Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model

Abstract

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.

Fig 1. Chemical structure of MS-275 (entinostat).

Fig 2. The effect of MS-275 on histone acetylation in mouse Kera-308 cells.

(A) Representative Western blots depicting dose dependent increases in histone H3K9 acetylation upon treatment with the indicated amount of drug for 24 h. H3K9ac and total H3 blots were run separately on different gels using aliquots from the same sample preparations and processed in parallel. Uncropped Western blots are available as S5A Fig. (B) H3K9 acetylation levels were normalized to total H3 and quantified by densitometry using ImageJ (n = 3, unpaired t test, *p < 0.05, ***p < 0.001).

Fig 3. Analysis of MS-275 efficacy when applied topically in an SKH-1 hairless mouse model of UVB-induced cutaneous squamous cell carcinoma.

(A) Total number of tumors per group by week for ten weeks as defined by lesions ≥ 1 mm in diameter (n = 30 mice per group). (B) Tumor multiplicity for each group by week as defined by the average number of tumors for all mice at risk. (C) Tumor incidence by week as defined by the percentage of mice with lesions. (D) Tumor burden as defined by the average tumor volume per mouse for all mice at risk. (E) Individual tumor volumes plotted by week and by group.

Fig 4. Analysis of tumor tissue obtained from mice at the end of the cutaneous carcinogenesis study.

(A) Representative Western blots showing increases in H3K9 acetylation in tumors treated with MS-275 as compared to vehicle. Tumor tissue was selected at random from 6 mice in each group (1 mouse from each cage). Mice were sacrificed and tissue was isolated 12–16 h after the last inhibitor dose. H3K9ac and total H3 blots were run separately on different gels using aliquots from the same sample preparations and processed in parallel. Uncropped Western blots are available as S5B Fig. (B) H3K9 acetylation levels were normalized to total H3 and quantified by densitometry using ImageJ (n = 3). (C) Average increase in histone H3K9 acetylation for MS-275 and vehicle treated groups (n = 6, unpaired t test, *p < 0.05).

Fig 5. The effect of MS-275 on the proliferation of patient derived cutaneous squamous cell carcinoma cell lines.

(A) cSCC-IC4 (IC₅₀ = 1.24 ± 0.05 μM) and (B) cSCC-MET4 (IC₅₀ = 0.10 ± 0.02 μM) cells were seeded in 96-well plates for 24 h prior to addition of the indicated amount of inhibitor in DMSO (final concentration of DMSO was 0.5%). Cells were cultured for an additional 72 h with [³H]thymidine being added 6 h prior to harvesting cells. Radioactivity was measured using a Microbeta scintillation counter and normalized to vehicle treated controls (IC₅₀ = mean ± SE, n = 4). Representative Western blots in (C) cSCC-IC4 and (D) cSCC-MET4 cell lines depicting dose dependent increases in p21 levels upon treatment with the indicated amount of drug for 24 h. GAPDH and p21 were detected in samples that were run on the same gel. After transfer to a nitrocellulose membrane, the membrane was cut at the 25 kDa marker and membrane sections were incubated with the indicated primary antibody. Quantitation of p21 levels by densitometry for (E) cSCC-IC4 and (F) cSCC-MET4 cell lines using ImageJ (n = 3, unpaired t test, *p < 0.05, **p < 0.01).