Harnessing the gatekeepers of glucocorticoids for chemoprevention of non-melanoma skin cancer

Abstract

Despite effective surgical methods for non-melanoma skin cancer (NMSC), patients suffer from tissue damage, scarring, or even disfigurement; thus, there is a need for chemopreventive approaches. Because of the complex interplay between glucocorticoids (GCs), inflammation, and cancer, we sought to determine the role of 11β-hydroxysteroid dehydrogenase 1 and 2 (11βHSD1 and 2) in regulating GCs during skin cancer development and progression. 11βHSDs modulate the activation of GCs in a tissue-specific manner and have been reported to play a role in development and progression of other types of cancer, but their role has not yet been reported in NMSC. Here, we found a significant upregulation of 11βHSD2 protein in skin cancer cells when compared to normal skin cells, suggesting a role for this enzyme in the multifactorial process of skin cancer development. In addition, inhibition of 11βHSD2 with siRNA resulted in significant reduction in colony formation in vitro. Finally, our in vivo study elucidated that inhibition of 11βHSD2 with pharmacological inhibitor, Glycyrrhetinic acid (GA) could significantly diminish tumorigenesis in a well-studied in vivo mouse model of NMSC. Overall, these studies highlight for the first time a potential novel role for 11βHSD2 in NMSC development and may allow for new GC treatment approaches capable of avoiding deactivation by the enzyme. If 11βHSD2 can be inhibited as we have done here, or circumvented using modified GCs, this may lead to more efficacious outcomes for NMSC patients by preventing deactivation of the GC and minimizing resistance.

Keywords: 11β-hydroxysteroid dehydrogenases; chemoprevention; glucocorticoids; glycyrrhetinic acid; non-melanoma skin cancer; phytochemicals.

Figure 1

Detailed animal treatment schedule to study the effect of Glycyrrhetinic acid on DMBA/TPA induced skin tumorigenesis. All treatments described were administered topically in acetone to the shaved dorsal area of mice. Group 1, the control group mice were treated with 200 μL acetone twice weekly throughout the treatment period, and at each instance where TPA was administered. In the DMBA/TPA group, DMBA administration was performed only once at week zero to groups 2, 6, 7, and 8 in a dose of 50 μg/200 μL. In each group where DMBA was administered, TPA was also given twice weekly at a dose of 2 μg/200 μL for the duration of the study. GA control, groups 3, 4, and 5, each received their respective dose of GA (at 0.25 μmol, 0.5 μmol, and 1 μmol concentrations of GA) twice weekly. For our experimental groups, 6, 7, and 8, GA was administered at the respective dose (0.25 μmol, 0.5 μmol, and 1 μmol) 30 min prior to TPA treatment, twice weekly for entire length of the study

Figure 2

A, Representative Western blot and quantification showing increased 11βHSD2 expression in TPA‐induced JB6 P+ cells as compared to basal 11βHSD2 levels in vehicle treated JB6 P+ cells; Representative Western blot and quantification showing transformed RT101 mouse epidermal keratinocyte cell lines have aberrant 11βHSD2 expression relative to normal or pre‐neoplastic cell lines (JB6 P + without TPA treatment); Representative Western blot and quantification showing increased 11βHSD2 expression in papilloma‐producing MT1/2, and carcinoma‐producing Ca3/7 cells as compared to non‐tumorigenic 3PC cell lines. B, Representative Western blot and quantification showing basal 11βHSD2 levels in PM1 dysplastic, pre‐cancerous skin cells and Met4 skin cancer cells are significantly higher than basal levels shown in normal HaCaT skin cells. C, Inhibition of 11βHSD2 via siRNA significantly decreases TPA‐induced JB6 P+ and RT101 colony formation in soft agar as determined by the anchorage independent colony formation assay. 11βHSD2 inhibition by siRNA pool of 3 different siRNA results in significantly decreased colony formation in TPA‐induced JB6 P+ cells and the same was observed in RT101 cells. These results were validated using the same 3 11βHSD2 siRNA individually in transformed RT101 cells, and the inhibition of 11βHSD2 by each siRNA resulted in significantly decreased colony formation as seen in the colony quantification of the last panel. P < 0.05 were considered statistically significant (*). Densitometry shown at right of all Western blots in this figure is the average result of 3 separate experiments. 11βHSD2 was detected using anti‐11βHSD2 antibody

Figure 3

A, Representative images of papillomagenesis in the indicated groups after 15 weeks of treatment. When compared to vehicle‐treated animals (top panel), GA treatment resulted in significant inhibition of tumorigenesis as can be seen in representative images of all three DMBA/TPA/GA groups (bottom panel). B, Representative images of development of papilloma and squamous cell carcinoma in the indicated groups after 26 weeks of treatment. When compared to vehicle‐treated animals (top panel), GA treatment resulted in significant inhibition of tumorigenesis as can be seen in representative images of all three DMBA/TPA/GA groups (bottom panel)

Figure 4

A, Chemopreventive effect of GA on DMBA/TPA‐induced skin cancer in FVB mice. GA treatment significantly reduces average tumor weight in the short‐term timeline when compared to DMBA/TPA control. Twice‐weekly topical application of GA 30 min prior to TPA‐treatment resulted in a significant decrease in average tumor weight in all three experimental DMBA/TPA/GA groups (low, medium, and high doses). B, Tumor incidence data over both phase I and phase II of our in vivo study (26 weeks) show significant decreases in tumor incidence in TPA/GA(L) and TPA/GA(H) group for weeks 6‐16 when compared to TPA control. C, Average number of tumors per mouse over both phase I and phase II of our in vivo study (26 weeks). All 3 groups elicited a significant reduction in average number of tumors/mouse when compared to the TPA alone group. D, Latency of tumors was increased in all experimental groups compared to TPA control by 2 weeks. Treatment with GA in all three experimental groups (low, medium, and high [GA]) resulted in an increase in percent tumor‐free mice, with TPA/GA(L) and TPA/GA(H) showing significant increases when compared to TPA control. E, Incidence of SCC is significantly decreased by GA treatment; Histological analysis of tissue samples after undergoing 26 weeks of TPA‐treatments reveals that treatment with GA significantly decreased the percent incidence of SCC in two out of three experimental groups (TPA/GA(L) and TPA/GA(H)). P < 0.05 were considered to be statistically significant (*)

Figure 5

11βHSD2 expression is significantly induced upon TPA treatment, and GA abrogates this increase in expression; (A) Representative Western blots from both short‐term and long‐term endpoints of our DMBA/TPA two stage carcinogenesis study and (B) Quantitative analysis of densitometry showing relative 11βHSD1 and 11βHSD2 expression normalized to β‐actin. P  <  0.05 were considered to be statistically significant (*)