Epigenetic changes in Basal Cell Carcinoma affect SHH and WNT signaling components

Abstract

Background: The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.

Objective: We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.

Results: Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).

Conclusions: We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.

Figure 1. Methylation analysis in BCC and normal skin of ten candidate genes.

A. Illustration of MSP results of ten candidate genes resolved on a 2% agarose gel. BCC: Basal cell carcinoma; NL, normal; IVD, In Vitro Methylated-DNA; Huvec, Human Umbilical Vein Endothelial Cells; U, unmethylated; M, methylated; H₂0o, water control outside PCR; H₂0i, water control inside PCR. B. Illustration of methylation frequencies of ten candidate genes in BCC (n = 112) and normal skin samples (n = 124). P-values represent the difference between percentage methylation in BCC an normal skin. C. Illustration of methylation frequencies of ten candidate genes in BCC, sun-exposed (SE) normal skin and sun-protected (SP) normal skin. P-values represent both the difference between percentage methylation in BCC and SE and SP. D. SHH sequence data of bisulfite treated genomic DNA from patients. Upper part shows the SHH promoter region starting 1000 base pairs (bp) upstream of the transcription start site (TSS) to 1000 bp downstream. White boxes indicate putative CpG islands (EMBOSS, http://emboss.sourceforge.net). The 256 bp region sequenced stretches from −51 bp from the TSS to +409 bp. Indicated with arrows are the forward and reverse methylation specific PCR (MSP) primers. Vertical bars represent CpG dinucleotides and pie -charts represent the percentage of methylated CpG sites (percentage over at leased 10 sequenced clones).

Figure 2. Expression of SHH, APC, SFRP5 and RASSF1A is reduced in BCC versus healthy skin control tissue.

A. Relative expression levels of APC and SFRP5 in BCC tissues as compared to the expression level in normal skin (n = 6) (2−^ΔΔCt). All reactions were done in triplicates, standard error of the mean (SEM) is shown as error bars. Expression levels were normalized to Cyclophilin A. U, unmethylated sample; M, methylated sample. *p≤0.05, **p≤0.001. B. Relative mRNA expression in unmethylated versus methylated BCC samples for either APC or SFRP5. C. Microphotographs of selected samples of SHH, APC and RASSF1A. Bar = 200 µm.

Figure 3. Low levels of nuclear ß-catenin coincide with high levels of E-cadherin in BCC.

A. Microphotographs of selected sample of ß-catenin, showing nuclear staining only at the periphery or the tumor. Bar = 200 µm. B. Microphotographs of selected sample of E-cadherin showing lowered expression of the tumor compared with the normal epidermis.