Deregulation of XPC and CypA by Cyclosporin A: An Immunosuppression-Independent Mechanism of Skin Carcinogenesis

Skin cancer in organ transplant recipients (OTR) is a rising and devastating disease burden threatening their long-term survival (1–3). In these patients, squamous cell carcinoma (SCC) is the most common skin cancer, occurring 65 to 250 times as frequently as in the general population, causing serious morbidity and mortality (1, 2). Many OTRs are given immunosuppressive agents, including cyclosporin A (CsA), to suppress the host immune response. The primary mediator of immunosuppression by CsA is cyclophilin A (CypA), a member of a phylogenetically conserved cyclophilin family that potentially regulates protein folding in cells (4). This CsA–CypA complex binds to and inhibits calcineurin (5, 6), a serine/threonine phosphatase dephosphorylating family of transcription factor nuclear factor of activated T-cells (NFAT), which mediates an immune response (7).

Therefore it has been generally assumed that increased skin cancer risk is caused by immune suppression. However, this assumption is challenged by the following epidemiologic findings: (i) in patients with acquired immunodeficiency syndrome (AIDS), who have severe damage to their immune systems, Kaposi's sarcoma, cervical cancer, and lymphomas are the most common malignancies, whereas the risk of skin cancer only shows a marginal increase (1- to 3-fold; refs. 8–13); (ii) in contrast, in OTRs, skin cancer derived from epidermal keratinocytes is the most common malignancy (65–250 fold relative risk for SCC); and (iii) skin cancer mostly occurs in sun-exposed areas, depends on the duration of immunosuppressive treatment and ultra violet (UV) exposure history after (or even before) transplantation (3), and harbors mutations in tumor suppressor genes including p53 (14–16) and xerodermal pigmentosum C (XPC; ref. 17). These findings suggest that CsA directly targets epidermal keratinocytes for tumorigenic transformation in an immunosuppression-independent but UV-dependent manner.

Cyclosporin A may be linked to keratinocyte transformation at several levels. CsA may interfere with DNA damage repair and/or checkpoint function, processes activated by UVB-induced DNA damage to prevent genetic mutations. In response to DNA damage, the cells activate a specific DNA repair mechanism, that is, global genome nucleotide excision repair (GG-NER; refs. 18–21). In addition, the damaged cells activate the DNA damage response (DDR) signal-transduction pathway to coordinate cell-cycle transitions, DNA replication, DNA repair, and apoptosis to prevent cancer development (22–27).

Although immunosuppression has been thought to be the major factor in the increased skin cancer risk in OTRs, the mechanism of skin carcinogenesis in OTRs has not been understood to date; specifically, it remains unknown whether these cancers are immunosuppression dependent or independent. Here, using both immunocompromised nude mice as an in vivo model and human keratinocytes as an in vitro model, we have shown that, following UVB-induced DNA damage, CsA inhibited DNA repair through phosphoinositide 3-kinase (PI3K)/AKT-dependent XPC downregulation, and DDR through PI3K/AKT-dependent CypA upregulation. Either blocking UVB radiation or inhibiting the PI3K/AKT pathway prevented CsA-promoted skin tumorigenesis.

All human specimens were studied after approval by the University of Chicago Institutional Review Board. Frozen tissues were obtained under consent (Department of Medicine, University of Chicago). Protein lysate was used to determine mRNA levels of XPC and CypA by real-time PCR and CypA protein levels by Western blotting.

All animal procedures have been approved by the University of Chicago Institutional Animal Care and Use Committee. Nude mice were obtained from Harlan. Mice were exposed to UVB (100 mJ/cm², dose selected to avoid visible sunburn) dorsally or sham-irradiated, 3 times a week for up to 25 weeks to monitor tumor formation and growth. Mice were treated with vehicle (olive oil) or CsA (20 mg/kg) daily by gavage. Sunscreen was topically applied to the mouse dorsal skin before each UVB exposure. The sunscreen was composed of titanium dioxide (7.5%) and zinc oxide (7.5%) with a SPF factor of 60. LY294002 (LY; in 200 μL acetone) or vehicle (acetone alone) was applied topically to the mouse dorsal skin 1 hour before each UVB irradiation. Mouse skin samples were fixed in formalin for histologic analysis or immunohistochemical analysis for Ki67-positive cells (Immunohistochemistry Core facility), or snap-frozen for immunoblotting analysis. Mice were housed 5 animals per cage, and there was no evidence of dorsal wounds caused by fighting or sunburn.

Statistical analyses were conducted using Prism 5 (GraphPad software). Data were expressed as the mean of 3 independent experiments and analyzed by Student's t-test and ANOVA. Log-rank tests were used to evaluate the tumor onset in mice. A P value of less than 0.05 was considered statistically significant.

The details for UVB treatment, cell culture, siRNA and XPC transfection, cytosolic and nuclear fractionation, Western blotting, immunohistochemical analysis, real-time PCR, luciferase reporter assays, and DNA repair analysis can be found in Supplementary Data.

To determine the role of CsA in skin carcinogenesis, we used immunocompromised nude mice and a UVB skin carcinogenesis model. Nude mice are defective in the T-cell-mediated immune system, which is a target of CsA for immunosuppression treatment in OTRs. In addition, nude mice have been widely used for xenograft models in investigating immunosuppression-independent mechanisms of CsA tumorigenesis in our work (28) and others (29–31). Mice were treated with CsA (20 mg/mL) daily by gavage for 1 week before the initial UVB exposure and throughout the experiment, as in our previous studies, and irradiated with UVB (100 mJ/cm²) 3 times a week for 25 weeks (28). This UVB radiation (100 mJ/cm²) is the maximal exposure that does not cause sunburn in nude mice.

Compared with the UVB-irradiated vehicle group, CsA treatment caused early onset of tumorigenesis (Fig. 1A; P < 0.05, log-rank test) and increased tumor multiplicity (Fig. 1B; P < 0.05, Student's t-test). However, CsA alone without UVB treatment was not tumorigenic (Fig. 1A). The number of both PAP (papillomas) and SCCs per mouse was higher in CsA-treated mice than in their vehicle counterparts (Fig. 1C). While formation of the differentiated nonspindle type of SCC was similar in both groups, the incidence of spindle cell SCC, the most aggressive form, was increased in the CsA-treated group as compared with the vehicle group (Fig. 1D—and E). Taken together, these data indicate that, while CsA alone is not tumorigenic, it increases UVB tumorigenesis in different stages, including tumor onset, multiplicity, and aggressiveness, resembling the observations made in human OTRs (3).

To determine the role of UV in CsA-sensitized skin tumorigenesis, we applied sunscreen topically to the mouse dorsal skin before each UVB irradiation. Sunscreen completely prevented the skin tumorigenesis induced by UVB and CsA (Fig. 2A; P < 0.05, log-rank test). As expected, sunscreen also completely blocked UVB-induced skin tumorigenesis (Fig. 2B; P < 0.05, log-rank test). These data further show that UVB radiation is the trigger for CsA-sensitized skin carcinogenesis.

To determine the mechanism of CsA's cocarcinogenic action, we analyzed the impact of CsA on repairing UVB-induced DNA damage products, cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone dimers (6-4PP; refs. 32, 33). In HaCaT keratinocytes, CsA significantly inhibited CPD repair (Fig. 3A and B), whereas it had no effect on 6-4PP repair (Fig. 3C). A similar effect of CsA on CPD and 6-4PP repair was detected in mouse skin and normal human epidermal keratinocytes (NHEK) cells post-UVB irradiation (Fig. 3D–F). To determine the molecular target of CsA, we measured the protein levels of key factors in GG-NER, including XPC and DNA damage-binding proteins 1 and 2 (DDB1 and DDB2; refs. 18–21). CsA downregulated the XPC levels in both HaCaT and NHEK cells, whereas it had no effect on either DDB1 or DDB2 (Fig. 3G—and H). Adding XPC to CsA-treated HaCaT cells restored the DNA repair capacity (Fig. 3I and J). These findings indicate that CsA impairs UVB-induced DNA damage repair through suppressing XPC.

To determine how CsA reduces XPC protein levels, we ascertained whether it occurs at the mRNA and/or transcriptional level. CsA significantly reduced the mRNA levels of XPC and the transcriptional activity of the XPC promoter (Fig. 4A and B). As compared with normal human skin, XPC mRNA levels were significantly downregulated in human SCCs and basal cell carcinomas (BCCs; Fig. 4C; P < 0.05, Student's t-test). To determine how CsA decreases XPC transcription, we assessed the effect of CsA on the nuclear accumulation of E2F4/p130, the repressor complex for XPC transcription (34). CsA increased the nuclear localization of both p130 and E2F4 (Fig. 4D). Removing the active E2F binding site in the XPC promoter diminished the inhibitory effect of CsA on XPC transcription (Fig. 4E). Inhibiting PI3K/AKT activation by LY (10 μmol) restored XPC protein levels (Fig. 4F), reduced the nuclear E2F4 level (Fig. 4G), and increased XPC transcription (Fig. 4H). These data indicate that CsA suppresses XPC transcription through PI3K/AKT-dependent activation of the E2F4/p130 nuclear localization.

In addition to repairing DNA damage, a proper DDR is also vital for maintaining genomic integrity following UVB damage. To determine the role of CsA on the DDR, we analyzed the role of CsA in UVB-induced activation of checkpoint kinase-1 (Chk1; refs. 35, 36) and checkpoint kinase-2 (Chk2; refs. 37–40), 2 pathways critical for DDR and checkpoint function. UVB alone activated both Chk1 and Chk2 (Fig. 5A), whereas CsA inhibited UVB-induced activation of Chk1 and Chk2 (Fig. 5A). CsA increased bromodeoxyuridine (BrdUrd) incorporation in both un-irradiated cells and UVB-irradiated cells (Fig. 5B), indicating that CsA promotes cell proliferation and impairs checkpoint function. In UVB-irradiated mouse skin, CsA increased the incidence of Ki67-positive cells (Fig. 5C), suppressed phosphatase and tensin homologue (PTEN) expression (Fig. 5D; Supplementary Fig. S1), and increased AKT activation (Fig. 5D), consistent with our recent in vitro findings (28). In addition, in mouse skin, HaCaT and NHEK cells, CsA also increased the levels of cyclophilin A (CypA; refs. Fig. 5D; Supplementary Fig. S2A–S2C). As compared with normal human skin, CypA mRNA and protein levels were increased in human SCCs and BCCs (Fig. 5E; Supplementary Fig. S2D), suggesting that CypA is an oncogene in human skin cancer. In CsA-treated cells, knockdown of CypA using siRNA increased the activation of Chk1 and Chk2 (Fig. 5F) and reduced BrdUrd incorporation in both un-irradiated and UVB-irradiated cells (Fig. 5G), whereas it had no effect on XPC downregulation, AKT activation (Supplementary Fig. S3A), or UVB-induced DNA damage repair (Supplementary Fig. S3B). CsA had no effect on ATF3, which has been shown to be induced by calcineurin inhibition (31; Supplementary Fig. S4A). Knockdown of Calcineurin B (CnB1) upregulated ATF3, whereas it did not affect XPC and CypA levels (Supplementary Fig. S4B), UVB-induced DNA damage repair (Supplementary Fig. S4C), or the activation of Chk1 and Chk2 (Supplementary Fig. S4D). However, inhibiting the PI3K/AKT pathway by LY (10 μmol) suppressed CsA-induced CypA upregulation (Fig. 5H), indicating that CypA is a downstream factor for PI3K/AKT. However, overexpression of AKT by infecting HaCaT cells with constitutively active AKT (A+) had no effect on CypA expression (Supplementary Fig. S2E), indicating that PI3K/AKT activation is required but not sufficient for CsA-induced CypA upregulation. Other molecular pathways altered by CsA may play an important role. These data indicate that the negative impact of CsA on UVB-induced checkpoint function is mediated through PI3K/AKT-dependent CypA upregulation.

To determine the specific role of epidermal PI3K/AKT activation in CsA-sensitized skin tumorigenesis in mice, we topically applied the widely used PI3K inhibitor LY (10 nmol) 1 hour before each UVB exposure. LY delayed tumor onset in CsA- and UVB-treated nude mice (Fig. 6A; P < 0.05, log-rank test). In addition, LY also reduced the number of tumors (Fig. 6B; P < 0.05, Student's t-test) and tumor volume (Fig. 6C; P < 0.05, Student's t-test) per mouse. The incomplete preventive effect by LY might be because of its limited penetration into the mouse skin, especially when the epidermis became hyperplastic and hyperkeratotic following chronic UVB irradiation. In the skin from CsA-treated mice, LY enhanced CPD repair (Fig. 6D), whereas it did not affect 6-4PP repair, which was more efficient than CPD repair (Fig. 6E). In nontumor skin from UVB-irradiated mice, LY decreased the incidence of Ki67-positive cells (Fig. 6F). These data show that PI3K/AKT activation drives the cocarcinogenesis of CsA by compromising DNA repair and cell growth control.

In CsA-treated transplant recipients, immunosuppression has been widely accepted as the major cause of increased skin carcinogenesis. However, consistent with epidemiologic data supporting the critical role of UV radiation, we have used controlled laboratory in vitro and in vivo models to show that CsA impairs the DNA repair and DDR in the response of keratinocytes to UVB. As a consequence, in immunocompromised nude mice, CsA increased skin tumorigenesis in which UVB damage and PI3K/AKT activation are required. At the molecular level, CsA downregulates the DNA repair protein XPC and upregulates the CsA-binding partner CypA through activating the PI3K/AKT pathway. Blocking UVB damage or inhibiting the PI3K/AKT pathway prevents CsA-sensitized skin tumorigenesis. Our findings further support a cell-autonomous action of CsA on skin tumorigenesis.

Following UVB damage, CsA inhibited UVB-induced DNA damage repair through suppressing XPC transcription. Consistent with recent studies using human keratinocytes and fibroblasts (41–44), we found that CsA reduced DNA repair, especially CPD repair, in human keratinocytes and mouse skin. In renal OTRs, UV-type p53 mutations were found to be prevalent, further supporting the negative role of CsA on DNA repair of UVB-induced damage (16). Although CsA may affect many molecular targets (45), we found that PI3K/AKT-dependent E2F4/p130 nuclear accumulation is critical for CsA-induced XPC downregulation. This is consistent with the effect of inhibiting PTEN by siRNA knockdown or by increasing its acetylation through SIRT1 inhibition on nucleotide excision repair (46, 47).

In addition to reducing DNA repair capacity, CsA also disrupted the UVB-induced DDR. In keratinocytes, CsA not only inhibited UVB-induced activation of Chk1 and Chk2, but also bypassed UVB-induced proliferation control. At the molecular level, we found that CsA induced CypA upregulation through a PI3K/AKT-dependent mechanism. The upregulation of CypA was required for CsA-mediated inhibition of checkpoint function but not DNA repair. Interestingly, CypA is found to be overexpressed in many types of cancer cells and contributes to cell proliferation and survival (48, 49). We found that CypA expression is increased in human cutaneous SCCs and BCCs, the 2 most common skin malignancies derived from keratinocytes and mainly caused by UV damage, suggesting the oncogenic role of CypA in skin tumorigenesis. It is possible that through increasing CypA levels in keratinocytes, CsA predisposes skin to UVB-induced skin tumorigenesis by inhibiting the proper DDR.

In summary, we have shown that UVB damage and PI3K/AKT activation are the principal drivers for CsA-sensitized skin tumorigenesis and that deregulation of XPC and CypA compromises UVB-induced DNA damage repair and checkpoint function. Our findings not only identified immunosuppression-independent molecular and cellular mechanisms by which CsA compromises genomic integrity, but also revealed UV damage and PI3K/AKT activation as essential triggers that can be used to improve skin cancer prevention and treatment in patients treated with CsA.

K. Soltani serves on the board of directors in Elorac Pharma and receives stocks from Elorac Pharma, DUSA, Winston and Gideon Pharmaceuticals, but none of them are relevant to this study. No potential conflicts of interest were disclosed by the other authors.

Conception and design: W. Han, K. Soltani, Y.-Y. He

Development of methodology: W. Han, Y.-Y. He

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Han, K. Soltani, M. Ming, Y.-Y. He

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Han, K. Soltani, Y.-Y. He

Writing, review, and/or revision of the manuscript: W. Han, K. Soltani, Y.-Y. He

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Ming, Y.-Y. He

Study supervision: Y.-Y. He

The authors thank Terri Li for the Ki67 immunohistochemistry, Dr. Pradip Raychaudhuri (University of Illinois at Chicago, Chicago, IL) for kindly providing the XPC promoter luciferase construct, Drs. Tom Gajewski and Ping Yu for helpful discussions, and Dr. Ann Motten for critical reading of the manuscript.

This work was supported by NIH grant ES016936 (Y.-Y. He), the University of Chicago Comprehensive Cancer Center Pilot program (P30 CA014599), the CTSA (NIH UL1RR024999), and UC Friends of Dermatology Research Funds.