Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-β−/−) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-β−/−/−− versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-β−/− mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-β−/− mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-β−/−, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-β.

Nicotinamide (NAM), an amide form of vitamin B3, replenishes cellular energy after ultraviolet radiation (UVR) exposure, thereby enhancing DNA repair and reducing UVR’s immunosuppressive effects. NAM reduces actinic keratoses and new keratinocyte cancers in high risk individuals, but its effects on melanoma are unknown. Melanomas arising on NAM or placebo within the ONTRAC skin cancer chemoprevention trial (Oral Nicotinamide To Reduce Actinic Cancer) were examined by immunohistochemistry. The effects of NAM (50 µM, 5 mM and 20 mM) on the viability, proliferation and invasiveness of four human melanoma cell lines and on the viability and proliferation of two human melanocyte lines, with and without UV irradiation were also investigated. 50 µM NAM did not affect viability, proliferation or invasion of melanoma or melanocyte cell lines, whereas concentrations too high to be achievable in vivo reduced viability and proliferation. Nicotinamide did not enhance melanoma viability, proliferation or invasiveness in vitro , providing additional confidence in its safety for use in clinical trials in high risk patients. Peritumoral and tumour infiltrating CD4 + and CD8 + lymphocytes were significantly increased in melanomas arising on NAM compared to those arising on placebo. Given the chemopreventive activity of nicotinamide against keratinocyte cancers, its DNA repair enhancing effects in melanocytes and now its potential enhancement of tumour-infiltrating lymphocytes and lack of adverse effects on melanoma cell growth and proliferation, clinical trials of nicotinamide for melanoma chemoprevention are now indicated

UV-induced DNA damage, suppression of antitumor immune responses, and promotion of cutaneous inflammation underly the skin tumor-promoting activities of sunlight. Targeting the immune regulatory pathways activated by UV is proving to be a highly effective therapeutic strategy in the treatment of both melanoma (Hodi et al., 2010Hodi F.S. O'Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. et al.Improved survival with ipilimumab in patients with metastatic melanoma.N Engl J Med. 2010; 363: 711-723Crossref PubMed Scopus (11264) Google Scholar) and keratinocyte cancers (Day et al., 2017Day F. Kumar M. Fenton L. Gedye C. Durable response of metastatic squamous cell carcinoma of the skin to ipilimumab immunotherapy.J Immunother. 2017; 40: 36-38Crossref PubMed Scopus (30) Google Scholar). However, many of these first-generation check-point inhibitors come with considerable side effects including an increased risk of autoimmune toxicities (Phan et al., 2003Phan G.Q. Yang J.C. Sherry R.M. Hwu P. Topalian S.L. Schwartzentruber D.J. et al.Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.Proc Natl Acad Sci USA. 2003; 100: 8372-8377Crossref PubMed Scopus (1384) Google Scholar) and rejection of allografts in some transplant recipients at a high risk of metastatic skin cancer (Abdel-Wahab et al., 2019Abdel-Wahab N. Safa H. Abudayyeh A. Johnson D.H. Trinh V.A. Zobniw C.M. et al.Checkpoint inhibitor therapy for cancer in solid organ transplantation recipients: an institutional experience and a systematic review of the literature.J Immunother Cancer. 2019; 7: 106Crossref PubMed Scopus (148) Google Scholar). Therefore, alternative approaches and new therapies targeting other immune pathways are needed. We discovered that a major way in which UV suppresses immunity in mice via the activation of a unique type of regulatory B cell (Byrne and Halliday, 2005Byrne S.N. Halliday G.M. B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity.J Invest Dermatol. 2005; 124: 570-578Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Kok et al., 2016Kok L.F. Marsh-Wakefield F. Marshall J.E. Gillis C. Halliday G.M. Byrne S.N. B cells are required for sunlight protection of mice from a CNS-targeted autoimmune attack.J Autoimmun. 2016; 73: 10-23Crossref PubMed Scopus (16) Google Scholar, Matsumura et al., 2006Matsumura Y. Byrne S.N. Nghiem D.X. Miyahara Y. Ullrich S.E. A role for inflammatory mediators in the induction of immunoregulatory B cells.J Immunol. 2006; 177: 4810-4817Crossref PubMed Scopus (70) Google Scholar). Adoptive transfer of B cells from UV-exposed mice suppresses cutaneous immune responses (Byrne and Halliday, 2005Byrne S.N. Halliday G.M. B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity.J Invest Dermatol. 2005; 124: 570-578Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Matsumura et al., 2006Matsumura Y. Byrne S.N. Nghiem D.X. Miyahara Y. Ullrich S.E. A role for inflammatory mediators in the induction of immunoregulatory B cells.J Immunol. 2006; 177: 4810-4817Crossref PubMed Scopus (70) Google Scholar) as well as experimental autoimmune encephalomyelitis (Kok et al., 2016Kok L.F. Marsh-Wakefield F. Marshall J.E. Gillis C. Halliday G.M. Byrne S.N. B cells are required for sunlight protection of mice from a CNS-targeted autoimmune attack.J Autoimmun. 2016; 73: 10-23Crossref PubMed Scopus (16) Google Scholar), a model of central nervous system autoimmunity. Indeed, UV was unable to protect mice from experimental autoimmune encephalomyelitis if B cells had been depleted with a mouse-anti-mouse CD20 mAb. Thus, UV-activated B cells play a key role in UV-induced immune suppression. Considering the central role that UV immune suppression plays in carcinogenesis, we hypothesized that depleting UV-activated immunoregulatory B cells will limit UV-induced skin tumor growth, enhance survival, and/or prevent metastasis to the skin-draining lymph nodes. To investigate this hypothesis, we used a murine photocarcinogenesis model, in which the shaved backs of 8-week-old female C57BL/6 mice (Australian BioResources, Moss Vale, Australia) were exposed, with the approval from the University of Sydney Animal Ethics Committee (#2014/630), to the increasing doses of solar-simulated UV radiation (maximum dose 3.64 J/cm2) four times per week for 30 weeks as we have described (Sarchio et al., 2014Sarchio S.N.E. Scolyer R.A. Beaugie C. McDonald D. Marsh-Wakefield F. Halliday G.M. et al.Pharmacologically antagonizing the CXCR4-CXCL12 chemokine pathway with AMD3100 inhibits sunlight-induced skin cancer.J Invest Dermatol. 2014; 134: 1091-1100Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The appearance, anatomical location, and size of any skin lesion were recorded twice a week. At the end point, all lesions were processed for hematoxylin and eosin and analyzed by histology with only those lesions histopathologically confirmed as squamous cell carcinomas being included in the final analysis. For the prophylactic treatment of mice, 20 weeks after the start of UV, which is before the macroscopic appearance of skin tumors, groups were randomized to receive either 100 μg of mouse anti-mouse CD20 mAb intraperitoneally (supplied by Genentech, San Francisco, CA) or 100 μg of InVivoPlus Mouse IgG2a Isotype control (BioXcell; West Lebanon, NH) (Kok et al., 2016Kok L.F. Marsh-Wakefield F. Marshall J.E. Gillis C. Halliday G.M. Byrne S.N. B cells are required for sunlight protection of mice from a CNS-targeted autoimmune attack.J Autoimmun. 2016; 73: 10-23Crossref PubMed Scopus (16) Google Scholar). Antibodies were injected every 2 weeks for another 20 weeks (Figure 1a). Prophylactic B-cell depletion before the appearance of skin lesions had no effect on UV-induced skin cancer growth (Figure 1b) or survival (Figure 1c). Next, we investigated the therapeutic effect of B-cell depletion in mice bearing a UV-induced tumor. Once an animal had developed a lesion (median 30 weeks, range 27–37 weeks after commencing UV irradiation), they were carefully monitored and continued to receive UV for an additional 4 weeks. If the lesion persisted during that time, then the mouse was randomized to receive either anti-CD20 or isotype. Antibodies were injected every 2 weeks for an additional 28 weeks or until the mice reached their end point (Figure 1d). We found that treating tumor-bearing mice with anti-CD20 antibodies placed significant limits on tumor growth (Figure 1e). The end point of this experiment was reached when a mouse lost more than 15% of their weight, was burdened with more than 10 tumors, or if any one tumor was larger than 100 mm2 in size. Mice treated with anti-CD20 reached this end point significantly later (P = 0.0016; linear regression) than isotype control-treated groups (Figure 1f). By 20 weeks after treatment, all of the control mice had exited the experiment, whereas only 50% of mice with depleted B cells had done so. Finally, to investigate the effect of B cell depletion on skin tumor metastasis, we analyzed the tumor-draining (inguinal) lymph nodes via immunohistology (Figure 2). Cytokeratin-expressing cells coming from the skin could only be detected in control (isotype)-treated mice. Some lymph nodes from these mice had distinct micrometastases (Figure 2a), whereas others had much larger macrometastases (Figure 2b). As expected, the lymph nodes of mice treated with anti-CD20 were devoid of B cells (Figure 2a). However, in contrast to control (isotype)-treated mice, none of the anti-CD20-treated mice had any detectable metastases. In this study, we have shown that B cells play an essential role in allowing the UV-induced skin tumors to grow and metastasize. If B cells are depleted before this carcinogenic cascade is complete (i.e., before macroscopic tumors appear), then there is no reduction in tumor size (Figure 1b), incidence (all mice developed tumors), or multiplicity (median of four and five tumors per mouse treated with isotype or anti-CD20, respectively). This failure to protect from the development of UV-induced keratinocyte cancer may be because nascent tumors, which are not reliant on B cells for their establishment, are permitted to grow and metastasize. In contrast, with UV-activated immunoregulatory B cells present throughout the carcinogenic process, the majority of skin tumors that establish have done so with a reliance on B cells for their growth and survival. Indeed, if that pro-tumorigenic environment is removed (e.g. by depleting B cells), then UV-induced skin tumors can no longer continue to grow (Figure 1e) and the chances of a tumor metastasizing is reduced (Figure 2), thereby leading to significantly enhanced survival (Figure 1f). UV-activated B cells suppress T cell-driven immune responses, in part by interfering with dendritic cells (Byrne and Halliday, 2005Byrne S.N. Halliday G.M. B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity.J Invest Dermatol. 2005; 124: 570-578Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and producing immunoregulatory IL-10 (Matsumura et al., 2006Matsumura Y. Byrne S.N. Nghiem D.X. Miyahara Y. Ullrich S.E. A role for inflammatory mediators in the induction of immunoregulatory B cells.J Immunol. 2006; 177: 4810-4817Crossref PubMed Scopus (70) Google Scholar). This B cell-driven suppression of antitumor immunity may explain our observations. However, when we investigated the tumor-immune infiltrate by multiplex Opal staining, we could not detect any considerable difference in the number of helper (CD4+) or cytotoxic (CD8+) T lymphocytes (Figure 1g). Most of the B220+ cells were also CD8+, which in the periphery may be a marker of T cell activation and/or apoptosis (Renno et al., 1996Renno T. Hahne M. Tschopp J. MacDonald H.R. Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.J Exp Med. 1996; 183: 431-437Crossref PubMed Scopus (125) Google Scholar). Hence, B cells were not readily detectable in any of the tumors, further suggesting that UV-activated B cells may be exerting their pro-tumorigenic effects in the tumor-draining lymph nodes. This absence of B cells in the diseased tissue is consistent with what others have found in viral models of skin carcinogenesis (de Visser et al., 2005de Visser K.E. Korets L.V. Coussens L.M. De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.Cancer Cell. 2005; 7: 411-423Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar) and what we previously observed in the mice exposed to experimental autoimmune encephalomyelitis-protecting UV (Kok et al., 2016Kok L.F. Marsh-Wakefield F. Marshall J.E. Gillis C. Halliday G.M. Byrne S.N. B cells are required for sunlight protection of mice from a CNS-targeted autoimmune attack.J Autoimmun. 2016; 73: 10-23Crossref PubMed Scopus (16) Google Scholar) where the lymph nodes draining the central nervous system, but not the spinal cord itself, contained numerous UV-activated B cells. UV-induced skin cancers are highly antigenic and will be immunologically rejected if transferred into an unirradiated host (Kripke, 1974Kripke M.L. Antigenicity of murine skin tumors induced by ultraviolet light.J Natl Cancer Inst. 1974; 53: 1333-1336Crossref PubMed Scopus (433) Google Scholar). We have shown that depleting B cells can enhance the survival of mice burdened with a UV-induced skin cancer. A role for immunoregulatory B cells in UV-induced keratinocyte cancer growth and metastasis has not been described before; however, our findings are consistent with what others have shown in nonmelanoma skin cancer models. Early studies showed that rendering mice B cell-deficient with anti-IgM antibodies made them resistant to skin tumor challenge, slowed tumor growth, and significantly reduced the incidence of experimental metastasis (Brodt and Gordon, 1978Brodt P. Gordon J. Anti-tumor immunity in B lymphocyte-deprived mice. I. Immunity to a chemically induced tumor.J Immunol. 1978; 121: 359-362PubMed Google Scholar). Genetically B cell-deficient (μMT) mice reject transplanted UV-induced skin tumors more frequently than controls (Monach et al., 1993Monach P.A. Schreiber H. Rowley D.A. CD4+ and B lymphocytes in transplantation immunity. II. Augmented rejection of tumor allografts by mice lacking B cells.Transplantation. 1993; 55: 1356-1361Crossref PubMed Scopus (65) Google Scholar), and B cells play a key role in promoting chemical- (Schioppa et al., 2011Schioppa T. Moore R. Thompson R.G. Rosser E.C. Kulbe H. Nedospasov S. et al.B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis.Proc Natl Acad Sci USA. 2011; 108: 10662-10667Crossref PubMed Scopus (247) Google Scholar) and viral- (de Visser et al., 2005de Visser K.E. Korets L.V. Coussens L.M. De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.Cancer Cell. 2005; 7: 411-423Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar) induced nonmelanoma skin carcinogenesis. In contrast, using murine models that have been adoptively transferred with weakly immunogenic tumor cell lines, B cells have been shown to play a role in mediating tumor regression (Li et al., 2009Li Q. Teitz-Tennenbaum S. Donald E.J. Li M. Chang A.E. In vivo sensitized and in vitro activated B cells mediate tumor regression in cancer adoptive immunotherapy.J Immunol. 2009; 183: 3195-3203Crossref PubMed Scopus (93) Google Scholar) and expanding antitumor T cells (Candolfi et al., 2011Candolfi M. Curtin J.F. Yagiz K. Assi H. Wibowo M.K. Alzadeh G.E. et al.B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.Neoplasia. 2011; 13: 947-960Crossref PubMed Scopus (73) Google Scholar). Thus, the role played by B cells in tumor growth and metastasis is complex and may differ depending on the location and antigenicity of the tumor. In conclusion, we have confirmed that targeting B cells with a depleting mAb is an effective therapeutic strategy in a mouse model of UV-induced skin cancer. Analogous CD20 targeting mAbs, including rituximab and ocrelizumab, exist for human B cells and are in clinical use for the treatment of leukemia and autoimmune diseases. If our findings in mice hold true for humans with UV-induced skin cancers, then these drugs could be rapidly deployed to tackle aggressive metastatic keratinocyte cancers. Raw data are available via Mendeley (https://doi.org/10.17632/r555sx5wft.1). Lai-Fong Kok: https://orcid.org/0000-0001-5983-9159 Angela L. Ferguson: https://orcid.org/0000-0001-5974-0423 Jacqueline E. Marshall: https://orcid.org/0000-0002-3263-3297 Benita C.Y. Tse: https://orcid.org/0000-0002-0056-8056 Gary M. Halliday: https://orcid.org/0000-0003-1040-2321 Scott N. Byrne: https://orcid.org/0000-00033029-1710 The authors state no conflict of interest. This work was supported by a Cancer Council NSW project grant (RG14-14). The authors gratefully acknowledge Sydney Microscopy & Microanalysis for their assistance with the multiplex staining. Conceptualization: LK, AF, GH, SB; Data Curation: LK, AF, JM, BT, SB; Formal Analysis: LK, AF, JM, BT, GH, SB; Funding Acquisition: GH, SB; Investigation: LK, AF, JM, BT; Methodology: LK, AF, GH, SB; Project Administration: LK, AF, GH, SB; Validation: LK, AF, JM, BT, GH, SB; Visualization: AF, JM, BT, SB; Writing - Original Draft Preparation: SB; Writing - Review and Editing: LK, AF, JM, BT, GH, SB.

Nicotinamide (NAM), an amide form of vitamin B3, has been recognized as an effective chemopreventive agent for keratinocyte cancers (KCs) (basal cell carcinoma and squamous cell carcinoma [SCC]) and actinic keratoses. The ONTRAC study (Oral Nicotinamide To Reduce Actinic Cancer, Australian New Zealand Clinical Trials Registry number ACTRN12612000625875) was a multicenter, phase 3, double-blinded, controlled trial in which NAM (500 mg) or placebo was given twice daily for 12 months to immunocompetent patients who had at least two KCs in the previous 5 years. In this high-risk population, NAM reduced the incidence of new KCs by 23%, with similar efficacy in preventing both SCC and basal cell carcinoma (Chen et al., 2015Chen A.C. Martin A.J. Choy B. Fernández-Peñas P. Dalziell R.A. McKenzie C.A. et al.A phase 3 randomized trial of nicotinamide for skin-cancer chemoprevention.N Engl J Med. 2015; 373: 1618-1626Crossref PubMed Scopus (363) Google Scholar). Chemopreventive efficacy has also been reported in immunocompromised transplant recipients (Bostom et al., 2016Bostom A.G. Merhi B. Walker J. Robinson-Bostom L. More than skin deep? Potential nicotinamide treatment applications in chronic kidney transplant recipients.World J Transpl. 2016; 6: 658-664Crossref PubMed Google Scholar, Drago et al., 2016Drago F. Ciccarese G. Parodi A. Nicotinamide for skin-cancer chemoprevention.N Engl J Med. 2016; 374: 789-790Crossref PubMed Scopus (14) Google Scholar). UVR diminishes antigen-presenting capability and induces immunosuppressive cytokines (Halliday, 2005Halliday G.M. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.Mutat Res. 2005; 571: 107-120Crossref PubMed Scopus (362) Google Scholar). The photoprotective effects of NAM on skin immunity (Damian et al., 2008Damian D.L. Patterson C.R. Stapelberg M. Park J. Barnetson R.S. Halliday G.M. UV radiation-induced immunosuppression is greater in men and prevented by topical nicotinamide.J Invest Dermatol. 2008; 128: 447-454Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) may be due to its ability to enhance DNA repair, thereby reducing DNA photolesions (Surjana et al., 2013Surjana D. Halliday G.M. Damian D.L. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.Carcinogenesis. 2013; 34: 1144-1149Crossref PubMed Scopus (86) Google Scholar), which are a trigger for UV-induced immunosuppression (Kripke et al., 1992Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.Proc Natl Acad Sci USA. 1992; 89: 7516-7520Crossref PubMed Scopus (463) Google Scholar). Understanding the immune mechanisms in skin cancer, including the roles of T and B lymphocytes, macrophages, and dendritic cells, can provide a basis for chemopreventive and therapeutic opportunities (Rangwala and Tsai, 2011Rangwala S. Tsai K.Y. Roles of the immune system in skin cancer.Br J Dermatol. 2011; 165: 953-965Crossref PubMed Scopus (130) Google Scholar). We analyzed KCs collected at the Royal Prince Alfred Hospital ONTRAC study site (Sydney, Australia) (n = 130), using a range of immunological markers to better understand the mechanisms by which NAM reduced skin carcinogenesis. We also assessed DNA damage in tumors arising at sun-exposed sites on NAM and on placebo by staining for cyclobutane pyrimidine dimers (CPDs) and 7,8-dihydro-8-oxoguanine (8oxoG). A total of 130 tumors from 78 patients were included in the study (70 and 60 arose in patients receiving placebo and NAM, respectively). The study was approved by the Ethics Committees of the University of Sydney and the Sydney Local Health District, and all patients provided written informed consent. Selection of tumors was made blinded to treatment allocation (see Supplementary Figure S1 online). To minimie the risk of false-p ositive results arising from multiple hypothesis testing, we a priori grouped the immunological markers, on the basis of their intercorrelation, to form four indices (see Supplementary Figure S2 online for statistical methods). Thus, the primary analyses of immunological markers comprised a family of four hypothesis tests. Index 1 reflected the abundance of lymphocyte markers and was derived from CD3 (Novocastra, Leica Biosystems, Newcastle, UK), CD4 (Cell Marque, Rocklin, CA), CD8 (Dako, Carpenteria, CA), and FoxP3 (Abcam, Melbourne, Australia). Index 2 reflected the abundance of macrophage markers and was derived from CD68 (Dako) and CD163 (Novocastra). Index 3 was the count of the dendritic cell marker CD11c (Novocastra). Index 4 was the count of the proliferative cell marker, Ki-67 (Ventana, Tuscan, AZ) (see Supplementary Materials and Methods). Tumors arising on sun-exposed sites (head, neck, elbows, forearms, and hands) were stained for DNA damage markers (CPDs and 8oxoG, n = 49 tumors). Comparisons between randomized groups were performed using a mixed linear modeling approach. The detailed methods are described in the Supplementary Materials and Methods and elsewhere (Thompson et al., 2014Thompson B.C. Surjana D. Halliday G.M. Damian D.L. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in primary melanocytes.Exp Dermatol. 2014; 23: 509-511Crossref PubMed Scopus (47) Google Scholar). Analysis of the immunological markers showed no convincing statistical evidence of a treatment effect on Index 1 (lymphocyte markers CD3, CD4, CD8, and FoxP3; P = 0.06), Index 3 (dendritic cell marker CD11c; P = 0.06), or Index 4 (proliferative cell marker Ki-67; P = 0.94 peritumoral and P = 0.77 intratumoral) (Figure 1). The effect of NAM on Index 2 (macrophage markers CD68 and CD163 counts) was significant (P = 0.0029). Secondary analyses showed this to be driven by a prominent reduction in CD68 in patients receiving NAM (P = 0.0018), although no clear treatment effect on CD163 was noted (P = 0.15). A post hoc analysis of the ratio of CD163 to CD68 provided some evidence of a NAM effect (P = 0.0482). This reflects the disproportionately larger decrease in CD68 cells compared with CD163 cells in the NAM group of tumors (Figure 1). Analysis of the DNA damage markers found nonsignificant reductions with NAM in (i) CPD lesions of 49% in the epidermis (P = 0.21) and 21% intratumorally (P = 0.73) (Figure 2a) and (ii) 8oxoG lesions in the epidermis of 10.5% (P = 0.76) and in the tumoral region of 28.5% (P = 0.37) (Figure 2b). CPDs and 8oxoG have previously been observed in human skin SCCs and actinic keratoses (Agar et al., 2004Agar N.S. Halliday G.M. Barnetson R.S. Ananthaswamy H.N. Wheeler M. Jones A.M. The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis.Proc Natl Acad Sci USA. 2004; 101: 4954-4959Crossref PubMed Scopus (447) Google Scholar). DNA repair, essential for the prevention of UV-induced carcinogenesis, is a highly energy dependent process. NAM is a precursor of nicotinamide adenine dinucleotide and an essential coenzyme in adenosine triphosphate production (Park et al., 2010Park J. Halliday G.M. Surjana D. Damian D.L. Nicotinamide prevents ultraviolet radiation-induced cellular energy loss.Photochem Photobiol. 2010; 86: 942-948Crossref PubMed Scopus (97) Google Scholar). Consistent with previously observed effects of NAM on DNA repair in ex vivo UV-irradiated human skin (Surjana et al., 2013Surjana D. Halliday G.M. Damian D.L. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.Carcinogenesis. 2013; 34: 1144-1149Crossref PubMed Scopus (86) Google Scholar), tumors arising in NAM-supplemented participants tended to have lower levels of CPD and 8oxoG photolesions, although this failed to reach significance. It is likely that there was high variability in intensity and time since sun exposure, as well as variability in DNA repair efficiency, in our study participants. This would have resulted in a large spread in the data. We found a significant decrease in the number of macrophages in KCs that arose in patients receiving NAM compared with placebo. This was restricted to a significant reduction in tumor-associated cells expressing CD68+, which is a marker identifying both M1 and M2 macrophages. This is consistent with the observation of higher numbers of CD68+ macrophages in SCC compared with normal skin (Pettersen et al., 2011Pettersen J.S. Fuentes-Duculan J. Suarez-Farinas M. Pierson K.C. Pitts-Kiefer A. Fan L. et al.Tumor-associated macrophages in the cutaneous SCC microenvironment are heterogeneously activated.J Invest Dermatol. 2011; 131: 1322-1330Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and the association of CD68+ cells with inflammation and higher 10-year uveal melanoma mortality (Mäkitie et al., 2001Mäkitie T. Summanen P. Tarkkanen A. Kivelä T. Tumor-infiltrating macrophages (CD68+ cells) and prognosis in malignant uveal melanoma.Invest Ophthalmol Vis Sci. 2001; 42: 1414-1421PubMed Google Scholar). CD163, which is associated with an M2 macrophage functional program, was not significantly reduced. This suggests that M1 macrophages are specifically depleted by NAM. NAM's effects on macrophages have been sparsely documented, although it has been reported that in diabetic patients NAM suppresses inflammatory cytokine production by monocytes and macrophages (Krętowski et al., 2000Krętowski A. Myśliwiec J. Szelachowska M. Kinalski M. Kinalska I. Nicotinamide inhibits enhanced in vitro production of interleukin-12 and tumour necrosis factor-α in peripheral whole blood of people at high risk of developing type 1 diabetes and people with newly diagnosed type 1 diabetes.Diabetes Res Clin Pract. 2000; 47: 81-86Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Inflammation and reactive oxygen/nitrogen species, which are produced by M1 macrophages (Tan et al., 2016Tan H.Y. Wang N. Li S. Hong M. Wang X. Feng Y. The reactive oxygen species in macrophage polarization: reflecting its dual role in progression and treatment of human diseases.Oxid Med Cell Longev. 2016; 2016: 2795090Crossref PubMed Scopus (326) Google Scholar), are recognized promoters of skin carcinogenesis (Halliday, 2005Halliday G.M. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.Mutat Res. 2005; 571: 107-120Crossref PubMed Scopus (362) Google Scholar), and NAM inhibition of inflammatory cytokine production by macrophages may therefore contribute to its chemopreventive effects on skin cancer. The tumors studied, however, are the subset that resisted chemoprevention by NAM, and the effects of NAM on cancers that failed to grow could not determined. The authors state no conflict of interest. This work was supported by funding from the Sydney Medical School Foundation, The Australasian College of Dermatologists, National Health & Medical Research Council project grant number 1026977. RM was supported by two University of Sydney scholarships: The Postgraduate Scholarship in Dermatology and the Walter Eberhard Schroeder Dermatology Research Scholarship. RAS is supported by an Australian National Health and Medical Research Council practitioner fellowship. We would also like to acknowledge the kind contribution of the patients who participated in the ONTRAC study. Assistance from colleagues in the Tissue Pathology and Diagnostic Oncology departments at the Royal Prince Alfred Hospital and the Melanoma Institute Australia's Melanoma Pathology Translational Research group is also gratefully acknowledged. Download .pdf (.81 MB) Help with pdf files Supplementary Data

Nicotinamide (vitamin B3) has photoprotective effects and reduces skin cancer incidence in high risk patients. Nicotinamide also improves cognition in animal models. As part of the ONTRAC (Oral Nicotinamide To Reduce Actinic Cancer) phase III placebo-controlled, randomized trial to assess nicotinamide's efficacy in skin cancer prevention, we included clinical neurocognitive function and patient-reported quality of life assessments at baseline and after 12 months of intervention in individuals with previous skin cancer in order to assess any effect of oral nicotinamide (500 mg po twice daily) on cognitive function and quality of life. In our sample of 310 participants who completed neurocognitive function testing at baseline and at 12 months, we were not able to detect any significant effect of oral nicotinamide on cognitive function nor on quality of life. Further studies of nicotinamide's effects on cognition in humans might include individuals with pre-existing mild cognitive impairment, and it may be that higher doses of nicotinamide are required to significantly influence cognitive function compared to doses required to reduce skin cancer.

Nicotinamide is a water-soluble vitamin B3 derivative that has many roles in medicine. This review examines the role of nicotinamide in dermatology and its actions in preventing photoageing and skin cancers in humans. Nicotinamide prevents ultraviolet radiation (UV) from reducing ATP levels and inhibiting glycolysis, thus preventing the UV radiation-induced energy crisis. This enhances DNA repair and reduces UV-induced suppression of immunity. Randomised controlled clinical trials have also shown that nicotinamide reduces transepidermal water loss and the development of new non-melanoma skin cancers in high-risk humans. This review also examines nicotinamide's safety profile.

BackgroundUltraviolet radiation (UVR) is the principal cause of keratinocyte skin cancers. Previous work found that levels of the chromatin remodelling protein, Brahma (Brm), are diminished during the progression from actinic keratoses to cutaneous squamous cell carcinomas in humans, and its loss in UV-irradiated mouse skin causes epidermal hyperplasia and increased tumour incidence.MethodsThe skins of mice and mouse and human keratinocytes deficient in Brm were exposed to UVR and evaluated for cell cycle progression and DNA damage response.ObjectiveTo identify the mechanisms by which loss of Brm contributes to UVR-induced skin carcinogenesis.ResultsIn both mouse keratinocytes and HaCaT cells, Brm deficiency led to an increased cell population growth following UVR exposure compared to cells with normal levels of Brm. Cell cycle analysis using a novel assay showed that Brm-deficient keratinocytes entered cell cycle arrest normally, but escaped from cell cycle arrest faster, enabling them to begin proliferating earlier. In mouse keratinocytes, Brm primarily affected accumulation in G0/G1-phase, whereas in HaCaT cells, which lack normal p53, accumulation in G2/M-phase was affected. Brm-deficient keratinocytes in mouse skin and human cell cultures also had higher levels of UVR-induced cyclobutane pyrimidine dimer photolesions. These effects occurred without any compensatory increase in DNA repair or cell death to remove photolesions or the cells that harbor them from the keratinocyte population.ConclusionThe loss of Brm in keratinocytes exposed to UVR enables them to resume proliferation while harboring DNA photolesions, leading to an increased fixation of mutations and, consequently, increased carcinogenesis.

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4(+) cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity.

The cornea sits at the anterior aspect of the eye and, like the skin, is highly exposed to ultraviolet radiation ( UVR ). The cornea blocks a significant proportion of UVB from reaching the posterior structures of the eye. However, UVA can penetrate the full thickness of the cornea, even reaching the anterior portion of the lens. Epidemiological data indicate that UVR is a contributing factor for a multitude of diseases of the cornea including pterygium, photokeratitis, climatic droplet keratopathy and ocular surface squamous neoplasia ( OSSN ), although the pathogenic mechanisms of each require further elucidation. UVR is a well‐known genotoxic agent, and its effects have been well characterized in organs such as the skin. However, we are only beginning to identify its effects on the cornea, such as the UVR signature C → T and CC → TT transversions identified by sequencing and increased proliferative and shedding rates in response to UVR exposure. Alarmingly, a single low‐dose exposure of UVR to the cornea is sufficient to elicit genetic, molecular and cellular changes, supporting the consideration of using protective measures, such as wearing sunglasses when outdoors. The aim of this review was to describe the adverse effects of UVR on the cornea.

Inadequately repaired post-UV DNA damage results in skin cancers. DNA repair requires energy but skin cells have limited capacity to produce energy after UV insult. We examined whether energy supply is important for DNA repair after UV exposure, in the presence of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), which reduces UV-induced DNA damage and photocarcinogenesis in a variety of models. After UV exposure of primary human keratinocytes, the addition of 1,25(OH)2D3 increased unscheduled DNA synthesis, a measure of DNA repair. Oxidative phosphorylation was depleted in UV-irradiated keratinocytes to undetectable levels within an hour of UV irradiation. Treatment with 1,25(OH)2D3 but not vehicle increased glycolysis after UV. 2-Deoxyglucose-dependent inhibition of glycolysis abolished the reduction in cyclobutane pyrimidine dimers by 1,25(OH)2D3, whereas inhibition of oxidative phosphorylation had no effect. 1,25(OH)2D3 increased autophagy and modulated PINK1/Parkin consistent with enhanced mitophagy. These data confirm that energy availability is limited in keratinocytes after exposure to UV. In the presence of 1,25(OH)2D3, glycolysis is enhanced along with energy-conserving processes such as autophagy and mitophagy, resulting in increased repair of cyclobutane pyrimidine dimers and decreased oxidative DNA damage. Increased energy availability in the presence of 1,25(OH)2D3 is an important contributor to DNA repair in skin after UV exposure.

Ultraviolet radiation (UVR) causes DNA damage in melanocytes by producing photolesions such as cyclobutane pyrimidine dimers and 8‐oxo‐7‐hydrodeoxyguanosine. The production of reactive oxygen species by UVR also induces inflammatory cytokines that, together with the inherent immunosuppressive properties of UVR, propagate carcinogenesis. Nicotinamide (Vitamin B3) enhances DNA repair, modulates the inflammatory environment produced by UVR, and reduces UV‐induced immunosuppression. As nicotinamide reduces the incidence of actinic keratoses and nonmelanoma skin cancers in high‐risk individuals and enhances repair of DNA damage in melanocytes, it is a promising agent for the chemoprevention of melanoma in high‐risk populations.

Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells.

The ultraviolet (UV) radiation contained in sunlight is a powerful immune suppressant. While exposure to UV is associated with protection from the development of autoimmune diseases, particularly multiple sclerosis, the precise mechanism by which UV achieves this protection is not currently well understood. Regulatory B cells play an important role in preventing autoimmunity and activation of B cells is a major way in which UV suppresses adaptive immune responses. Whether UV-protection from autoimmunity is mediated by the activation of regulatory B cells has never been considered before. When C57BL/6 mice were exposed to low, physiologically relevant doses of UV, a unique population of B cells was activated in the skin draining lymph nodes. As determined by flow cytometry, CD1dlowCD5–MHC-IIhiB220hi UV-activated B cells expressed significantly higher levels of CD19, CD21/35, CD25, CD210 and CD268 as well as the co-stimulatory molecules CD80, CD86, CD274 and CD275. Experimental autoimmune encephalomyelitis (EAE) in mice immunized with MOG/CFA was reduced by exposure to UV. UV significantly inhibited demyelination and infiltration of inflammatory cells into the spinal cord. Consequently, UV-exposed groups showed elevated IL-10 levels in secondary lymphoid organs, delayed EAE onset, reduced peak EAE score and significantly suppressed overall disease incidence and burden. Importantly, protection from EAE could be adoptively transferred using B cells isolated from UV-exposed, but not unirradiated hosts. Indeed, UV-protection from EAE was dependent on UV activation of lymph node B cells because UV could not protect mice from EAE who were pharmacologically depleted of B cells using antibodies. Thus, UV maintenance of a pool of unique regulatory B cells in peripheral lymph nodes appears to be essential to prevent an autoimmune attack on the central nervous system.