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. 2018 Feb 8;9(1):577.
doi: 10.1038/s41467-018-03025-1.

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin

Manuel Rodríguez-Paredes  1 , Felix Bormann  1 , Günter Raddatz  1 , Julian Gutekunst  1 , Carlota Lucena-Porcel  2   3 , Florian Köhler  1 , Elisabeth Wurzer  4 , Katrin Schmidt  4 , Stefan Gallinat  4 , Horst Wenck  4 , Joachim Röwert-Huber  5 , Evgeniya Denisova  6 , Lars Feuerbach  6 , Jeongbin Park  7 , Benedikt Brors  6   8   9 , Esther Herpel  2   3 , Ingo Nindl  5 , Thomas G Hofmann  1 , Marc Winnefeld  4 , Frank Lyko  10
Affiliations

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin

Manuel Rodríguez-Paredes et al. Nat Commun. .

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and usually progresses from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain partially understood. Here, we use Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status in healthy, AK and cSCC epidermis samples. Importantly, we show that AK methylation patterns already display classical features of cancer methylomes and are highly similar to cSCC profiles. Further analysis identifies typical features of stem cell methylomes, such as reduced DNA methylation age, non-CpG methylation, and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature is detected only in half of the samples, while the other half shows patterns more closely related to healthy epidermis. These findings suggest the existence of two subclasses of AK and cSCC emerging from distinct keratinocyte differentiation stages.

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Conflict of interest statement

E.W., K.S., S.G., H.W. and M.W. are employees of Beiersdorf AG. F.L. has received consultation fees from Beiersdorf AG.

Figures

Fig. 1
Fig. 1
Actinic keratosis (AK) and cutaneous squamous cell carcinoma (cSCC) show similar aberrant methylation patterns. a Principal component analysis of 12 healthy, 16 AK, and 18 cSCC epidermis samples using all 850k CpG probes. b Scatter plots comparing the epidermis methylomes of healthy and AK samples (left panel), and healthy and cSCC samples (right panel). Differentially (P < 0.05, F-test) methylated probes are shown in blue. c Scatter plot comparing the epidermis methylomes of AK and cSCC samples. No significantly (P < 0.05, F-test) differentially methylated probes were detected
Fig. 2
Fig. 2
AK and cSCC methylomes show cancer-specific features. a Methylation status of the different epigenomic substructures in the epidermis of AK (left) and cSCC (right) patients compared to healthy controls. The box plots indicate highly significant (P ≤ 9.1E−77, two-sided t-test) hypermethylation of the CpG islands and hypomethylation of the Open Sea probes in both AK and cSCC samples. b Fractions of hyper and hypomethylated CpGs in AK and cSCC epidermis, within different epigenomic substructures and in comparison to healthy skin. c Probes within lamina-associated domains (LADs) are significantly (P ≤ 3.4E−288, two-sided t-test) hypomethylated in AK and cSCC when compared to healthy epidermis
Fig. 3
Fig. 3
AK and cSCC display typical features of stem cell methylomes. a Mean difference between chronological and methylation-based biological age for healthy, AK, and cSCC samples. b Box plots indicating the average methylation levels of the 210 (left panel) and 263 (right panel) differentially (P < 0.05, F-test) methylated non-CpG probes in AK and cSCC. c The DNA methyltransferase DNMT3B gene is significantly upregulated in AK (P < 0.002, two-sided t-test) and cSCC (P < 3.43E−06, two-sided t-test). RNA-seq gene expression data from 7 healthy, 10 AK, and 9 cSCC epidermis samples was obtained from Chitsazzadeh et al.
Fig. 4
Fig. 4
AK and cSCC express epidermal stem cell markers. The figure shows representative hematoxylin and eosin stainings, as well as p63, keratin K5, and keratin K14 immunostainings in epidermis from healthy donors, AK and cSCC patients, respectively. Ectopic expression of the three epidermal stem cell markers was observed in AK and cSCC samples. Scale bars, 200 µm
Fig. 5
Fig. 5
A specific methylation signature at keratin gene clusters identifies two distinct subclasses of AK/cSCC. a UCSC genome browser tracks showing significant DNA methylation differences (Δβ, P < 0.05, F-test, vertical green lines) at the keratin gene cluster on chromosome 12 for AK (upper part) and cSCC (lower part) in comparison to healthy epidermis. b Specific DNA methylation patterns of the genes encoding keratins K5, K14, K15, and K80 (KRT5, KRT14, KRT15, and KRT80, respectively). Heatmaps show DNA methylation levels (in β values, from blue (= 0) to red (= 1)) of probes (columns) located in the promoter and gene body, for individual healthy, AK, and cSCC samples (rows). Filled and empty circles denote the two distinct subclasses of AK (purple) and cSCC (black), respectively. c Principal component analysis of healthy, AK, and cSCC epidermis samples based on 1364 keratin-associated methylation probes. For comparisons, keratin methylation patterns from 26 additional tumor entities were also included. d Hierarchical clustering of the dataset shown in c. The 26 tumor entities depicted in this graph, and also used in c, are from left to right: testicular germ cell tumors, liver hepatocellular carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, lung squamous carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, lung adenocarcinoma, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, bladder urothelial carcinoma, colon adenocarcinoma, rectum adenocarcinoma, skin cutaneous melanoma, adrenocortical carcinoma, glioblastoma multiforme, sarcoma, thyroid carcinoma, kidney renal papillary cell carcinoma, cholangiocarcinoma, prostate adenocarcinoma, breast invasive carcinoma, pancreatic adenocarcinoma, kidney renal clear cell carcinoma, and mesothelioma
Fig. 6
Fig. 6
Enhancer methylation patterns separate AK and cSCC into keratinocyte-like and epidermal stem cell-like subtypes. a Venn diagram displaying the total number of enhancer regions defined for human embryonic stem cells and keratinocytes, respectively, . As expected, both cell types only share about a quarter of their enhancers (b) Hierarchical clustering of AK and cSCC epidermis samples based on the methylation status of the 850k probes contained in ESC (left) and keratinocyte (right) enhancers. Methylation profiles of ESCs epidermis samples were used as reference. Filled and empty circles denote the two distinct subclasses of AK (purple) and cSCC (black), respectively. c Heatmaps showing the methylation profiles of enhancers gained (n = 109, left) and lost (n = 11,825, right) during EpSC differentiation, in AK and cSCC epidermis samples, respectively. Colors represent the normalized average methylation levels of each enhancer region
Fig. 7
Fig. 7
Model describing the emergence of AK/cSCC subtypes from different stages of epidermal differentiation. Mutations giving rise to AK can result in the transformation of distinct epidermal differentiation stages, resulting in two distinct subclasses of AK/cSCC
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References

    1. Alam M, Ratner D. Cutaneous squamous cell carcinoma. N. Engl. J. Med. 2001;344:975–983. doi: 10.1056/NEJM200103293441306. - DOI - PubMed
    1. Mittelbronn MA, Mullins DL, Ramos-Caro FA, Flowers FP. Frequency of pre-existing actinic keratosis in cutaneous squamous cell carcinoma. Int. J. Dermatol. 1998;37:677–681. doi: 10.1046/j.1365-4362.1998.00467.x. - DOI - PubMed
    1. Glogau RG. The risk of progression to invasive disease. J. Am. Acad. Dermatol. 2000;42:23–24. doi: 10.1067/mjd.2000.103339. - DOI - PubMed
    1. Siegel JA, Korgavkar K, Weinstock MA. Current perspective on actinic keratosis: a review. Br. J. Dermatol. 2016;177:350–358. doi: 10.1111/bjd.14852. - DOI - PubMed
    1. Chitsazzadeh V, et al. Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates. Nat. Commun. 2016;7:12601. doi: 10.1038/ncomms12601. - DOI - PMC - PubMed

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