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HaCat cells, a human keratinocyte line, are commonly utilised as an in vitro cell model for toxicity testing and the discernment of processes of chemically induced skin carcinogenesis. Here, as part of an ongoing program of carcinogenesis research, we tested the genomic transcriptional response of two keratinocyte cell lines HaCat (human) and Pam212 (mouse) to 12-O-tetradecanoylphorbol 13-acetate (TPA), one of the most studied skin carcinoma promoting agents, and compared this with the response in primary keratinocytes. Differences in the genomic response profile indicated an insufficiency in the MEK/ERK pathway signalling in HaCat but not Pam212 cells compared to primary keratinocytes. TPA can also activate NFkappaB and so we tested whether this was also deficient in the HaCat cells using TNFalpha which signals directly to NFkappaB. By this method NFkappaB was found to be equally active in both HaCat and Pam212 cells. Analysis of ERK phosphorylation showed that while TPA mediated ERK phosphorylation occurred in both cell lines it was more robust and difficult to inhibit in Pam212 cells suggesting that there may be an insufficiency in this step in HaCat cells leading to a reduced response. Overall these data indicate that caution should be employed when using HaCat cells as an in vitro skin model for biochemical research or toxicological evaluation.

To the Editor, Non-melanoma skin cancer (NMSC) is the most prevalent cancer in man. For reasons that are unclear NMSC is dramatically increased in organ transplant recipients (OTR) compared to immunocompetent patients (non-OTR) (Euvrard et al, 2003). Increased risk of metastasis and the presence of multiple tumors are a significant source of morbidity in OTR. These patients present with different types of NMSC with squamous cell carcinoma (SCC), and keratoacanthoma (KA) being particularly prevalent. The genetic aberrations driving these cancers in OTRs and non-OTRs are poorly understood. Receptor tyrosine kinases (RTKs) are frequently activated by somatic mutations and/or amplification in human cancers, particularly in epithelial tumors. To determine whether RTKs are mutated in SCC, we searched the literature to identify RTKs that play a role in epidermal homeostasis and thus could be candidate oncogenes in squamous lesions. We chose to analyze epidermal growth factor receptor (EGFR) that is highly expressed in a small subset of metastatic cutaneous SCCs (Bauknecht et al, 1985; Shimizu et al, 2001; Maubec et al, 2005); FGFR3 that is mutated in familial acanthosis nigricans and Crouzon’s syndrome, a type of craniosynostosis (Berk et al, 2007) and induces acanthosis and benign tumors in transgenic mice (Logie et al, 2005); FGFR2, that is also mutated in Crouzon’s syndrome and in this disease is associated with acanthosis nigricans (Meyers et al, 1995). We included the insulin like growth factor receptor 1(IGF1R); mice lacking this receptor have hypoplastic skin (Liu et al, 1993; De Moerlooze et al, 2000), and MET the receptor for the ligand HGF. Mice over expressing the MET receptor exhibit enhanced numbers of hair follicles and accelerated hair follicle morphogenesis (Lindner et al, 2000), a feature associated with cyclosporine use in OTRs. Finally, we assessed ERBB2 that induces SCCs when targeted to mouse skin (Kiguchi et al, 2000). We determined the mutation status of the kinase domains of EGFR, IGF1R, MET and ERBB2 and the regions of FGFR2 and FGFR3 that are mutated in Crouzon’s syndrome in a cohort of 95 tumors that consisted of 70 SCCs and 25 KAs from 55 OTR and 40 non-OTR tumors; not every tumor was analyzed for every gene. Genomic DNA was extracted from archival formalin fixed paraffin embedded samples and amplified with M13 sequence-tailed primers (supplementary table 1). Mutations were found in EGFR, FGFR2 and FGFR3 but not in ERRB2, MET and IGF1R (Table 1). The somatic nature of the mutations was confirmed by sequencing the adjacent normal skin in all three cases in which mutations were found. EGFR was mutated in one of 40 (2.5%) SCCs, a frequency not dissimilar to that detected in head and neck SCCs (7.3%) (Willmore-Payne et al, 2006). The particular Y727H mutation we found in exon 18 of EGFR has been observed in SCC of the lung (Pallis et al, 2007). In addition to mutational activation, amplification of wild-type EGFR can drive tumorigenesis in a variety of cancers and in head and neck SCC cell lines (Weichselbaum et al, 1989). In a dataset of array-based comparative genomic hybridization (CGH) of a larger cohort of SCCs (n=268, 173 OTR tumors and 95 non-OTR tumors) and KAs (n=46, 27 OTR tumors and 19 non-OTR tumors) we found amplifications of the EGFR region on chromosome 7 in three SCCs (1.1%) but not in KAs (Figure 1a) (Ridd et al. manuscript in preparation). Interestingly, all of the tumors harboring an EGFR amplification were from non-OTRs. The amplifications were confirmed by fluorescence in situ hybridization (FISH) (Figure 1b). The tumor with the EGFR mutation had no amplification of EGFR, and the three tumors with amplification did not show any mutations. We also analyzed EGFR protein levels in 275 SCCs (157 OTR tumors and 118 non-OTR tumors) and 69 KAs (28 OTR tumors and 41 non-OTR tumors) using tissue microarrays. EGFR was over-expressed compared to adjacent normal skin in 19 of the 275 SCCs (6.9%) tumors and none of the KAs (Figure 1c and 1d). EGFR was significantly over-expressed in a greater number of non-OTR SCCs (14/118 (11.8%) compared to OTR SCCs (5/157 (3.1%)) (p

Studies have suggested that somatic events in tumors can depend on an individual's constitutional genotype. We used squamous cell carcinomas (SCC) of the skin, which arise in high multiplicity in organ transplant recipients, as a model to compare the pattern of somatic alterations within and across individuals. Specifically, we performed array comparative genomic hybridization on 104 tumors from 25 unrelated individuals who each had three or more independently arisen SCCs and compared the profiles occurring within patients to profiles of tumors across a larger set of 135 patients. In general, chromosomal aberrations in SCCs were more similar within than across individuals (two-sided exact-test p-value ), consistent with the notion that the genetic background was affecting the pattern of somatic changes. To further test this possibility, we performed allele-specific imbalance studies using microsatellite markers mapping to 14 frequently aberrant regions of multiple independent tumors from 65 patients. We identified nine loci which show evidence of preferential allelic imbalance. One of these loci, 8q24, corresponded to a region in which multiple single nucleotide polymorphisms have been associated with increased cancer risk in genome-wide association studies (GWAS). We tested three implicated variants and identified one, rs13281615, with evidence of allele-specific imbalance (p-value = 0.012). The finding of an independently identified cancer susceptibility allele with allele-specific imbalance in a genomic region affected by recurrent DNA copy number changes suggest that it may also harbor risk alleles for SCC. Together these data provide strong evidence that the genetic background is a key driver of somatic events in cancer, opening an opportunity to expand this approach to identify cancer risk alleles., Author Summary Tumors exhibit DNA copy number gains and losses, many of which alter the dosage of genes that promote or suppress tumorigenesis. Evidence from familial cancer syndromes and animal models have shown that DNA copy number changes acquired somatically during tumor progression can be controlled by the constitutional genotype. The genetic heterogeneity among humans makes it difficult to systematically assess the extent of this effect. We used a unique clinical scenario of squamous cell carcinoma (SCC), which can arise in high multiplicity within patients, to compare the pattern of somatic alterations on a homogeneous genetic background. We examined the genome-wide pattern of DNA copy number changes of tumors from individuals who had three or more independent SCCs. We identified multiple chromosomal regions that showed higher frequency of change in SCCs within patients than across patients, suggesting that the genetic background of the individual is important in driving these changes. We further confirmed this by identifying eight regions with strong evidence for a selection of loss or gain of a particular allele within patients. Together these data demonstrate that the genetic background of an individual influences the pattern of somatic alterations in tumors, offering a novel approach to map susceptibility alleles.

Purpose: Nonmelanoma skin cancer incidence is enhanced >50-fold in patients taking antirejection drugs (ARD) following organ transplantation. Preclinical studies suggest that ARD treatment increases transforming growth factor-β1 (TGF-β1) levels, which contribute to enhanced tumor susceptibility independent of the immunosuppressive effects of ARDs. This study investigates whether TGF-β signaling is elevated in transplant patients. Experimental Design: Immunohistochemical tissue microarray analysis was used to determine the levels of TGF-β1, TGF-β2, TGF-β3, TβRII, and activated P-Smad2/3 and P-Smad1/5/8, which are phosphorylated directly by distinct TGF-β/BMP receptor complexes. We analyzed >200 cutaneous lesions and adjacent nonlesional skin samples from 87 organ transplant recipients, and 184 cutaneous lesions and adjacent skin samples from 184 individuals who had never received ARDs. Results: We found significantly higher levels of P-Smad2 in both nonlesional and lesional tissue from transplant recipients compared with those not exposed to ARDs (P ≤ 0.001). In contrast, P-Smad1/5/8, a marker of activation of the bone morphogenetic protein signaling pathway, was generally not expressed at higher levels in patients taking ARDs, including analysis of nonlesional skin, actinic keratoses, carcinoma in situ, or squamous cell carcinoma but was differentially expressed between keratoacanthoma from transplant recipients compared with those from non–transplant recipients (P ≤ 0.005). Conclusions: Observation of elevated P-Smad2 levels in transplant recipients is consistent with the notion that elevated TGF-β signaling may contribute to malignancy in organ transplant recipients. Disparate P-Smad1/5/8 expression levels between keratoacanthoma from the two patient groups might reflect the distinct BMP-responsive cell of origin for this hair follicle–derived lesion. (Clin Cancer Res 2009;15(16):5101–7)

Microphthalmia-associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient-matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix-loop-helix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia-associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

To the Editor, Non-melanoma skin cancer poses a significant clinical problem to organ transplant recipients (OTRs). These patients present with a plethora of different types of skin tumors, in particular squamous cell carcinomas (SCCs) and keratoacanthomas (KAs). KA is considered a benign variant of SCC with the propensity to grow rapidly and regress spontaneously (Karaa et al, 2007). The molecular defects that give rise to SCCs and KAs are poorly understood. However, patients with familial incontinentia pigmenti (IP) develop KAs, particularly subungually (Montes et al, 2004). IP is a rare X-linked disease that is lethal in males and in females leads to a transient inflammatory reaction followed by hyperpigmentation in a distribution pattern that depends on X-chromosome inactivation. IP is caused by mutation or genomic rearrangement of NEMO (NF-κB essential modifier) also referred to as IKBKG or IKKγ. NEMO functions as the regulatory subunit of the IKKα and IKKβ complex and is essential for their kinase activity (Rothwarf et al, 1998). Together, these kinases activate NF-κB in response to a variety of external stimuli including TNFα and IL-1 (Rothwarf et al, 1998). NEMO is required for this response, as embryonic fibroblasts from IP patients cannot activate NF-κB in response to TNFα (Smahi et al, 2000). Under resting conditions, NF-κB subunits are held in the cytoplasm through tethering to IκB proteins. Once stimulated, IKK proteins phosphorylate IκB resulting in its ubiquitination, with subsequent translocation of NF-κB proteins to the nucleus where they activate gene transcription. There is increasing evidence from mouse models that NF-κB signaling is deregulated in skin cancer (van Hogerlinden et al, 1999; Dajee et al, 2003; Park et al, 2007). Silencing of the NF-κB pathway is required for RAS-induced tumorigenesis in a xenograft model (Dajee et al, 2003). Moreover, IκBα is elevated in human SCCs concomitant with the retention of p65, a subunit of NF-κB in the cytoplasm (Dajee et al, 2003). Finally, IKKα deficient mice are more susceptible to chemical induced skin carcinogenesis than wild type mice (Park et al, 2007). Thus, there are several lines of evidence that support the loss of NF-κB activity promotes skin cancer. In light of the role of the deregulated NF-κB pathway in squamous neoplasia and the increased frequency of KAs in IP patients we assessed the status of NEMO in KA. Sequencing of the NEMO gene is complicated by the presence of a pseudogene (ΔNEMO) located in inverted orientation 22kb away on the X chromosome (Aradhya et al, 2001) (Aradhya et al, 2001). The sequence of ΔNEMO is completely identical with exons 3–10 of NEMO (Aradhya et al, 2001). Thus, female genomic DNA from formalin fixed paraffin embedded tumors (FFPE) contains four identical copies of exons 3–10, two from each gene, which, especially in the presence of DNA from stromal tissue, can make detection of somatic mutations challenging. For this reason, we chose to investigate the presence of somatic mutations in NEMO in KAs from male patients only. Hematoxylin and eosin sections were reviewed by a dermatopathologist and regions of tumor tissue were identified for dissection. New sections of 30μm thickness were cut from the blocks and the corresponding tumor tissue and, where possible, adjacent normal tissue was collected by microdissection. Genomic DNA was isolated (Bastian et al, 1998) and the concentration determined (Ginzinger et al, 2000). Genomic DNA (5ng) was used to amplify exons 2 to 9 and the coding portion of exon 10 from 21 KAs; 11 from immunocompetent and 10 from OTR patients. The primer sequences are listed in Table 1 Table 1 Primers used to amplify exons 2–9 and the coding portion of exon 10 of NEMO We detected one tumor with a non-synonymous mutation resulting in a M407V substitution (Fig. 1a). This mutation has been described previously in IP patients and was thought to be associated with a lethal male phenotype. However, as this mutation was also detected in the normal tissue adjacent to the tumor it is likely to be a germline event in our patient (Fig. 1b). NEMO protein expression was analyzed by immunohistochemistry using a tissue microarray consisting of adjacent normal skin (n=15) and KAs (n=57) (Fig. 2). No significant difference in expression was found between neoplastic keratinocytes and the adjacent histopathologically normal appearing epidermis (p

Differential gene expression in two established initiation and promotion skin carcinogenesis models during promotion and tumor formation was determined by microarray technology with the purpose of distinguishing the genes more associated with neoplastic transformation from those linked with proliferation and differentiation. The first model utilized dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion in the FVB/N mouse, and the second TPA promotion of the Tg.Ac mouse, which is endogenously initiated by virtue of an activated Ha-ras transgene. Comparison of gene expression profiles across the two models identified genes whose altered expression was associated with papilloma formation rather than TPA-induced proliferation and differentiation. DMBA suppressed TPA-induced differentiation which allowed identification of those genes associated more specifically with differentiation rather than proliferation. EASE (Expression Analysis Systemic Explorer) indicated a correlation between muscle-associated genes and skin differentiation, whereas genes involved with protein biosynthesis were strongly correlated with proliferation. For verification the altered expression of selected genes were confirmed by RT-PCR; Carbonic anhydrase 2, Thioredoxin 1 and Glutathione S-transferase omega 1 associated with papilloma formation and Enolase 3, Cystatin beta and Filaggrin associated with TPA-induced proliferation and differentiation. In situ analysis located the papillomas Glutathione S-transferase omega 1 expression to the proliferating areas of the papillomas. Thus we have identified profiles of differential gene expression associated with the tumorigenesis and promotion stages for skin carcinogenesis in the mouse.