Genomic instability, mutations and expression analysis of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in actinic keratosis

doi:10.1016/j.canlet.2008.01.042

Cancer Letters

Volume 264, Issue 1, 8 June 2008, Pages 145-161

https://doi.org/10.1016/j.canlet.2008.01.042 Get rights and content

Abstract

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14^ARF, p15^INK4b, p16^INK4a and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16^INK4a (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14^ARF gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16^INK4a and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15^INK4b, independently from the intracellular pathway mediated by p16^INK4a, and of p14^ARF in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.

Introduction

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). SCCs of the skin are the most prevalent keratinocyte-derived tumours, second to basal cell carcinomas (BCCs) [1]. Clinically AKs appear primarily on chronically sun-exposed areas [2], and sites adjacent to AKs may contain significant histological alterations, suggesting extensive pre-neoplastic alterations in sun-damaged skin [3]. It has been found that 82.4% of SCCs arise either within (26.7%) or in close proximity to AKs (55.7%) [4], and the risk of AK progression to SCC has been reported as being between 0.025% and 16% [5]. This progression is thought to be due to chronic sun exposure, specifically ultraviolet B sunlight. Additionally, 72% of SCCs are associated with contiguous AKs [6].

UV radiation (UVR) is a prevailing factor implicated in the etiology of SCCs and AKs. Solar radiation (UVB and UVA) can mutagenise DNA, which often produces UV landmark mutations (two tandem CC:GG to TT:AA transitions and two C:G to T:A transitions at dipyrimidic sites). When these mutations affect the function of sufficient oncogenes, tumour suppressor genes (TSGs) and important housekeeping genes, they result in an uncontrolled cell cycle and the transformation of keratinocytes [7].

TSG inactivation is recognised as a pivotal event in the initiation and progression of neoplasia. The 9p21 chromosomal band, at which the INK4a/ARF (CDKN2A) and INK4b (CDKN2B) gene loci are located, is one of the most frequently altered genomic regions in human cancers [8], and the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus is the second most commonly altered gene locus in human cancer after p53 [9]. The locus CDKN2A encodes two TSGs, p16^INK4a and p14^ARF, both involved in the negative control of cell proliferation. They share common exons 2 and 3, but have alternatively spliced first exons (exon 1a for p16^INK4a and 1b for p14^ARF). These first exons are under the control of distinct promoters and uniquely create two proteins that have no sequence homology at the amino acid level [10]. p16^INK4a is a CDK4 inhibitor that specifically acts at the G1/S phase of the cell cycle by negatively controlling retinoblastoma (Rb) phosphorylation status [11], while p14^ARF binds to MDM2 preventing both MDM2-mediated p53 degradation and MDM2-mediated Rb inactivation thus causing the arrest of the G1 and G2 phases of the cell [12]. Consequently, stabilised p53 can induce temporary and permanent growth arrest, DNA repair, terminal differentiation or apoptosis in response to oncogenic signals and DNA damage [13]. p15^INK4b, another cyclin-dependent kinase inhibitor gene located in chromosome region 9p21.2, has a strong structural and functional homology to p16^INK4a [10] and is transcriptionally activated by transforming growth factor-β (TGF-β) [14]. p15^INK4b protein binds to the cdk4-Cyclin D complex, displacing p27 and freeing it to bind to and inhibit the cdk2-Cyclin E complex required for entry into the S phase of the cell cycle [15].

Knockout mice experiments suggest a possible role for p14^ARF in the initiation of cutaneous SCC development [8], [11] and for p16^INK4a in the determination of the differentiation status of skin squamous tumours [16]. In primary cutaneous SCC, loss of heterozygosity (LOH) at the 9p21 locus and CDKN2A mutations have been reported, but with disparate result [17], [18], [19], [20]. Although AK shares many of the same loci as SCC, LOH in the CDKN2A locus appears to be less common in AK lesions than it is in SCC [21].

Inactivation of the Rb and p53 tumour suppressor pathways is observed in most human cancers [8]. By virtue of its close involvement in both pathways, the p14^ARF–p15^INK4b–p16^INK4a gene cluster at chromosome 9p21 may be a nexus in the cellular-growth-control network, the inactivation of which results in the collapse of the tumour suppression system [28]. The loss of the CDKN2A, CDKN2B and p53 genes at the gene level (point mutation, deletion or promoter hypermethylation) or at the mRNA expression level in pre-tumour and tumour cells is, perhaps, the key event that provides considerable growth stimuli leading to uncontrolled proliferation and destabilisation.

In this study, we investigated the integrity of the CDKN2A and CDKN2B loci in 26 AK samples, by analysing the presence of allelic imbalance/loss of heterozygosity (AI/LOH) in microsatellite markers and observed a low percentage of LOH (4%) and a significant incidence of MSI (15%) in at least one marker of the 9p21 locus. Furthermore, exons 1a, 1b and 2 of the CDKN2A and exon 1 of the CDKN2B loci were screened for mutations in 26 AK samples, and novel mutations apart from previously reported nucleotide changes were detected. Mutation analysis and genomic instability in SCC has been extensively studied in the past. Since many of our patients with AK presented contiguous SCC, we considered it interesting to study the expression levels of the TSGs p14^ARF, p15^INK4b, p16^INK4a and p53 in AK and SCC, in comparison with normal adjacent tissue. Our results suggest that p16^INK4a and p53 mRNA down-regulation in SCC compared to AK, might lead in collapse of the tumour suppressor system. Furthermore, we suggest a possible role of p15^INK4b, independently from the intracellular pathway mediated by p16^INK4a, and of p14^ARF in AK development, as well as in the progression of AK to SCC. Apart from mutations and LOH/MSI at 9p21, the deregulation of the expression profile of the TSGs may play a significant role in AK appearance and progression of AK to SCC.

Section snippets

Tumour specimens and DNA extraction

Twenty-six AK with 22 adjacent normal tissues and 12 SCC samples with six adjacent normal tissues, were obtained from patients treated at ‘A. Sygros’ Hospital (Athens, Greece) with the approval of the donors and the institute’s ethical committee. Patients with AK lesions were between the ages of 63 and 92 years (average 76; SD ± 7.2; 16 males and 10 females). Patients with SCC lesions were between the ages of 63 and 88 years (average 75; SD ± 7.5; 7 males and 5 females). A total of eight AK patients

Results

Mutation analysis and genomic instability for the genes p14^ARF, p15^INK4B, p16^INK4a and p53 in SCC, has been extensively studied in the past [19], [20], [21], [25], [26], [27], [28], [29]. Considering the fact that many of our AK patients had contiguous SCCs, and that so far few reports have focused on the expression of these TSGs in SCC, it was prompting also to evaluate their mRNA levels.

Discussion

Inactivation of the Rb and p53 tumour suppressor pathways is observed in most human cancers [8]. Due to its close participation in both pathways, the p14^ARF–p15^INK4b–p16^INK4a gene cluster at chromosome 9p21 may be a nexus of the cellular-growth-control network, the inactivation of which results in the collapse of the tumour suppression system [28]. To date, a vast amount of data has demonstrated multiple types of genetic alterations in the 9p21 region, the prevalence of which varies with the

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The specific DNA mutations that have been characterized in AKs include those seen in keratinocyte carcinoma, including mutations in TP53, the gene encoding the p53 protein, known to have an important role in tumor suppression for a variety of malignancies, including SCC.36,37 Additional mutations that are thought to be pathogenic in the development of AK include mutations in p16, ras family members, NFKB, CDKN2A, telomerase, and TNF alpha.38-43 Estimates of the risk of progression of AK to SCC vary from less than 0.1% to 20%.27,30,44,45
Actinic keratoses (AK) are rough scaly patches that arise on chronically ultraviolet-exposed skin and can progress to keratinocyte carcinoma.
This analysis examined the literature related to the management of AK to provide evidence-based recommendations for treatment. Grading, histologic classification, natural history, risk of progression, and dermatologic surveillance of AKs are also discussed.
A multidisciplinary Work Group conducted a systematic review to address 5 clinical questions on the management of AKs and applied the Grading of Recommendations, Assessment, Development, and Evaluation approach for assessing the certainty of the evidence and formulating and grading clinical recommendations. Graded recommendations were voted on to achieve consensus.
Analysis of the evidence resulted in 18 recommendations.
This analysis is based on the best available evidence at the time it was conducted. The pragmatic decision to limit the literature review to English language randomized trials may have excluded data published in other languages or limited identification of relevant long-term follow-up data.
Strong recommendations are made for using ultraviolet protection, topical imiquimod, topical 5-fluorouracil, and cryosurgery. Conditional recommendations are made for the use of photodynamic therapy and diclofenac for the treatment of AK, both individually and as part of combination therapy regimens.
The Genomic Landscape of Actinic Keratosis
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Apart from an early study that suggested that AKs have more LOH than cSCC (Rehman et al., 1996), our findings are contrary to those of more recent studies that found that AKs have relatively few CNAs (García-Díez et al., 2019; Hameetman et al., 2013). However, most studies agree on the most common sites of chromosomal instability, with 9p loss—the region where CDKN2A is located—ubiquitous among AK studies (Ashton et al., 2003; García-Díez et al., 2019; Kanellou et al., 2008; Rehman et al., 1996). Interrogating published expression datasets against our SMG list found KIF24, KCNK5, EPB41L2, and ABI3BP downregulated in AK compared with those in the normal skin, suggesting tumor suppressor roles.
Actinic keratoses (AKs) are lesions of epidermal keratinocyte dysplasia and are precursors for invasive cutaneous squamous cell carcinoma (cSCC). Identifying the specific genomic alterations driving the progression from normal skin to skin with AK to skin with invasive cSCC is challenging because of the massive UVR-induced mutational burden characteristic at all stages of this progression. In this study, we report the largest AK whole-exome sequencing study to date and perform a mutational signature and candidate driver gene analysis on these lesions. We demonstrate in 37 AKs from both immunosuppressed and immunocompetent patients that there are significant similarities between AKs and cSCC in terms of mutational burden, copy number alterations, mutational signatures, and patterns of driver gene mutations. We identify 44 significantly mutated AK driver genes and confirm that these genes are similarly altered in cSCC. We identify azathioprine mutational signature in all AKs from patients exposed to the drug, providing further evidence for its role in keratinocyte carcinogenesis. cSCCs differ from AKs in having higher levels of intrasample heterogeneity. Alterations in signaling pathways also differ, with immune-related signaling and TGFβ signaling significantly more mutated in cSCC. Integrating our findings with independent gene expression datasets confirms that dysregulated TGFβ signaling may represent an important event in AK‒cSCC progression.
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Vibrio cholerae, the causative agent of cholera, tend to colonize the small intestine as a Gram-negative pathogen. The intestinal mucus layer forms mucin physical barrier, consisted of high molecular weight proteins. Regarding the role of toxin–coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including toll like receptors (TLRs) and Nucleotide-binding oligomerization domain-containing proteins (NODs) using Caco2- PBMC co-cultures as an interactive model. The rTcpA protein of V. cholerae was expressed in BL21 Escherichia coli, purified using Ni-column chromatography and confirmed by western blotting. Nontoxic doses of rTcpA was determined on Caco-2 cell lines and different concentrations of rTcpA (1, 5, 10 and 50 μg/mL) showed a statistically significant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner. This finding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection. NOD2 was significantly increased which suggests that it may contribute in pro-inflammatory responses observed in cholera disease. No change in NOD1 expression was seen which might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen. In conclusion, the rTcpA protein of V. cholerae showed a statistically significant modulatory effect on the human gut epithelium gene expression which would help promising protection in prophylaxis applications.
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Genomic instability, mutations and expression analysis of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in actinic keratosis

Abstract

Introduction

Section snippets

Tumour specimens and DNA extraction

Results

Discussion

Cancer Lett.

Cell

Cell

J. Invest. Dermatol.

Biochem. Biophys. Res. Commun.

Cancer Lett.

J. Invest. Dermatol.

J. Invest. Dermatol.

J. Dermatol. Sci.

FEBS Lett.

J. Invest. Dermatol.

J. Urol.

Mol. Cell

J. Biol. Chem.

J. Invest. Dermatol.

J. Invest. Dermatol.

Mod. Pathol.

Cell

Am. J. Pathol.

Cancer Lett.

J. Invest. Dermatol.

J. Invest. Dermatol.

J. Invest. Dermatol.

Precursors to skin cancer

Cancer

Solar keratoses

Br. J. Dermatol.

Frequency of pre-existing actinic keratosis in cutaneous squamous cell carcinoma

Int. J. Dermatol.

The risk of progression to invasive disease

J. Am. Acad. Dermatol.

The majority of cutaneous squamous cell carcinomas arise in actinic keratoses

J. Cutan. Med. Surg.

Radiation-induced activation of transcription factors in mammalian cells

Radiat. Environ. Biophys.

Role of the p16 tumor suppressor gene in cancer

J. Clin. Oncol.

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2)

J. Mol. Evol.

Mutations associated with familial melanoma impair p16INK4 function

Nat. Genet.

The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2

EMBO J.

p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest

Nature

The INK4a/ARF network in tumour suppression

Nat. Rev. Mol. Cell Biol.

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14^ARF, p15^INK4b, p16^INK4a and p53 in actinic keratosis