACF in colon carcinogenesis and to validate their predictive value of rectal aberrant crypt foci (ACF) for colorectal cancer (CRC) and adenomas.The Hedgehog (Hh) signaling pathway is known to have a paramount role in the proper development of the embryo. In the past few years, it has become clear that The Hh pathway can have a crucial role in tumorigenesis when reactivated in adult tissues.There are 3 vertebrate homologues of Hh ligands:Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH). Of the three Hh-family genes in mammals, SHH has been the most studied, mainly because of the prevailing paradigm that SHH is expressed in various tissues and experiments with SHH protein are generally also applicable to other Hh homologues. However, recent studies challenge the functional redundancy of three Hh molecules by showing that IHH is not a functional homologue of SHH activity during tumor formation. SHH and IHH, required in early endoderm and several essential aspects of gastrointestinal development,seem to be the only Hh ligands expressed in the colon.Briefly, when Hh ligands bind the transmembrane receptor Patched (PTCH), PTCH-mediated suppression of Smoothened (SMO) receptor is relieved, leading to GLI entering the nucleus and activating transcription of Hh target genes. Vertebrate cells contain 3 Gli genes, Gli-1,-2 and-3. Glil, the final effector molecule of Hh signaling, activates transcription of most downstream target genes and is itself a transcriptional target of the pathway.Aberrant activation of the Hh pathway in cancers is driven by either mutations(ligand independent) or ligand overexpression (ligand dependent). Mutations of PTCH, SMO and Suppressor of Fused (SUFU) have been reported to be responsible for ligand independent activation of the pathway in basal cell carcinoma, medulloblastoma and rhabdomyosarcoma[28]. On the other hand, ligand-dependent activation of Hh pathway has been shown in a number of digestive tract malignancies, including liver cancer, pancreatic adenocarcinoma,[30] oesophageal, stomach and biliary tract cancers Moreover, rare mutations of pathway components in digestive tract tumors also suggest that the Hh pathway is activated ligand dependently in these tumors.Now, by observing the number of rectal ACF, collecting the results of colonoscopy and histology of patients, investigating the information of possible related factors for CRC, we descript the distribution and prevalence of ACF, evaluated the relationship between ACF and CRC or advanced adenoma, and screened the significant risk factors of ACF in the rectum.Much work has been done to understand the exact role of this pathway in colorectal cancer (CRC). Therefore, there is a strong need to clarify the role of ACF in colon carcinogenesis and to validate their relevance as biomarkers of tumorigenesis. Thus, understanding the roles and mechanisms of Hh signaling in ACF should give novel insights into potential treatments for this disease.Materials and Methods一,The predictive value for colorectal cancer1. PatientsFrom March 2009 to September 2009 in Nanfang Hospital, native patients who underwent colonoscopy for the first time and agreed to accept a rectal ACF quantification in vivo using high-magnification-chromoscopic-colonoscopy were recruited in our study and asked to complete a questionnaire. Those patients with severe rectitis or multiple ulcers in rectum, familial adenomatous polyposis and rectectomy were excluded.2. Data collectionThe contents of questionnaire included demographic information, sex, age, patient history of cancer or other diseases (including colorectal disease, gallstone history, appendectomy, diabetes mellitus, hypertension, coronary artery disease). Subjects with missing responses were telephoned by a researcher and the missing data were collected over the telephone. Information on the size, location, and macroscopic appearance of all polypoid lesions and CRC observed during colonoscopy was also recorded. When biopsy or polypectomy was performed, pathological findings were also recorded.3. Colonoscopy, Mucosal Staining and EvaluationBowel preparation included ingesting 2L of polyethylene glycol(PEG) solution in the morning 4 hours before examination. During colonoscopy, the rectal mucosa was washed with 50 ml of distilled water.After that,0.4% indigo carmine solution was applied to the mucosa with a spray catheter. After excess pools of indigo carmine were suctioned away, the number of ACF in the rectum was counted. This counting was conducted in the lower rectal region, extending from the middle Houston valve to the dentate line, approximately 13-15cm from the anal verge. The colonoscopic examination for detection of ACF was done using magnifying colonoscope. ACF were defined as lesions in which two or more crypts had dilated, often with oval or slit-like openings, and which were raised above the surrounding mucosa. Patients with no ACF were classified as ACF grade 0, and those with 1-4,5-9 and more than 10 were classified as ACF gradeⅠ,Ⅱ,Ⅲrespectively.4. Histologic ExaminationAll the specimens were reviewed by at least tow pathologists with special expertise in gastrointestinal pathology. Dysplasia was classified as low-grade dysplasia or high-grade dysplasia, on the basis of the degree of cytologic atypia and the presence of any architectural abnormality in the gland. Adenoma with low-grade dysplasia included adenoma cases with either mild or moderate dysplastic changes. Both adenoma with severe dysplasia and carcinoma in situ were categorized as adenoma with high-grade dysplasia. Cancer was diagnosed only if there was a clear histological demonstration of penetration of the carcinoma through the muscularis mucosa into the submucosa, which we confirmed from the histological result of resected bowel. Patients were classified by their most advanced histological lesion at the baseline colonoscopy. ACF specimens obtained were immediately fixed in 10% buffered formalin solution and were embedded in paraffin, and stained with hematoxylin and eosin (H&E). ACF were classified as nondysplastic and dysplastic.二.The activity of the Hh signaling pathway in ACF.1. 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.2. We investigated the mRNA levels of key molecules (SHH, PTCH1, Smo, GLI1 of the Hh signaling by quantitative real-time PCR in specimens.3.We also examined the protein expression of these molecules by immunostaining in ACF. Immunoreactivity was estimated semiquantitatively.4.Methylation specific PCR (MSP) We also examined the methylation status of SHH in primary colorectal tumors by methylation specific PCR (MSP). Bisulfite modified DNA was used as a template for MSP using primers specific for either the methylated or unmethylated sequences. The amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining and UV transillumination.三.Statistical AnalysisAll statistical tests were performed using the software SPSS Version 13.0 (SPSS, Chicago, IL, USA). A P-value less than 0.05 was considered statistically significant. Results for categorical variables were presented as Number (%). Two-group differences in categorical variables were determined by the x 2 or Fisher exact tests. All significance tests are two-tailed.Results1. The number of patients with colonic lesions macroscopically detected by high-magnification-chromoscopic-colonoscopy according to rectal ACF grading is shown in Table 3.96 patients were classified as ACF Grade 0,149 as Grade 1, 128 as Grade 2,128 as Grade 3.23 Grade 0 patient (24.0%) had colorectal lesions detected by colonoscopy compared with 55 (36.9%) Grade 1,55 (43.0%) Grade 2, and 79 (61.7%) Grade 3 patients.9 Grade 0 patient (9.5%) had a advanced neoplasm compared with 16 Grade 1 (16.8%),18 Grade 2(18.9%), and 52 Grade 3 (54.7%) patient. For statistical analyses, ACF was the predictors of Age, colonic advanced neoplasms and colorectal lesion. Female were less suffered from ACF than male. The prevalence of colonic advanced neoplasms in each ACF grade were significantly different from each other (P<0.001).2. Histopathologic Analysis of Aberrant Crypt Foci Of the 405 ACF biopsies taken for histopathologic analysis,54(13.3%) showed dysplasia,139 (34.3%) showed hyperplasia, and 212 (52.3%) showed neither hyperplasia nor dysplasia.3. Female is the protect factor for advanced neoplasia(P,0.002; OR,0.439;95%CI, 0.260-0.742), Age is the risk factor for advanced neoplasia (P,0.012; OR, 2.755;95%CI,1.247-6.089)。ACF is the risk factor for advanced neoplasia (P< =0.001;OR,1.866;95%CI,1.452-2.397)。4. Hh pathway members expression in normal colon and ACFSHH stained the cytoplasm in most of the dysplastic ACF, about 61.1%(33/54). In 93 (66.9%) hyperplastic ACF and 160 (75.5%) ACF with no dysplasia or hyperplasia, membranous expression, not cytoplasmic or nuclear expression, of SHH was observed. Of the 139 hyperplastic ACF,20 (14.4%) showed cytoplasmic expression(P<0.001). In the immunohistochemistry of PTCH1, the negative rate was 85.2% (46/54) in the dysplastic ACF. On the other hand, the positive rate was 61.2% (85/139) and 64.2%(136/212) in hyperplastic ACF and ACF with no dysplasia or hyperplasia respectively (P<0.001). Expression of SMO were more frequently in dysplastic ACF than in hyperplastic ACF and ACF with no dysplasia or hyperplasia (P<0.001). In dysplastic ACF 40 of 54 (74.1%) expressed SMO,whereas only 4 of 139 (2.9%) showed that in hyperplastic ACF and 1 of 212 (0.47%) in ACF with no dysplasia or hyperplasia. Immunostaining analysis revealed that GLI-1 protain was also frequently detected in dysplastic ACF, about 66.7% (36/54), compared with hyperplastic ACF and ACF with no dysplasia or hyperplasia,33.1% (46/139) and 30.7% (65/212),respectively. The GLI-1 expression levels of dysplastic ACF were significantly different from that of the others(P<0.001).5.mRNA lever of SHH is higher in d-ACF than in normal tissue,about 66.7%,and reversely in n-ACF (P<0.001). mRNA lever of SMO is higher in d-ACF than in normal tissue,about 61.1%,and reversely in n-ACF (P<0.001). mRNA lever of GLI-1 is higher in d-ACF than in normal tissue,about 72.2%,and reversely in n-ACF (P<0.001). mRNA lever of PTCH1 is lower in d-ACF than in normal tissue,about 66.7%,and reversely in n-ACF (P<0.001).6. Hypermethylation of PTCH1 promoter promoter in ACF by MSPPTCH1 hypermethylation was frequently observed in d-ACF(64.8%) and n-ACF(18.8%). (P=0.001)7. Hypermethylation of PTCH1 promoter in ACF by MS-HRMPTCH1 hypermethylation was frequently observed in d-ACF(63.3%) and n-ACF(26.7%). (P=0.009) 8.The relationship between the espression of mRNA and methylation in d-ACFPTCH1 hypermethylation was 51.9%(28/54) in d-ACF,which were low expression in mRNA (P=0.007)Conclusions1. Subjects with colon adenoma or cancer were more apt to have rectal ACF.Rectal ACF has predictive value for colonic lesions and advanced neoplasms. Man had higher mean number of rectal ACF than women.2. The ligands dependent activity of the Hh pathway is altered in d-ACF, which may play a role in the carcinogenesis of primary CRCs.3. Aberrant methylation of promoter plays a major role in the expression regulation of PTCH1 in d-ACF.