Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin

Abstract

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

Figure 1. Solar simulated light(SSL) induces mTOR/Akt-related signaling events in mouse epidermis

SKH-1 hairless mice (n=3) were acutely irradiated with 105 kJ/m² UVA/6.4 kJ/m² UVB and sacrificed at the indicated times (0 hr refers to mice that were irradiated and immediately sacrificed). Epidermal proteins were analyzed by Western blotting (A) or reverse phase protein microarray (RPPA, B). The heat map represents the average of the three independently analyzed samples per timepoint (0hr, 30 min, 1hr, 2hr, 4hr, 8hr and 24hr). The signaling diagram represents some of the proposed signaling pathways activated by solar-simulated light based on data obtained through the RPPA analysis.

Figure 2. Treatment schedule of topical rapamycin application affects the outcome of solar simulated light-induced skin tumorigenesis

SKH-1 hairless female mice were treated with solar simulated light (SSL) 3 times a week for 15 weeks (n = 20). Control mice were treated on their back skin with vehicle (acetone) 1hr prior to each UV exposure, and continued with 3× weekly acetone treatments after UV stopped. Mice in the “Prevention” group were treated 3 times a week with rapamycin (50nmol/back) 1 hr prior to UV exposure, and continued receiving this treatment after SSL stopped. Mice in the “Intervention” group received only vehicle (acetone) 1hr prior to each SSL treatment, but switched to treatments with rapamycin after SSL stopped. All mice were observed for tumor number and size until sacrifice at week 25, at which point tissues were harvested. Tumor multiplicity (average number of tumors per mouse) is shown in A. Tumor burden (average area per mouse) is shown in B. Data are shown as mean values + SEM.

Figure 3. Topical treatment with rapamycin potentiates acute SSL-induced apoptotic signaling in vivo

3A. Top row: Volume rendering displays of ^99mTc-duramycin SPECT images in SKH-1 mice representative of untreated control (A), vehicle control with SSL (B), and rapamycin treatment with SSL (C). Mice were imaged 48 hours after SSL exposure. Bottom row: Autoradiograph images of the back skin (right) and belly skin (left) collected from animals presented above in the top row. The images labeled by D, E, and F are collected from corresponding A, B, and C mice, respectively. Multiple lesions (arrows) of increased radioactive uptake (hot spots) were predominantly visualized on the back skin of the mice receiving SSL exposure with rapamycin treatment (F), less in that of mice receiving SSL with vehicle treatment (E), and not on the skin of control mice. 3B. Results of ^99mTc-duramycin ex vivo biodistribution measurements of control, SSL with vehicle, and SSL with rapamycin treatment groups at 2 hours after radiotracer injection. * = P < 0.01 compared to Control; † = P < 0.01 compared to SSL.

Figure 4. Rapamycin treatment causes increased Akt signaling in cultured keratinocytes which is blocked by PHT-427

Primary human keratinocytes were exposed to either 100nM rapamycin over a timecourse (A), or differing doses of Rapamycin for 24hr (B) and then harvested for Western blot analysis, 20ug per lane. Blots were probed for p-Akt (S473), p-S6 Ribosomal Protein, or beta tubulin as a loading control. HaCaT human keratinocytes were grown to 70% confluence and then treated with rapamycin, PHT-427, or both in serum-free media at the indicated doses. After 24hr the cells were lysed and processed for Western blot analysis, 20ug per lane (C). Blots were probed for p-Akt (S473), p-S6 Ribosomal Protein or beta tubulin as a loading control.

Figure 5. Topical treatment with rapamycin prior to SSL causes hyperphosphorylation of Akt (S473) in mouse epidermis which is inhibited by PHT-427

Female SKH-1 hairless mice (n = 3) were pretreated three times (48hr, 24hr, and 1hr) with rapamycin (50nmol/back) prior to acute solar simulated light (SSL) treatment at the dose used in Figure 1. Mice were then sacrificed along a timecourse up to 4hr and epidermal proteins were extracted for Western blot analysis (0hr indicates sacrifice immediately after UV exposure, A). To confirm the delayed hyperphosphorylation of Akt (S473) after SSL in the presence of rapamycin, new mice were treated as described above and harvested at 6hr and 24hr post SSL for Western blot analysis of epidermal lysates (B, C). A third acute mouse experiment was performed to test the effects of PHT-427 on p-Akt (S473) in SSL-exposed epidermis. Mice were treated with rapamycin or PHT-427 (3.7umol/back) or both as described above and harvested 6hr post SSL exposure. Epidermal lysates were blotted for p-Akt (S473), p-S6 Ribosomal Protein or beta-tubulin as a loading control.

Figure 6. PHT-427 additively contributes to skin tumor inhibition when combined with rapamycin

SKH-1 hairless female mice were treated 3 times a week for 15 weeks with SSL. After 15 weeks UV treatments stopped and mice were split into four groups (n = 20). Mice were then treated topically on their backs three times a week for an additional 10 weeks with either vehicle (acetone), rapamycin (50 nmol/back), PHT-427 (3.7umol/back) or both agents (same doses). All mice were observed for tumor number and size until sacrifice at week 25. Tumor multiplicity (average number of tumors per mouse) is shown in A. Tumor burden (average area per mouse) is shown in B. Data are shown as mean values + SEM.