Identification and Validation of Potentially Clinically Relevant CpG Regions within the Class 2 Tumor Suppressor Gene SFRP1 in Pancreatic Cancer

Abstract

In pancreatic cancer treatment, tumor stage-dependent chemotherapies are used to prolong overall survival. By measuring DNA promoter hypermethylation in the plasma of patients with stage IV pancreatic cancer, it was recently shown that promoter DNA methylation of the tumor suppressor gene SFRP1 has a high value for predicting failure of drug treatment with gemcitabine. In this study, we therefore aimed to identify as precisely as possible the region in the SFRP1 promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the SFRP1 transcription start site that were significantly more methylated in pancreatic cancer compared to normal pancreatic acinar tissue. A core CpG island was identified that exhibited abundant tumor DNA methylation and anti-correlation of SFRP1 mRNA expression. To validate our in silico results, we performed bisulfide conversion followed by DNA pyrosequencing of 28 matched formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer cases and six pancreatic cancer cell lines. A defined block of seven CpG sites within the core CpG island was identified, which confirmed our in silico results by showing significantly higher SFRP1 methylation in pancreatic cancer specimens than in normal pancreatic tissue. By selecting this core CpG island, we were able to determine a median overall survival benefit for the low SFRP1 methylation group compared to the high SFRP1 methylation group (702 versus 517 days, p = 0.01) in the TCGA pancreatic cancer cohort. We propose a compact pyrosequencing assay that can be used in the future to further investigate the prognostic value of SFRP1 promoter hypermethylation in predicting pancreatic cancer chemoresistance. Therefore, instead of DNA analysis from blood (liquid biopsy), DNA easily extractable from cancer tissue blocks (FFPE material) could be used.

ITIH5 as a multifaceted player in pancreatic cancer suppression, impairing tyrosine kinase signaling, cell adhesion and migration

“…Subsequently to the analysis of the TCGA cohort, we used a recently described PDAC patient cohort from our hospital [ 38 ] and analyzed it for ITIH5 promoter hypermethylation. The cohort included matched patient tissues with 28 normal, 28 tumor, and 7 metastasis samples.…”

“…For FFPE patient cohort, a previously described study population from Aachen was analyzed [ 38 ], including FFPE tissue samples of 28 PDAC patients with corresponding adherent normal acinar pancreas tissues and seven corresponding metastases. Briefly, as prior described in [ 38 ], 2 μm sections were prepared for each tissue sample and stained with hematoxylin and eosin (HE) and assessed by an experienced pathologist to highlight relevant areas of the tissues.…”

“…Sections of FFPE tissue were processed as previously described [ 38 ]. Genomic DNA from FFPE tissue and from cell lines was isolated using the QIAamp ® DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.…”

“…Total cellular RNA from cultured cells was extracted using NucleoSpin RNA plus (Macherey-Nagel, D€ uren, Germany) according to the manufacturer's instructions likewise. RNA from FPPE tissue was extracted using the ReliaPrep TM FFPE Total RNA Miniprep System (Promega) as previously described [38]. The concentration of nucleic acids was measured with the NanoDrop ND-1000 Spectrophometer (VWR).…”

Promoter hypermethylation of SFRP1 as a prognostic and potentially predictive blood-based biomarker in patients with localized pancreatic ductal adenocarcinoma

“…A recent paper described a detailed molecular analysis of crucial regions of SFRP1 CpG site hypermethylation with identification of a core CpG island (CGI2) of particular interest (50). The investigators were able to show a strong inverse correlation between DNA methylation status of the CGI2 and SFRP1 mRNA expression both in silico and in vitro, providing stronger evidence for the regulatory mechanism of methylation in PDAC.…”