Downregulation of the hedgehog receptor PTCH1 in colorectal serrated adenocarcinomas is not caused by PTCH1 mutations

Abstract

Colorectal serrated adenocarcinoma forms about 15-20% of colorectal carcinomas. We have previously shown that downregulation of PTCH1 is distinctive for this type of colorectal cancer. In several other tumor types, somatic inactivating PTCH1 mutations have been shown to lead to aberrant Hedgehog signaling, but in colorectal cancer the role of PTCH1 mutations has not been thoroughly studied. Here, we have analyzed the mutation status of PTCH1 in a series of 33 colorectal serrated adenocarcinomas by sequencing all 23 coding exons. We detected 11 previously known SNPs and eight new alterations. The latter included five synonymous changes and two previously unknown missense variations, somatic M319V, and germline V1231A. V1231A was also present in population controls and likely represents polymorphism. The somatic M319V variant does not appear to be an attractive candidate for a disease-associated mutation because in silico analyses did not support the pathogenic nature of the change. A somatic, intronic 1-bp deletion was detected in a short poly(T) stretch in two microsatellite unstable tumors. None of the three changes had predicted effect on splicing when analyzed in silico. Our results did not reveal any clearly deleterious inactivating PTCH1 mutations in our collection of colorectal serrated adenocarcinomas. This suggests that other mechanisms are involved in the observed downregulation of the PTCH1 gene. These might include, e.g., constantly active MAPK signaling by KRAS or BRAF mutations or silencing of PTCH1 by hypermethylation, and further studies are needed to reveal these mechanisms.

Clinicopathological Features of Serrated Adenocarcinoma Defined by Mäkinen in Dukes’ B Colorectal Carcinoma

“…Tuppurainen et al [16 ]reported in 2005 that SAC was not directly related to microsatellite instability and Laiho et al [24] identified in 2007 the ephrin receptor B2, hypoxia-inducible factor 1-alpha and patched (PTCH1) as proteins important for the genesis of SAC. Stefanius et al [25,26 ]did not report any deleterious inactivating PTCH1 mutation in SAC but, in 2011, they observed that SAC is characterized by BRAF and KRAS mutations and it is conceivable that the loss of the PTCH1 protein expression may result from constant activation of the mitogen-activated protein kinase pathway, caused by either KRAS or BRAF mutations.…”

Hedgehog signaling pathway in colorectal cancer: function, mechanism, and therapy

“…The study by Stefanius et al revealed no correlation between Hh and colorectal serrated adenocarcinomas 59. Hu et al also reported that Hh signals were more frequently expressed in the microsatellite instable group, compared with the microsatellite stable group in CRC 60.…”

The Prognostic Significance of the Hedgehog Signaling Pathway in Colorectal Cancer

“…Although there were studies in which 2 independent pathologists reviewed the slides, 24,33,[37][38][39][40]43,49,53 increasing the validity of the assessment, in most of the reports this information was missing 22,23,28,36,38,42,45,47,48,51 or only 1 pathologist reviewed the specimens. 32,40 The antibodies and methodological conditions used during IHC also varied significantly between studies.…”

“…In Table 2 the antibodies used for Gli1, Shh, Smo, and Ptch IHC and the positive and negative controls are summarized. 3,[22][23][24]32,33,[36][37][38][39][40][41]43,47,48,[51][52][53] Comparisons of the different antibodies is beyond the scope of this review but the effect of different antibody concentrations in the interpretation of results as been highlighted in the past. 66 In most cases the absence of primary antibody was used as a negative control but there were cases in which the primary antibody was replaced with phosphateor Tris-buffered saline.…”

“…46 Interestingly, in a study by Stefanius et al, who aimed to determine whether the decreased expression of Ptch1 was accompanied by gene mutation, no polymorphisms or mutations in the gene were found. 47 Ptch1 and Smo were found to be expressed significantly more frequently in cancer specimens compared with HPs and adenomas. 32 Similar results were observed for Shh and Gli1 at the protein level but not for Gli1 mRNA, which was equally expressed between CRCs, HPs, and adenomas.…”