Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080638
ISSN1525-2191
AutoresJustin G. Madson, David T. Lynch, Jessica Svoboda, Rebecca Jangraw Ophardt, Jodi K. Yanagida, Sumanth K. Putta, Andrew Bowles, Carol S. Trempus, Raymond W. Tennant, Laura A. Hansen,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoThe Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-rasHa transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser280. Since Akt phosphorylates Chk1-Ser280, the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism. The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-rasHa transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser280. Since Akt phosphorylates Chk1-Ser280, the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism. Activation of signaling pathways following UV radiation, known as the UV response, resembles the response of cells to growth factors. Interestingly, the receptor tyrosine kinase Erbb2 (HER2/Neu) is rapidly activated following UV exposure of skin and cultured keratinocytes.1Madson JG Lynch DT Tinkum KL Putta SK Hansen LA Erbb2 regulates inflammation and proliferation in the skin after ultraviolet irradiation.Am J Pathol. 2006; 169: 1402-1414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Erbb2 activity is increased following UV irradiation by two distinct mechanisms: reactive oxygen species-dependent inactivation of tyrosine phosphatases2Coffer PJ Burgering BM Peppelenbosch MP Bos JL Kruijer W UV activation of receptor tyrosine kinase activity.Oncogene. 1995; 11: 561-569PubMed Google Scholar, 3Huang RP Wu JX Fan Y Adamson ED UV activates growth factor receptors via reactive oxygen intermediates.J Cell Biol. 1996; 133: 211-220Crossref PubMed Scopus (253) Google Scholar, 4Rosette C Karin M Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.Science. 1996; 274: 1194-1197Crossref PubMed Scopus (943) Google Scholar and an activator protein 2α-dependent increase in Erbb2 expression.5Han CY Lim SC Choi HS Kang KW Induction of ErbB2 by ultraviolet A irradiation: potential role in malignant transformation of keratinocytes.Cancer Sci. 2008; 99: 502-509Crossref PubMed Scopus (11) Google Scholar Our recent gene profiling and cell biology experiments demonstrated that the UV-induced activation of Erbb2 increases inflammation and cell proliferation,1Madson JG Lynch DT Tinkum KL Putta SK Hansen LA Erbb2 regulates inflammation and proliferation in the skin after ultraviolet irradiation.Am J Pathol. 2006; 169: 1402-1414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar suggesting Erbb2 may contribute to UV-induced skin tumorigenesis. Indeed, transgenic overexpression of Erbb2 in the skin results in epidermal and follicular hyperplasia and spontaneous tumor formation.6Bol D Kiguchi K Beltran L Rupp T Moats S Gimenez-Conti I Jorcano J DiGiovanni J Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter.Mol Carcinog. 1998; 21: 2-12Crossref PubMed Scopus (46) Google Scholar, 7Xie W Wu X Chow LT Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar In addition, Erbb2 overexpression is detected in some human non-melanoma skin cancers, where it is associated with more aggressive disease.8Krahn G Leiter U Kaskel P Udart M Utikal J Bezold G Peter RU Coexpression patterns of EGFR. HER2, HER3 and HER4 in non-melanoma skin cancer.Eur J Cancer. 2001; 37: 251-259Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Maubec E Duvillard P Velasco V Crickx B Avril MF Immunohistochemical analysis of EGFR and HER-2 in patients with metastatic squamous cell carcinoma of the skin.Anticancer Res. 2005; 25: 1205-1210PubMed Google Scholar Investigation of the influence of the UV-induced activation of Erbb2 on skin tumorigenesis, however, has not been previously reported. Identification of the importance of the Erbb2 family member and signaling partner epidermal growth factor receptor (EGFR) on UV-induced skin tumors strongly supports a role for Erbb2 in skin tumorigenesis as well. The UV-induced activation of EGFR blocks cell cycle arrest, increases cell proliferation, suppresses apoptotic cell death, and increases skin tumorigenesis.10El Abaseri TB Putta S Hansen LA Ultraviolet irradiation induces keratinocyte proliferation and epidermal hyperplasia through the activation of the epidermal growth factor receptor.Carcinogenesis. 2006; 27: 225-231Crossref PubMed Scopus (165) Google Scholar, 11Jean C Hernandez-Pigeon H Blanc A Charveron M Laurent G Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells.J Invest Dermatol. 2007; 127: 2418-2424Crossref PubMed Scopus (17) Google Scholar, 12Peus D Vasa RA Meves A Beyerle A Pittelkow MR UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival.Photochem Photobiol. 2000; 72: 135-140Crossref PubMed Scopus (74) Google Scholar, 13Wan YS Wang ZQ Shao Y Voorhees JJ Fisher GJ Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGF receptors in human skin in vivo.Int J Oncol. 2001; 18: 461-466PubMed Google Scholar, 14El Abaseri TB Fuhrman J Trempus C Shendrik I Tennant RW Hansen LA Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the epidermal growth factor receptor.Cancer Res. 2005; 65: 3958-3965Crossref PubMed Scopus (82) Google Scholar The effects of EGFR on apoptosis and cell cycle arrest result, at least in part, from its activation of phosphatidyl inositol-3-kinase (PI3K)/Akt signaling.11Jean C Hernandez-Pigeon H Blanc A Charveron M Laurent G Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells.J Invest Dermatol. 2007; 127: 2418-2424Crossref PubMed Scopus (17) Google Scholar, 13Wan YS Wang ZQ Shao Y Voorhees JJ Fisher GJ Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGF receptors in human skin in vivo.Int J Oncol. 2001; 18: 461-466PubMed Google Scholar For example, EGFR-dependent PI3K/Akt activation blocks the activation of signaling downstream from ataxia telangiectasia and Rad3-related (ATR) to block cell cycle arrest.11Jean C Hernandez-Pigeon H Blanc A Charveron M Laurent G Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells.J Invest Dermatol. 2007; 127: 2418-2424Crossref PubMed Scopus (17) Google Scholar, 15Shtivelman E Sussman J Stokoe D A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle.Curr Biol. 2002; 12: 919-924Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Bortul R Tazzari PL Billi AM Tabellini G Mantovani I Cappellini A Grafone T Martinelli G Conte R Martelli AM Deguelin. A PI3K/AKT inhibitor, enhances chemosensitivity of leukaemia cells with an active PI3K/AKT pathway.Br J Haematol. 2005; 129: 677-686Crossref PubMed Scopus (79) Google Scholar, 17Box AH Demetrick DJ Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines.Carcinogenesis. 2004; 25: 2325-2335Crossref PubMed Scopus (57) Google Scholar Activation of the ATR cell cycle checkpoint following UV-induced DNA damage allows time for DNA repair (reviewed in18Cimprich KA Cortez D ATR: an essential regulator of genome integrity.Nat Rev Mol Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1324) Google Scholar). ATR phosphorylates and activates Chk1, and to a lesser extent Chk2, kinases that phosphorylate the cell cycle regulator Cdc25a.19Falck J Mailand N Syljuasen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (872) Google Scholar, 20Lee CH Chung JH The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation.J Biol Chem. 2001; 276: 30537-30541Crossref PubMed Scopus (129) Google Scholar Phosphorylation by Chk1/2 inactivates Cdc25a and targets it for rapid, ubiquitin-directed degradation.19Falck J Mailand N Syljuasen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (872) Google Scholar, 21Mailand N Falck J Lukas C Syljuasen RG Welcker M Bartek J Lukas J Rapid destruction of human Cdc25A in response to DNA damage.Science. 2000; 288: 1425-1429Crossref PubMed Scopus (651) Google Scholar The Cdc25a phosphatase activates cyclin-dependent kinase (CDK)2 by removal of inhibitory phosphate groups at CDK2-Tyr15Shtivelman E Sussman J Stokoe D A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle.Curr Biol. 2002; 12: 919-924Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar and CDK2-Thr14El Abaseri TB Fuhrman J Trempus C Shendrik I Tennant RW Hansen LA Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the epidermal growth factor receptor.Cancer Res. 2005; 65: 3958-3965Crossref PubMed Scopus (82) Google Scholar.19Falck J Mailand N Syljuasen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (872) Google Scholar, 21Mailand N Falck J Lukas C Syljuasen RG Welcker M Bartek J Lukas J Rapid destruction of human Cdc25A in response to DNA damage.Science. 2000; 288: 1425-1429Crossref PubMed Scopus (651) Google Scholar, 22Xiao Z Chen Z Gunasekera AH Sowin TJ Rosenberg SH Fesik S Zhang H Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents.J Biol Chem. 2003; 278: 21767-21773Crossref PubMed Scopus (320) Google Scholar Loss of Cdc25a activity following ATR activation thus blocks activation of cyclin/CDK complexes, resulting in cell cycle arrest.22Xiao Z Chen Z Gunasekera AH Sowin TJ Rosenberg SH Fesik S Zhang H Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents.J Biol Chem. 2003; 278: 21767-21773Crossref PubMed Scopus (320) Google Scholar Cell cycle arrest allows time for the repair of DNA damage and reduces mutagenesis. If cell cycle arrest and DNA repair mechanisms are inadequate, cells acquire mutations that lead to cancer. EGFR promotes G2/M-phase progression by blocking the activation of this cell cycle checkpoint through PI3K/Akt-dependent inhibitory phosphorylation of Chk1,11Jean C Hernandez-Pigeon H Blanc A Charveron M Laurent G Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells.J Invest Dermatol. 2007; 127: 2418-2424Crossref PubMed Scopus (17) Google Scholar a potential mechanism for its role in promoting UV-induced skin tumorigenesis.14El Abaseri TB Fuhrman J Trempus C Shendrik I Tennant RW Hansen LA Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the epidermal growth factor receptor.Cancer Res. 2005; 65: 3958-3965Crossref PubMed Scopus (82) Google Scholar In contrast to the extensive literature documenting the effects of EGFR activation on the response of the skin to UV, little investigation of the importance of other EGFR family members in UV-induced skin carcinogenesis has been undertaken. We hypothesized that repeated activation of the Erbb2 receptor resulting from chronic exposure to UV might also contribute to UV-induced skin tumorigenesis by deregulating cell cycle checkpoint control. This paradigm does not require oncogenic activation of Erbb2, but rather depends on repeated cycles of activation of normal physiological levels of proto-oncogenic Erbb2. We found that inhibition of the UV-induced activation of Erbb2 substantially reduced skin tumorigenesis in a transgenic mouse model. Using both mouse skin and cell culture models, we also determined that UV-induced skin tumorigenesis was associated with Erbb2-dependent inhibitory phosphorylation of Chk1, maintenance of Cdc25a, and decreased cell cycle arrest. These data demonstrate that activation of Erbb2 on UV irradiation increases UV-induced skin tumorigenesis by suppressing a DNA damage-induced cell cycle checkpoint. The dorsal hair of homozygous v-rasHa transgenic Tg.AC mice on an FVB/N background was clipped one day before treatment using electric clippers (Wahl, Sterling, IL) and on the day of treatment with a Remington Microscreen shaver (Madison, NC). Four mg AG825 (AG Scientific, San Diego, CA and Calbiochem, San Diego, CA) dissolved in 200 μl dimethyl sulfoxide (DMSO), or the vehicle alone, was applied topically to the shaved back of the mice 2 hours before exposure to 10 kJ/m2 UV or sham irradiation. This concentration of AG825 applied over the shaved dorsal surface of the mouse did not significantly absorb UV. The UV-B TL 40W/12 RS bulbs (Philips, Somerset, NJ) used emitted approximately 30% UVA, 70% UVB, and <1% UVC, with a total output of 470 μW/cm2, as measured with radiometric photodetector probes (Newport, Irvine, CA). Tumor number, tumor volume using calipers, and skin-fold thickness using calipers (Mitutoyo, Aurora, IL) were assessed weekly. All animal procedures were performed in accordance with American Association of Laboratory Animal Care guidelines and approved by Creighton University's Institutional Animal Care and Use Committee. Primary newborn mouse keratinocytes from CD-1 mice were isolated as described previously.23Lichti U Anders J Yuspa SH Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.Nature Protocols. 2008; 3: 799-810Crossref PubMed Scopus (346) Google Scholar In brief, the skins were floated overnight on 0.25% trypsin at 37°C, the epidermis separated, minced, centrifuged in S-MEM (Invitrogen, Carlsbad, CA) with 8% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), and plated in this medium. The next day, cells were refed S-MEM with 8% chelexed serum and a calcium concentration adjusted to 0.05 mmol/L. The cells were grown in this medium to 70% to 80% confluence, the medium replaced with a thin layer of PBS containing 0.05 mmol/L calcium, and exposed to 600 J/m2 UV or sham irradiated as described previously.1Madson JG Lynch DT Tinkum KL Putta SK Hansen LA Erbb2 regulates inflammation and proliferation in the skin after ultraviolet irradiation.Am J Pathol. 2006; 169: 1402-1414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Some keratinocytes were treated with 45 μmol/L AG825 (or with the concentrations indicated elsewhere)(AG Scientific, San Diego, CA), 15 μmol/L PI3K inhibitor LY294002 (Calbiochem, La Jolla, CA), 15 μmol/L Akt inhibitor IL-6-hydroxymethyl-chiro-inositol-2–20-methyl-3-O-ocadecylcarbomate (Calbiochem) or the vehicle alone. Plasmids with Cdc25a24Hassepass I Voit R Hoffmann I Phosphorylation at serine 75 is required for UV-mediated degradation of human Cdc25A phosphatase at the S-phase checkpoint.J Biol Chem. 2003; 278: 29824-29829Crossref PubMed Scopus (70) Google Scholar and enhanced green fluorescent protein (GFP) cDNA were cotransfected with Lipofectamine Transfection Reagent (Invitrogen, Carlsbad, CA). Transfection efficiency was quantified by determining the proportion of the cells that incorporated the GFP 1 day post-transfection. To obtain keratinocytes with genetic ablation of Erbb2, loxP sites were inserted flanking exon 1 of Erbb2, as described in.25Leu M Bellmunt E Schwander M Farinas I Brenner HR Muller U Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development.Development. 2003; 130: 2291-2301Crossref PubMed Scopus (74) Google Scholar Keratinocytes homozygous for the Erbb2 loxP mutation were infected with Cre recombinase-expressing or empty adenoviral vectors in polybrene (Sigma, St. Louis, MO). For in vivo analysis of cell cycle distribution, sections of formalin-fixed skin from paraffin embedded blocks were digested in PBS containing 0.5% pepsin (Sigma, St. Louis, MO). Cultured keratinocytes or pepsin-digested sections were suspended in Vindelov's solution (3.5 mmol/L Tris base, pH 7.6, 10 mmol/L NaCl, 10 μg/ml ribonuclease A, 75 μg/ml propidium iodide, 1.0 μl/ml Ipegal),26Vindelov LL Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei Virchows.Arch B Cell Pathol. 1977; 24: 227-242PubMed Google Scholar run on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and analyzed using ModFit LT 3.1 software (Verity Software House, Topsham, ME). Some cells were treated with 10 μmol/L 5-bromo-2′-deoxyuridine (BrDU, Sigma) before harvest, treated with hydrochloric acid and trypsin, incubated with a fluorescein isothiocyanate-conjugated anti-BrDU antibody (BD Biosciences, San Diego, CA), and propidium iodide added before analysis. Data from at least 10,000 cells from each sample were collected using the flow cytometer. Flash frozen skin was ground with a mortar and pestle on dry ice then homogenized with a polytron, or cells lysed in buffer containing 10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1 mmol/L EDTA, Complete Protease Inhibitor Cocktail (Roche, Germany), 1 mmol/L Na3VO4, 1.5 μmol/L EGTA, and 10 μmol/L NaF. Protein was measured using a BioRad assay (Sigma) and equal amounts of protein run on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Equal loading and transfer were confirmed using Ponceau S staining (Sigma). Membranes were immunoblotted with antibodies recognizing actin (Sigma), phosphorylated ATR/M substrates (Cell Signaling, Danvers, MA), Cdc25a (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc25b (Cell Signaling), Cdc25c (Santa Cruz Biotechnology), phospho-Chk1-Ser296 (Cell Signaling), phospho-Chk1-Ser280 (gift of Ramon Parsons), phospho-Chk1345 (Santa Cruz), phospho-Chk2387 (Cell Signaling), and phospho-Akt (Cell Signaling, Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and visualized using chemiluminescent reagents (Pierce, Rockford, IL). H&E staining was performed on paraffin-embedded sections following standard protocols. Following antigen retrieval, paraffin-embedded sections were incubated with antibodies to keratin 1, keratin 6, or filaggrin (all from Covance, Princeton, NJ), Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Carlsbad, CA), and 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Promega, Madison, WI). The tumor experiment was analyzed using two-way analysis of variance (ANOVA). Statistical significance in other experiments was assessed using two-way ANOVA or Student's t-test with Bonferroni post-test. All experiments, excluding the tumor experiment, were replicated several times and consistent results obtained. To test our hypothesis that the inhibition of Erbb2 prevents UV-induced skin tumorigenesis, groups of mice were topically treated with the Erbb2 inhibitor AG825 before UV irradiation using a published method.1Madson JG Lynch DT Tinkum KL Putta SK Hansen LA Erbb2 regulates inflammation and proliferation in the skin after ultraviolet irradiation.Am J Pathol. 2006; 169: 1402-1414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar This skin tumorigenesis experiment was performed in v-rasHa transgenic Tg.AC mice, because of their enhanced sensitivity to UV-induced skin tumorigenesis,14El Abaseri TB Fuhrman J Trempus C Shendrik I Tennant RW Hansen LA Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the epidermal growth factor receptor.Cancer Res. 2005; 65: 3958-3965Crossref PubMed Scopus (82) Google Scholar, 27Trempus CS Mahler JF Ananthaswamy HN Loughlin SM French JE Tennant RW Photocarcinogenesis and susceptibility to UV radiation in the v-Ha-ras transgenic Tg.AC mouse.J Invest Dermatol. 1998; 111: 445-451Crossref PubMed Scopus (28) Google Scholar according to the timeline shown in Figure 1A. Inhibition of Erbb2 before UV irradiation blocked the development of more than half of the tumors, with 34 tumors per vehicle-treated mouse and only 15 tumors per inhibitor-treated mouse by the end of the experiment (Figure 1B). While the vehicle-treated mice continued to accrue tumors throughout the duration of the experiment, the inhibitor-treated group reached a plateau in tumor number by 7 weeks after the first UV exposure (Figure 1B). By the end of the experiment, mean tumor volume was also 70% less in the AG825-treated group when compared with the vehicle control (Figure 1B, axis on right). Representative tumors from each treatment group were examined histologically and all were benign squamous papillomas (see supplemental Figure S1A at http://ajp.amjpathol.org) with similar differentiation status (see supplemental Figure S1B at http://ajp.amjpathol.org). These data demonstrate that blocking the UV-induced activation of Erbb2 suppresses mouse skin tumorigenesis. Because AG825 only transiently inhibits Erbb2 activity, the effects of the inhibitor were examined during the first few days after inhibitor treatment and UV irradiation. The influence of the Erbb2 inhibitor on epidermal hyperplasia was measured during the first 2 weeks of the tumor experiment regimen outlined in Figure 1A. Little hyperplasia was induced in the first week after UV irradiation, and the effect of the Erbb2 inhibitor on this response was minimal (Figure 2A, left panel). Following the second UV irradiation, epidermal hyperplasia was significantly suppressed by inhibition of Erbb2 (Figure 2A, left and right panels, day 9). Thus, the UV-induced activation of Erbb2 augments epidermal hyperplasia, a response that becomes more pronounced with multiple UV exposures. The influence of Erbb2 on both apoptosis and cell proliferation was assessed in vivo to determine the mechanisms of Erbb2's effect on hyperplasia in the skin after UV irradiation. The percentage of basal cells positive for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was increased at 24 hours after UV in vehicle-treated skin (Figure 2B), consistent with previous observations in this animal model (14). Inhibition of Erbb2 did not increase apoptotic cell death at this time point (Figure 2B). Erbb2 reportedly suppresses keratinocyte differentiation,28De Potter IY Poumay Y Squillace KA Pittelkow MR Human EGF receptor (HER) family and heregulin members are differentially expressed in epidermal keratinocytes and modulate differentiation.Exp Cell Res. 2001; 271: 315-328Crossref PubMed Scopus (59) Google Scholar a process leading to keratinocyte death that is mechanistically and morphologically distinct from apoptosis. Inhibition of Erbb2 before UV irradiation did not alter the localization of expression of early (keratin 5) or late (loricrin) markers of differentiation in the skin (see supplemental Figure S2A at http://ajp.amjpathol.org). These data demonstrate that Erbb2's influence on hyperplasia and tumor development does not result from the suppression of cell death via apoptosis or terminal differentiation following UV exposure but rather must be a consequence of an effect on cell proliferation. Mean proliferating cell nuclear antigen expression, a marker of cell proliferation, was less in Erbb2 inhibitor treated sham-irradiated skin and in UV-exposed skin when compared with the vehicle-treated controls (see supplemental Figure S2B at http://ajp.amjpathol.org). Cell cycle analysis revealed that the percentage of cells in S-phase increased on inhibition of Erbb2 before UV irradiation (Figure 2C). These data suggest that Erbb2 affects cell cycle regulation in the skin, a hypothesis that was further investigated using both inhibition and genetic ablation of the receptor in cultured keratinocytes. Inhibition and genetic ablation of Erbb2 in cultured keratinocytes were used to determine the mechanisms through which Erbb2 contributes to skin tumorigenesis. To test our hypothesis that the UV-induced activation of Erbb2 blocks cell cycle arrest after UV irradiation, DNA synthesis was examined after UV irradiation of cultured primary keratinocytes lacking Erbb2 activity. Forty-five μmol/L AG825 was required to block the UV-induced activation of Erbb2 in primary keratinocytes, as shown in Figure 3A. BrDU incorporation was significantly reduced in sham-irradiated keratinocytes lacking Erbb2 activity (Figure 3, B and C). By 12 hours after UV irradiation, BrDU incorporation was reduced to nearly zero in keratinocytes lacking Erbb2 activity while substantial BrDU incorporation occurred in the irradiated controls with intact Erbb2 signaling (Figure 3, B and C). Thus, Erbb2 activation was necessary for DNA synthesis following UV irradiation. While apoptosis was significantly increased in the inhibitor-treated and sham-irradiated cells compared with the vehicle-treated controls (see supplemental Figure S3 at http://ajp.amjpathol.org), the decrease in DNA synthesis after UV irradiation was not an indirect result of increased apoptosis. Inhibition of Erbb2 did not increase UV-induced apoptosis (see supplemental Figure S3 at http://ajp.amjpathol.org). UV irradiation caused S-phase accumulation of keratinocytes (Figure 3, D and F), consistent with previous reports.29Hirose Y Katayama M Mirzoeva OK Berger MS Pieper RO Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence.Cancer Res. 2005; 65: 4861-4869Crossref PubMed Scopus (136) Google Scholar The influence of Erbb2 activation on S-phase progression was determined using both pharmacological and genetic models to interfere with Erbb2 signaling. Pharmacological inhibition of Erbb2 before UV exposure increased the percentage of cells in S-phase by 10% (Figure 3D). Erbb2 was also ablated by Cre recombinase expression in Erbb2fl/fl cultured primary keratinocytes (Figure 3E). Genetic ablation of Erbb2 increased UV-induced S-phase arrest by 18% after UV irradiation (Figure 3F). These data demonstrate that abrogation of Erbb2 signaling contributes to S-phase arrest in both sham-irradiated keratinocytes and after UV irradiation. Erbb2 promotes cell cycle progression by maintaining Cdc25a To understand how Erbb2 regulates S-phase progression, signaling pathways known to regulate progression into and through S-phase were examined. As a key mediator of S-phase arrest after UV irradiation, the ATR DNA damage response pathway was examined. Cdc25a is degraded in response to ATR activation and its degradation, in turn, triggers S-phase arrest.19Falck J Mailand N Syljuasen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed
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