MIF antagonist (CPSI-1306) protects against UVB-induced squamous cell carcinoma

Abstract

Macrophage migration inhibitory factor (MIF) is a homotrimeric proinflammatory cytokine implicated in chronic inflammatory diseases and malignancies, including cutaneous squamous cell carcinomas (SCC). To determine whether MIF inhibition could reduce UVB light-induced inflammation and squamous carcinogenesis, a small-molecule MIF inhibitor (CPSI-1306) was utilized that disrupts homotrimerization. To examine the effect of CPSI-1306 on acute UVB-induced skin changes, Skh-1 hairless mice were systemically treated with CPSI-1306 for 5 days before UVB exposure. In addition to decreasing skin thickness and myeloperoxidase (MPO) activity, CPSI-1306 pretreatment increased keratinocyte apoptosis and p53 expression, decreased proliferation and phosphohistone variant H2AX (γ-H2AX), and enhanced repair of cyclobutane pyrimidine dimers. To examine the effect of CPSI-1306 on squamous carcinogenesis, mice were exposed to UVB for 10 weeks, followed by CPSI-1306 treatment for 8 weeks. CPSI-1306 dramatically decreased the density of UVB-associated p53 foci in non-tumor-bearing skin while simultaneously decreasing the epidermal Ki67 proliferation index. In addition to slowing the rate of tumor development, CPSI-1306 decreased the average tumor burden per mouse. Although CPSI-1306-treated mice developed only papillomas, nearly a third of papillomas in vehicle-treated mice progressed to microinvasive SCC. Thus, MIF inhibition is a promising strategy for prevention of the deleterious cutaneous effects of acute and chronic UVB exposure.

Implications: Macrophage migration inhibitory factor is a viable target for the prevention of UVB-induced cutaneous SSCs.

Figure 1

Schematic of chronic UVB exposure and CPSI-1306 treatment schedule. Female Skh-1 mice were exposed to UVB three times per week on nonconsecutive days for 10 weeks. UVB treatments were then halted and mice were gavaged with 20 mg/kg/d of CPSI-1306 or vehicle 5 days per week for 4 weeks, then 3 days per week for an additional 4 weeks then left untreated for the final 7 weeks, after which they were sacrificed.

Figure 2

Inhibition of MIF increases keratinocyte apoptosis, while decreasing proliferation. Female Skh-1 mice were pretreated with vehicle or CPSI-1306 for 5 days, exposed to 1 MED of UVB, and sacrificed at 30 minutes or 6, 24, or 48 hours after UVB exposure. Dorsal skin was examined for expression of the proapoptotic protein cleaved caspase-3 or the proliferation marker Ki67 by IHC. The percentage of positively staining epidermal cells was determined (CPSI-1306 vs. vehicle). A, left: CPSI-1306 significantly enhanced epidermal cleaved caspase—mediated apoptosis at 6, 24, and 48 hours compared with vehicle (*, P < 0.0001). Right, representative cleaved caspase-3 IHC images (×60) at 24 hours post UVB exposure when the maximum difference was observed between CPSI-1306 and vehicle-treated mice. Arrows indicate cleaved caspase-3–positive epidermal cells. B, left: CPSI-1306 significantly decreased epidermal proliferation as examined by Ki67 IHC at both 24 (^*, P = 0.0206) and 48 hours (^*, P = 0.0010), relative to vehicle. Right: representative Ki67 IHC images (×60 magnification) at 48 hours post UVB exposure when the maximum difference was observed between CPSI-1306 and vehicle-treated mice. Bars represent SD; ^*, P < 0.05.

Figure 3

CPSI-1306 treatment decreases UVB-induced DNA damage. Epidermal DNA isolated from the dorsal skin of mice treated with vehicle or CPSI-1306 for 5 days before a single UVB exposure was examined by dot blot analysis to determine levels of CPD. A, left, at all time points examined, CPSI-1306 significantly decreased UVB-induced CPD levels compared with vehicle-treated skin (P = 0.0001). Right, dot blot for CPD; each dot represents DNA from one mouse and the various treatment groups are labeled and outlined for comparison between CPSI-1306 and vehicle-treated mice. Dorsal skin was also examined immunohistochemically to determine changes in levels of phosphohistone H2A.X and p53. B, left, CPSI-1306 treatment decreased epidermal levels of phosphohistone H2A.X compared with vehicle-treated mice at baseline and at all time points following UVB exposure (P < 0.0001). Right, representative phosphohistone H2A.X IHC images (×60 magnification) at 6 hours post UVB exposure when the maximum difference was observed between CPSI-1306 and vehicle-treated mice. C, CPSI-1306 significantly increased epidermal p53 levels at both 30 minutes (P = 0.0009) and 6 hours (P = 0.0001) following UVB exposure relative to vehicle. Right, representative p53 IHC images (×60 magnification) at 6-hour post UVB exposure when the maximum difference is observed between CPSI-1306 and vehicle-treated mice. Bars represent SD; ^*, P < 0.05.

Figure 4

Inhibition of MIF decreases UVB-induced squamous carcinogenesis. Skh-1 mice were exposed to UVB three times per week on nonconsecutive days for 10 weeks. UVB treatments were halted, then mice were gavaged 5 days per week with 20 mg/kg/d CPSI-1306 for 4 weeks then 3 days per week for an additional 4 weeks, and finally monitored for an additional 7 weeks without further treatment. All tumors were counted and measured weekly after cessation of UVB exposure, excised at the end of the experiment, and histologically graded by a board certified veterinary pathologist to determine the grade and extent of invasion. A, inhibition of MIF decreased tumor multiplicity compared with vehicle; B, CPSI-1306 treatment delayed tumor onset relative to vehicle; C, treatment with CPSI-1306 decreased tumor burden compared with vehicle; D, CPSI-1306 treatment inhibited tumor progression compared with vehicle; P1-P3, benign papilloma 1–3; MI1-MI3, malignant microinvasive squamous cell carcinoma.

Figure 5

CPSI-1306 treatment reduces proliferation and p53 foci formation in nontumor epidermis. Non–tumor-bearing dorsal skin harvested from chronically UVB-irradiated mice treated with either vehicle or CPSI-1306 was examined immunohistochemically to determine the percentage of Ki67-positive epidermal keratinocytes as well as the number of p53 foci. A, left, treatment with CPSI-1306 significantly inhibited epidermal proliferation in non–tumor-bearing skin compared with vehicle, relative to no UV exposure (P = 0.0168); right; representative Ki67 IHC images (×60 magnification) of chronic UVB-exposed CPSI-1306 and vehicle-treated mice. B, left, MIF inhibition after chronic UVB exposure significantly decreased the density of epidermal p53 foci (P = 0.0014); right, representative p53 IHC images (×20 magnification) of chronic UVB-exposed CPSI-1306 and vehicle-treated mice. Bars represent SD. ^*, P < 0.05.

Figure 6

A proposed model for the role of MIF in UVB-mediated inflammation and skin carcinogenesis. A, acute UVB exposure induces MIF expression and trimerization in the skin, which inhibits p53 function by enhanced degradation of p53, increased inflammation and epidermal proliferation, and decreased apoptosis. CPSI-1306 treatment reverses the above-mentioned effects of MIF, decreasing the accumulation of DNA damage. B, chronic UVB exposure results in increased dermal MIF expression and trimerization, leading to increased epidermal proliferation and decreased apoptosis. As a consequence of genomic DNA damage and loss of p53 function, there is increased tumor formation and progression in the vehicle-treated mice. Treatment with CPSI-1306 prevents the trimerization of MIF, thus reducing the accumulation of DNA damage and eventually the squamous carcinogenesis.