Alan Storey

Models for Skin Cancer

The Cancer Handbook, Jan 1, 2007

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“Licensed to kill”: Tyrosine dephosphorylation and Bak activation

Cell Cycle, Jan 1, 2011

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Cutaneous Squamous Cell Carcinoma (SCC) and the DNA Damage Response: pATM Expression Patterns in Pre-Malignant and Malignant Keratinocyte Skin …

PloS one, Jan 1, 2011

Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage resp... more Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage response that evokes a counter-response which arrests the growth of, or eliminates, damaged cells. Early precursor lesions express markers of an activated DNA damage response in several types of tumour, with a diminishing response in more advanced cancers. An important marker of DNA damage is ATM which becomes phosphorylated (pATM) upon activation. We have investigated pATM expression patterns in cultured keratinocytes, skin explants and a spectrum of pre-malignant to malignant keratinocyte skin lesions by immunohistochemistry. We found that pATM was mainly localised to the Golgi apparatus, which contrasts with its nuclear localisation in other tissues. Upon UV irradiation there is transient formation of pATM in nuclear foci, consistent with recruitment to the sites of DNA damage. By immunohistochemistry we show pATM expression in precancerous keratinocyte lesions is greater and predominantly nuclear when compared to the invasive lesions where pATM is weaker and predominantly cytoplasmic. Our results are consistent with the hypothesis that the DNA damage response acts as a barrier to cutaneous tumour formation, but also suggests that ATM expression in skin is different compared to other tissues. This may be a consequence of the constant exposure of skin to UVR, and has implications for skin carcinogenesis.

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Interaction Between Ultraviolet Radiation and Human Papillomavirus

Skin Cancer after Organ Transplantation, Jan 1, 2009

Human papillomaviruses (HPV) are small double-stranded DNA viruses that predominantly form hyperp... more

Proteomic analysis reveals a novel role for the actin cytoskeleton in human papillomavirus type 8 induced keratinocyte transformation

Virology, Jan 1, 2009

Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.

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Cutaneous HPV5 E6 causes increased expression of Osteoprotegerin and Interleukin 6 which contribute to evasion of UV-induced apoptosis

Carcinogenesis, Jan 1, 2010

Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at d... more Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at different body sites. The association between cutaneous squamous cell carcinoma (SCC) formation, ultraviolet (UV) irradiation and infection with a high-risk subset of cutaneous HPVs has been postulated although the underlying molecular mechanisms by which HPV may play a role in SCC development are not yet fully elucidated. Expression of the viral E6 oncoprotein has been shown to interfere with DNA damage responses and inhibit UV-induced apoptosis, suggesting HPV can contribute to early stages in tumorigenesis. However, cutaneous SCCs, in contrast to HPV-associated anogenital cancers do not harbor HPV DNA in every tumor cell. Here, we show that expression of E6 from the prototypic skin cancer-associated HPV type 5 induced the secretion of factors that were able to inhibit UV-induced apoptosis in non-HPV-expressing cell lines and primary human keratinocytes. The anti-apoptotic effect of HPV E6 expression was found to be mediated in part by upregulation of osteoprotegerin (OPG) and interleukin 6 (IL6). Purified OPG and IL6, when added to cells together, but not individually, reduced apoptosis following UV irradiation. We provide evidence that OPG and IL6 inhibit the extrinsic and intrinsic apoptotic pathways, respectively. Furthermore, we show by immunohistochemistry of HPV-typed SCC sections that IL6 protein is upregulated in HPV-positive tumors compared with HPV-negative cancers. These findings support the hypothesis that a small number of HPV-infected cells influence UV-induced apoptosis in the skin and contribute to tumorigenesis.

format_quoteIL6 is upregulated in HPV-positive SCC, indicating its role in modulating apoptosis and supporting tumorigenesis.format_quote

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The E2 protein of human papillomavirus type 8 increases the expression of matrix metalloproteinase-9 in human keratinocytes and organotypic skin cultures

Medical microbiology …, Jan 1, 2011

Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Altho... more Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.

format_quoteOverexpression of MMP9 in HPV8 E2-expressing epidermis shows E2 protein activates MMP9 transcription directly.format_quote

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Tyrosine dephosphorylation is required for Bak activation in apoptosis

The EMBO …, Jan 1, 2010

Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial ... more Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.

format_quoteThe study identifies a previously unrecognized dual-control model of Bak activation involving Y108 dephosphorylation and conformational change.format_quote

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Axl Promotes Cutaneous Squamous Cell Carcinoma Survival through Negative Regulation of Pro-Apoptotic Bcl-2 Family Members

Journal of Investigative …, Jan 1, 2010

Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma ... more Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma (SCC). Examination of a series of cutaneous SCC tumors revealed positive phospho-Akt (P-Akt) staining accompanied by weak TUNEL staining in Axl-positive tumors, suggesting an anti-apoptotic role for Axl in SCC survival. The role of Axl in UV-induced apoptosis was investigated in a cutaneous SCC cell line using retroviral short hairpin RNA sequences enabling stable Axl knock-down. We show that, although Axl knock-down has no effect on cell proliferation, it sensitizes cells to UV-induced apoptosis through increased activation of the pro-apoptotic protein Bad, a change in the conformation of Bax and Bak, release of cytochrome c into the cytosol, and activation of caspases. These events are accompanied by faster Akt dephosphorylation in UV-treated Axl knockdown cells and correlate with the degree of Axl knock-down. Treatment with the pan-caspase inhibitor zVADfmk partially rescued cells from UV-induced apoptosis but did not affect Bid cleavage or cytochrome c release, suggesting that cells die via the mitochondrial-mediated pathway. Thus, Axl confers resistance of SCC cells to apoptosis and displays potential as a target for therapeutic intervention in cutaneous SCC.

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Upregulation of lipocalin-2 in human papillomavirus-positive keratinocytes and cutaneous squamous cell carcinomas

Journal of General …, Jan 1, 2011

It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) typ... more It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs.

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Human papillomavirus 5 and 8 E6 downregulate interleukin-8 secretion in primary human keratinocytes

Journal of General …, Jan 1, 2010

Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early ... more Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.

format_quoteHigh viral load and prevalence of bHPV types in precancerous lesions suggest a critical role of HPV in early skin cancer progression.format_quote

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Proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8

Virology, Jan 1, 2009

Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.

format_quoteTransgenic mice expressing HPV8's complete early region developed spontaneous skin tumors without external carcinogenic treatment, highlighting HPV8's strong oncogenic potential.format_quote

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Papers by Alan Storey