B. L. Rademacher1, L. M. Meske1, K. A. Matkowskyj2, E. D. LaCount1, E. H. Carchman1 1University Of Wisconsin,Department Of Surgery, Division Of General Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Pathology And Laboratory Medicine,Madison, WI, USA
Introduction: Patients with anogenital human papilloma virus (HPV) infection are at high risk of developing squamous cell dysplasia that can progress to squamous cell carcinoma of the anal canal (SCCA). We have previously shown that dual PI3K/mTOR inhibition results in decreased dysplasia and SCCA with systemic drug administration in our HPV mouse model of anal carcinogenesis. Here we sought to investigate the effect of local, topical application of a dual PI3K/mTOR inhibitor, BEZ235, on tumor free survival, histopathologic changes and autophagy.
Methods: K14E6/E7 mice were given no treatment (Control), topical BEZ235 (BEZ), the carcinogen DMBA (DMBA), or both DMBA and BEZ for a total of 20 weeks. Mice were assessed weekly for tumor development. At 20 weeks they were euthanized and their anal samples examined for histopathologic changes at the anal transition zone (ATZ). Slide sections of the ATZ were assessed for mTOR and PI3K activity by staining for pS6 and pAKT expression (immunohistochemistry), respectively, and evidence of autophagic function via LC3β and p62 expression (immunofluorescence). Tumor free survival analysis was conducted used Kaplan Meier statistics, and all comparisons of mean differences in histopathologic score or protein signal were conducted using a one-way ANOVA.
Results: Regarding tumor free survival, mice receiving DMBA alone survived, on average, 16.9 weeks prior to tumor onset, whereas mice receiving both DMBA and BEZ survived, on average, 19.3 weeks (P<0.000001). Histopathological analysis revealed a significant decrease in mean score comparing DMBA with DMBA plus BEZ (P<0.000001). Comparing DMBA versus DMBA plus BEZ, IF revealed efficacy of topically applied dual PI3K/mTOR inhibitor, via significant decreases in both pS6 and pAKT (P<0.001 for both comparisons). Compared to Control mice, both BEZ and DMBA plus BEZ treated mice had significantly higher LC3β expression, signifying autophagic induction (P<0.005 for both comparisons), whereas DMBA, BEZ, and DMBA plus BEZ treated mice had significantly lower p62 expression, signifying increased autophagic function (P<0.0005 for all comparisons).
Conclusion: Consistent with systemic delivery of a dual PI3K/mTOR inhibitor, topical application of BEZ235 shows prolonged tumor free survival. Furthermore, this finding is confirmed via targeted inhibition of the PI3K/mTOR pathway resulting in activation of autophagy and decreased carcinogenesis.
