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There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-rasLA1). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-rasLA1 mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.

The current study was designed to explore the role of signal transducer and activator of transcription 1 (Stat1) during tumor promotion using the mouse skin multistage carcinogenesis model. Topical treatment with both 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-methyl-1,8-dihydroxy-9-anthrone (chrysarobin or CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Y701) and serine (S727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. CHRY treatment also led to upregulation of interferon regulatory factor 1 (IRF-1) mRNA and protein, which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNγ) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNγ signaling. Stat1 deficient (Stat1(-/-) ) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1(-/-) mice and wild-type littermates with TPA as the promoter. Maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNγ signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1, IRF-1 and uStat1.

Proton radiotherapy is becoming more common as protons induce more precise DNA damage at the tumor site with reduced side effects to adjacent normal tissues. However, the long-term biological effects of proton irradiation in cancer initiation compared with conventional photon irradiation are poorly characterized. In this study, using a human familial adenomatous polyposis syndrome susceptible mouse model, we show that whole-body irradiation with protons are more effective in inducing senescence-associated inflammatory responses (SIRs), which are involved in colon cancer initiation and progression. After proton irradiation, a subset of SIR genes (Troy, Sox17, Opg, Faim2, Lpo, Tlr2 and Ptges) and a gene known to be involved in invasiveness (Plat), along with the senescence-associated gene (P19Arf), are markedly increased. Following these changes, loss of Casein kinase Iα and induction of chronic DNA damage and TP53 mutations are increased compared with X-ray irradiation. Proton irradiation also increases the number of colonic polyps, carcinomas and invasive adenocarcinomas. Pretreatment with the non-steroidal anti-inflammatory drug, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid-ethyl amide (CDDO-EA), reduces proton irradiation-associated SIR and tumorigenesis. Thus exposure to proton irradiation elicits significant changes in colorectal cancer initiation and progression that can be mitigated using CDDO-EA.

The cellular and molecular mechanisms that underlie promotion by diverse tumor promoting agents remain to be fully elucidated. Studies using Stat1 knockout (KO) mice have shown that they were highly resistant to two-stage skin carcinogenesis using chrysarobin as the promoter. In contrast, Stat1 KO mice treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) as the tumor promoter exhibited no significant differences in tumor formation. To further investigate mechanism(s) whereby Stat1 mediates skin tumor promotion by chrysarobin Stat1 KO mice and wild-type (WT) controls were treated with the respective tumor promoters and Stat1-dependent signaling pathways were evaluated. Mice deficient in Stat1 displayed an attenuated induction of epidermal Cox-2 mRNA and protein as well as reduced PGE2 levels compared to WT controls following topical application of chrysarobin, whereas, mice treated with TPA exhibited no significant differences in Cox-2 levels or PGE2 levels. Other results also suggest that Stat1 may regulate various cytokine/chemokines involved in the inflammatory response following chrysarobin treatment. Preliminary experiments have also shown that Stat1 KO mice exhibit reduced induction of interferon regulatory factor-1 (IRF-1) following chrysarobin treatment, compared to WT controls. In contrast, treatment with TPA caused no significant differences in expression of IRF-1. Collectively, these findings suggest that chrysarobin may elicit a Stat1-dependent signaling response that is dependent on IRF-1, whereas TPA-mediated tumorigenesis is independent of this signaling pathway. These and other ongoing studies aimed at characterizing the role of Stat1 and IRF-1 signaling in chrysarobin-mediated tumor promotion will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2556. doi:1538-7445.AM2012-2556

Inflammation is a key component of cancer development in many tissues. Emerging clinical observations support the idea of a strong association of chronic inflammation and cancer. The JAK-STAT pathway is one of the major signaling pathways involved in modulating both pro- and anti-inflammatory responses. Aberrant expression of STATs has been reported in multiple human cancers and in animal models of tumorigenesis. Previous work performed in our lab demonstrated a role for Stat1 in mediating the promotion stage of epithelial multi-stage carcinogenesis in mouse skin when chrysarobin was used as the promoting agent. To further investigate the role of Stat1 in chrysarobin-mediated skin tumor promotion, Stat1 knockout (KO) mice and wild-type controls were treated with chrysarobin and the inflammatory response was assessed. Stat1 deficiency caused a reduction in the dermal inflammatory response following a single topical application of chrysarobin. In this regard, Stat1 KO mice had reduced dermal immune cell influx following chrysarobin treatment. In particular, Stat1 KO mice exhibited reduced influx of macrophages following a single application. Other results also suggest that Stat1 may regulate various cytokine/chemokines involved in the inflammatory response following chrysarobin treatment. Mice deficient in Stat1 displayed an attenuated induction of Cox-2 mRNA and protein compared to wild-type controls following a single application of chrysarobin (200 nmol per mouse). This latter data suggest that Stat1 may be involved in the regulation of Cox-2 in keratinocytes. Collectively, these findings suggest that Stat1 may contribute to chrysarobin-mediated skin tumor promotion by regulating, in part, levels of Cox-2, PGE2 and ultimately skin inflammation. These and other ongoing studies aimed at identifying the mechanistic basis for the role of Stat1 in chrysarobin-mediated tumor promotion will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2011-2730

Aberrant expression and/ or activity of STATs 1, 3, and 5 have been reported in multiple human cancers as well as in murine models of tumorigenesis. Previous work performed in our lab demonstrated a critical role for Stat3 in mediating the initiation and promotion stages of epithelial multi-stage carcinogenesis in mouse skin. In light of these findings, we have begun investigating the role of other STATs, particularly Stat1, in multistage skin carcinogenesis using both wild-type and Stat1 knockout (KO) mice. Western blot analyses revealed that topical application of the skin tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and chrysarobin induced phosphorylation of Stat1 in mouse epidermis. In addition, following treatment with chrysarobin, the level of total Stat1 protein was also dramatically elevated. To further investigate the role of Stat1 during epithelial carcinogenesis, Stat1 KO mice and wild-type littermates were initiated with 25 nmol DMBA and promoted with either 6.8 nmol TPA or 220 nmol chrysarobin. No significant differences were observed in either tumor incidence or multiplicity when TPA was used as the promoting agent, suggesting that Stat1 does not play a significant role in TPA-mediated skin tumor promotion. In contrast, Stat1 KO mice exhibited a dramatic reduction in both tumor multiplicity and incidence as compared to wild-type littermates when chrysarobin was used as the promoting agent. Additionally, following topical treatment with chrysarobin, Stat1 KO mice had a reduced epidermal proliferative response. To further investigate the impact of Stat1 deletion on chrysarobin-induced tumor promotion, Stat1 KO mice and wild-type controls were treated topically with chrysarobin and the inflammatory response was assessed. Stat1 KO mice displayed a significant reduction in Cox-2 induction compared to wild-type controls. Preliminary findings also showed that Stat1 KO mice have reduced immune cell influx following chrysarobin treatment. To investigate the potential role of reactive oxygen species (ROS) in the effects of chrysarobin on Stat1 activation, the dorsal skin of FVB mice were pre-treated topically with the anti-oxidants ascorbyl palmitate and α-tocopherol acetate. Topical application of α-tocopherol acetate significantly attenuated the epidermal proliferative response and Stat1 activation suggesting that chrysarobin-mediated generation of ROS may play a role in its effects on Stat1 during tumor promotion. Additional studies are underway to further determine the mechanistic basis for the role of Stat1 in chrysarobin-mediated tumor promotion. Supported by NIH grant CA 76520. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2480.