Role of autophagy pathways in PDT, a ROS-based anti-cancer therapy

The identification of druggable signalling components of adaptation and survival pathways evoked by a specific cancer treatment paradigm, leads the way to the development of novel strategies with a higher therapeutic efficacy. A promising approach to induce cancer cell killing utilizes agents or protocols capable of generating excessive amount of reactive oxygen species (ROS), leading to loss of viability. However, accumulating evidence indicate that ROS, along with cell death, can concurrently stimulate autophagy pathways. Both macroautophagy (or autophagy) and chaperone-mediated autophagy (CMA), two major lysosomal pathways for the in bulk degradation/recycling of cytoplasmic materials (proteins and organelles) and specific clearance of damaged/aberrant proteins, respectively, have been implicated in the modulation of ROS-mediated cellular damage. Most studies dealing with ROS signaling, however, did not address their cross-talk and the significance of CMA in ROS-mediated cell death. Moreover, several studies have used exogenous H2O2 as source of oxidizing agent, while it seems very likely that the type and location of the generated ROS may affect signaling mechanisms and the functional outcome of autophagy pathways. Thus, a clear understanding of the molecular players connecting these key cellular processes under conditions of oxidative stress and their significance in ROS-based anticancer treatments are still missing. This study focussed on the identification and functional significance of autophagy pathways stimulated by ROS generated by the light activation of hypericin, an ER-associated photosensitizer displaying interesting applications in photodynamic therapy (PDT). PDT is a well established paradigm of anticancer treatment utilizing the photochemical generation of cytotoxic ROS to kill the cancer cells. A distinguished molecular aspect of PDT is the ability to generate an initial ROS burst at a specific subcellular site, from which oxidative stress can further propagate. In this study we show that autophagy and CMA are stimulated after hypericin-PDT and function to mitigate ROS-mediated cell death. We found that autophagy mitigation, by Atg5 knock down or the autophagy inhibitor 3MA, reduces clearance of oxidatively damaged proteins and increased apoptosis, thus revealing the cytoprotective role of this catabolic process in hypericin-PDT. Paradoxically, genetic loss of autophagy caused by atg5-deficiency improves the clearance of terminally oxidized proteins and reduced photokilling. Furthermore, this study discloses that these functional differences are explained by the constitutive upregulation of CMA in unstressed Atg5-/- cells. CMA stimulation can efficiently compensate for autophagy loss, thereby increasing cellular resistance to subsequent photo-oxidative damage. In contrast, autophagy upregulation in CMA-deficient cells cannot forestall hypericin-PDT induced cell death, while it is cytoprotective against the canonical ER stressor thapsigargin, thus unraveling a stress specific recruitment of autophagy pathways. The existence of a tight functional connection between these two autophagy pathways revealed by this study urges the translation of this molecular knowledge in to in vivo conditions. It would be crucial to evaluate whether pharmacological/genetic inhibition of one autophagy pathway (i.e. macroautophagy by 3MA/chloroquine or by oncogene expression) may be compensated by the stimulation of another degradation mechanism (i.e. CMA), a condition that could specifically compromise the therapeutic outcome of ROS-based treatments. Additionally, an important conclusion from this study is that although autophagy is cytoprotective, CMA is the predominant defense mechanism against PDT-mediated ROS damage. The reason for such a functional hierarchy is not completely understood but could entail the more toxic character of soluble ROS-damaged proteins, which are specific targets of CMA, as compared to the ROS-damaged cytoplamic components (i.e. organelles, aggregates) cleared by autophagy. This hypothesis should spur new studies utilizing mutants of signaling components with a known function in formation of aggregates and/or in their degradation through autophagy, such as the adaptor protein p62, as well as the identification of signaling mechanisms regulating CMA in ROS-damaged cells. The preliminary studies shown here, indeed indicate that oxidative stress generated by hypericin-PDT promotes the transcriptional upregulation of p62 and provide molecular evidence that p38alpha MAPK, a key coordinator of the cellular antioxidant responses governed by Nrf2, identified by our previous work, is implicated in this process. p62 upregulation in PDT-treated cells regulates the formation of ubiquitinated protein-aggregates and their degradation through the autophagy machinery. Although, further investigations are required to disclose whether p62 is a central signalling node involved in the activation of Nrf2 signaling, the receptor and signalling functions of p62 implicate p38alpha MAPK as an attractive and druggable target during hypericin-PDT. However, this hypothesis will require further detailed investigations as a direct knock down of p62 did not have any pronounced cell death effects.nrpages: 126status: publishe