E. Carchman1, K. Matkowskyj2, P. Lambert3 1University Of Wisconsin,General Surgery,Madison, WI, USA 2University Of Wisconsin,Pathology,Madison, WI, USA 3University Of Wisconsin,Oncology,Madison, WI, USA
Introduction: The incidence and death rates from anal cancer are increasing 2% a year. The human papillomavirus (HPV) is the major etiologic risk factor for the development of squamous cell carcinoma (SCC) of the anus, with greater than 90% of anal cancers being HPV positive (high-risk subtypes HPV16 and 18). With HPV infection, two viral oncoproteins (E6 and E7) are expressed and their intracellular molecular changes enhance cellular growth and inhibit cell death, promoting an environment for carcinogenesis. We have developed a double transgenic mouse model (K14E6/K14E7 mice) of anal carcinogenesis in which the two oncoproteins are expressed in the stratified squamous epithelium. Topical treatment with the carcinogen dimethylbenz[a]anthracene (DMBA) induces histologic changes progressing from dysplasia to SCC of the anus. Using this model, we have previously described the up-regulation of the mammalian target of rapamycin (mTOR) pathway. mTOR is an upstream inhibitor of the protective cellular pathway called autophagy. Autophagy is important in the removal of damaged organelles/proteins through lysosomal degradation to maintain cellular health. The goal of this project is to examine the autophagic response throughout anal carcinogenesis. There are two autophagic proteins that are examined in this proposal, first is LC3 that is attached to the autophagosome membrane and second is the autophagic-specific substrate p62.
Methods: Cohorts (3-14 mice/cohort) of K14E6/K14E7 mice were treated topically with DMBA for 5, 10, 15 or 20 weeks. Control mice were not treated with DMBA for the same time points. Anal tissue was collected at the end of treatment, fixed, and serially sectioned. Every 7th section was stained with hemotoxylin and eosin and analyzed by a trained pathologist for the presence of dysplasia or SCC. Immunofluorescence (IF) was performed to detect autophagic proteins, LC3 and p62. Electron microscopy (EM) was performed at each time point.
Results:IF demonstrated an increase in LC3 protein levels throughout neoplastic progression, whereas p62 levels were noted to be high in dysplastic lesions and low in invasive SCC. EM demonstrated an accumulation of lysosomes and mitochondria as lesions progressed from low-grade to high-grade dysplasia, and normalization of lysosome numbers and late autophagosome appearance in SCC.
Conclusion:This study provides evidence of late autophagic flux inhibition during anal carcinogenesis in an HPV mouse model as indicated by increased p62 levels in the setting of increased LC3. This indicates that autophagy is induced, but then the autophagosome is inhibited from fusing to the lysosome resulting in an accumulation of p62. In anal SCC, the levels of p62 expression decreased with the continued increase in LC3 expression, demonstrating that autophagy is able to go to completion in established tumors. These findings support our hypothesis that inhibition of autophagy plays an important role in anal cancer development.