Abstract 130: Molecular basis of autophagy-mediated resistance to radiation and Apo2L/TRAIL therapy of prostate cancer

Abstract

Abstract Cell death and survival signaling plays an important role in the response to irradiation (IR) and antitumor chemotherapy. IR and other apoptotic insults, such as the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL), are used as prostate cancer therapeutics, however cellular resistance may hinder their effectiveness. PC3 cells were more sensitive to Apo2L/TRAIL-mediated cell death compared to LNCaP-derived C4-2 prostate cancer cells. The cell death observed showed both apoptotic as well as nonapoptotic features, raising the possibility that apart from classical apoptosis, autophagy might also contribute to cytotoxicity. Following Apo2L/TRAIL treatment of PC3 cells, microtubule associated protein 1 light chain LC3 (ATG8), a classical marker for autophagy, was recruited into autophagosomes following its cleavage (LC3 I) and lipid modification (LC3 II). In contrast, LC3 failed to associate with the autophagosomes in C4-2 cells. IR also resulted in increased LC3 II in PC3 cells whereas in C4-2 LC3 was predominantly unmodified. Inhibition of autophagosome and lysosome fusion with chloroquine resulted in accumulation of LC3 II in both PC3 and C4-2 cells suggesting that lipid modification of LC3 is intact in C4-2 cells. To address the rate of autophagic flux in detail we ectopically overexpressed GFP-mCherry-LC3 fusion protein and observed its localization upon Apo2L/TRAIL and IR treatment. C4-2 cells showed more LC3 II in the lysosomal compartment compared to autophagosomes, whereas PC3 had predominantly autophagosomal LC3 II. This indicates that the rate of autophagic outflux is higher in C4-2 cells upon Apo2L/TRAIL treatment. We next examined the expression and sub-cellular distribution of other autophagy related proteins. ATG5 is decreased in the membrane fraction of PC3 cells treated with Apo2L/TRAIL and IR. In C4-2 cells ATG5 is increased in the soluble and decreased in the membrane fractions upon IR while decreased in both cellular fractions upon Apo2L/TRAIL treatment. p62/SQSTM1 levels increased in poly-ubiquitinated aggregates in PC3 but not in C4-2 cells upon Apo2L/TRAIL treatment. Moreover, we observed differences in ATG5 and p62 associated native protein complexes among PC3 and C4-2 cells upon Apo2L/TRAIL and IR treatment. Characterization of these complexes would give new insights into autophagic signaling and Apo2L/TRAIL response. Our results suggest that in Apo2L/TRAIL or IR-sensitive PC3 cells accumulation of autophagosomes triggers apoptosis resulting in effective cell death whereas a more efficient autophagic response in C4-2 cells might be the reason for their resistance against Apo2L/TRAIL or IR. Further studying function of autophagy regulatory genes will help to understand their role in mediating cell death or survival outcomes in a cell context dependent manner, which may lead to the design of better therapies for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 130.