Ligands Dependent Activity Of Hedgehog Signaling Pathway Is Altered In Colorectal Tumors Correlating To Epigenetic Regulation Mechanism

Background and aimsThe 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.Gli1,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.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,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.Much work has been done to understand the exact role of this pathway in colorectal cancer(CRC).However,the published data on the Hh pathway activation in CRC are conflicting,and the effect of Hh pathway inhibitor on the viability of colon cancer cells is controversial.Furthermore,the details of Hh pathway regulation in CRC are still being unravelled.Thus,understanding the roles and mechanisms of Hh signaling in CRC should give novel insights into potential treatments for this disease.Epigenetic modification is one of the most important mechanisms in gene expression regulation.The most widely studied epigenetic abnormality in tumorigenesis is DNA methylation.There are a range of widely used methodologies in methylation studies,and each has its own advantages and disadvantages.The choice of proper technique is crucial in methylation analysis.However,an overall assessment for most commonly used techniques in methylation studies is lacking, especially in China.The present study was undertaken to investigate the ligands dependent activity of Hh pathway in colorectal tumors,and to attempt to clarify the regulatory mechanism of the Hh ligands expression during colorectal carcinogenesis.In addition, we assessed the commonly used methods for methylation measurement,in an attempt to provide information on methylation analysis.Materials and MethodsThe activity of the Hh signaling pathway in primary colorectal tumors.1.21 ADs and 18 CRCs were used in the study.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,IHH,GLI1) of the Hh signaling by semi-quantitative RT-PCR in tumor specimens.3.We also examined the protein expression of these molecules by immunostaining in neoplastic colonic tissues.Immunoreactivity was estimated semiquantitativelyThe activity of the Hh signaling pathway in colon cancer cell lines.The mRNA levels of key molecules of Hedgehog sinaling,including SHH,IHH, PTCH,SMO,and GLI1 were examined by semi-quantitative RT-PCR in 4 colon cancer cell lines.The regulatory mechanism of the ligands dependent activity of the Hh pathway during colon carcinogenesis.1.Demethylation treatment We screened for epigenetically silenced genes of the Hh pathway in 4 colon cancer cell lines by demethylation assay.For demethylation experiments,all the 4 colon cancer cells were treated with 10μM of 5-aza-2'-deoxycytidine(5-aza-dCyd) for 5 days.PBS-treated cells were cultured similarly and used as a control.All experiments were done in triplicates.2.Bisulfite sequencing(BSP) We investigated the methylation status of the SHH and IHH promoters in the colon cancer cell lines by BSP.Genomic DNA was extracted from the colon cancer cell lines and tissue samples.Bisulfite modification of 2μg of genomic DNA was performed.The bisulfite-modified DNA was amplified by seminested PCR.For sequence analysis,the PCR products were subcloned into the pGMT-T vector using a TA cloning kit.At least 5 clones were sequenced for each cell line and 10 clones for each primary tumor tested.3.Methylation specific PCR(MSP) We also examined the methylation status of SHH and IHH 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.The preliminary assessment of commonly used methods for methylation measurement.1.Plasmids derived from hypermethylated clone and unmethylated clone for SHH gene were served as template,to assess commonly used methods for methylation measurement,including BSP,MSP,combined bisulfite restriction analysis(COBRA) and direct sequencing of PCR products.2.BSP:The PCR products were subcloned into the pGMT-T vector using a TA cloning kit.Clones were sequenced using reverse primers.3.MSP:Variant proportion of methylated sequence was used as template,to determine the sensitivity of MSP for detecting methylated sequence.4.COBRA:Variant proportion of methylated sequence was amplified by BSP primers,followed by restriction enzyme digestion using Hha I.5.Conventional bisulfite genomie sequencing:Variant proportion of methylated sequence was amplified by BSP primers,and the products were sequenced directly using reverse primers.6.Tagged bisulfite genomie sequencing:Variant proportion of methylated sequence was amplified by tagged primers,and the products were sequenced directly using tagged primers.Statistical analysisResults for continuous variables were presented as Mean±SD.Results for categorical variables were presented as Number(%).Two-group differences in continuous variables were assessed by independent-samples T test or Mann-Whitney U-test.Two-group differences in categorical variables were determined by the x~2 or Fisher exact tests.Differences were considered significant if P＜0.05.All significance tests are two-tailed.All statistical tests were performed using the software SPSS Version 13.0.Results1.The Hh pathway is activated SHH-dependently in primary CRCs.There was no significant difference.in mRNA expression of SHH between CRCs and ADs(t＝-0.225,P＝0.823).Both IHH transcripts(Mann-Whitney U test, Z＝-3.197,P＝0.001) and GLI1 transcripts(t＝2.787,P＝0.008) were significantly decreased in CRCs compared with those in ADs.However,Protein expressions of SHH and GLI1 were increased significantly in primary CRCs compared with those in colorectal adenomas(ADs)(Mann-Whitney U test,Z＝-4.407,P＜0.001;t＝-2.213, P＝0.033,respectively).IHH was not expressed or expressed at a very low level in primary colorectal tumors,and no significant difference was found between the 2 groups(t＝0.696,P＝0.491).In addition,positive staining for IHH was frequently observed in the adjacent morphologically normal tissue of ADs.2.Absence of Hh ligands transcriptions and their induction after DNA demethylation treatment in colon cancer cell lines.SHH and IHH mRNA showed a complete absence of expression in all of the colon cancer cells,except expression of SHH in HCT8.Most components were silenced in poorly differentiated Lovo cells.After treatment with 5-aza-dCyd for 5 days,induction of SHH and IHH mRNA expression occurred in HCT8,SW1116,and SW480 cells,but not in Lovo cells.In addition,significant increase of PTCH and SMO expression was not observed after demethylation treatment.3.Promoters of SHH and IHH were hypermethylated in colon cancer cell lines.We failed to amplify BSP products from Lovo cells.In HCT8,SW1116,and SW480 cells,the 5'CpG island of SHH was densely methylated,and the island of IHH was partially methylated. 4.Hypomethylation of SHH promoter and hypermethylation of IHH promoter in primary CRCs.IHH hypermethylation was frequently observed in ADs(50.0%) and CRCs (70.6%);SHH was hypermethylated in ADs(80.0%),but hypomethylated in CRCs (23.5%,P＝0.001).5.In the range of currently widely used methodologies in methylation studies, each method has its own advantages and disadvantages.a.BSP revealed the methylation status of each CpG dinuclotide for a gene of interest.b.MSP had the advantage of high sensitivity for methylated sequences,and elevating annealing temperature overcame false positivity in MSP analysis.c.There was a bias towards the amplification of unmethylated DNA in COBRA analysis.d.Quantitative analysis by direct sequencing of the PCR products from bisulfite-treated DNA implicated several challenges:poor signal quality and overscaled cytosine signals.Conclusions1.The SHH dependent activation of the Hh pathway may play a role in the carcinogenesis of primary CRCs.2.Absence or downregulation of IHH expression may be an early event in the course of colonic tumor progression.3.Ligand dependent activity of the Hh pathway is lost in colon cancer cell lines.4.Aberrant methylation of promoter plays a major role in the expression regulation of SHH and IHH in primary colorectal tumors and colon cancer cell lines.5.In the range of currently widely used methodologies in methylation studies, each method has its own advantages and disadvantages.a.BSP can reveal the methylation status of each CpG dinuclotide.Nevertheless, this technique is relatively expensive and elaborate.b.MSP had the advantage of high sensitivity for methylated sequences,but the annealing temperature should be optimized to overcome false positivity. c.There was a bias towards the amplification of unmethylated DNA in COBRA analysis,although this technique is relatively easy to perform.d.Direct sequencing of the PCR products from bisulfite-treated DNA implied a potential high-throughput sequencing,but this technology need to be improved according to our experience.