Autophagy enables tumors to survive metabolic stress in a dormant state for prolonged periods of time and as such, this pathway is an emerging target for the creation of new anticancer therapies. Hydroxychloroquine (HCQ) is a drug that blocks autophagy, raising intralysosomal pH and inhibiting the final step of this pathway, impairing autophagic protein degradation. This results in autophagosome accumulation, potentially disturbing the survival of tumor cells reliant on autophagy and shunting them toward apoptotic and/or necrotic cell death. Preliminary results in melanoma cell lines indicate that treatment with chloroquine (CQ), a similar drug to HCQ used readily in the laboratory, increases autophagosome accumulation, and increased cell death was noted from treatment with CQ at varying concentrations. Additionally, CQ induced autophagosome accumulation in a mammalian tumor allograft model. Given that the autophagy pathway may be active in melanoma, we hypothesize that administration of HCQ can modulate the process of autophagy in melanoma tumor tissue samples in humans in vivo. To test this hypothesis, a phase 0 trial of HCQ in patients with stage III or IV melanoma planned for palliative or curative resection is proposed. Specifically, we will look for signs of autophagy inhibition by HCQ, comparing the mean number of autophagic vesicles per cell seen on electron microscopy (EM) in pre- versus post- treatment tumor specimens. Pre- and post- treatment skin samples and PBMCs will similarly be assayed to explore the potential of these samples as surrogate markers for treatment effect. While autophagosome accumulation on EM is a reliable method for detection of autophagy inhibition, it is expensive, tedious and requires a skilled individual to perform and hence, is not widely applicable to clinical specimens to detect autophagy inhibition. Thus, as part of this trial we will examine several proteins shown to detect inhibition of autophagy in laboratory models and in some preliminary studies of human tumor tissue, with the hope that these simpler assays will facilitate autophagy detection in human samples. Markers that will be examined include Beclin1, an essential autophagy regulator, and LC3, which is cleaved into LC3-I and LC3- II, with the latter translocated to the autophagosome membrane during autophagy. In addition, our group has discovered that autophagy deficient tumors under stress accumulate p62, a polyubiquitin binding protein that delivers aggregates to the lysosome for degradation, as well as several chaperone proteins including glucose related protein 70 (GRp170). It is expected that treatment with HCQ in melanomas with functional autophagy will result in increased autophagosome formation on EM and increased levels of LC3-II, p62 and GRp170 by immunohistochemistry and Western blot. Potential proapoptotic and/or ant proliferative effects of HCQ will also be explored through the use of pre- and post-treatment activated caspase-3, TUNEL, Ki-67 and mitotic rate assays. The goal of this proposal is to modulate autophagy for the first time in human tumors, using results from these experiments to guide development of markers of autophagy in future clinical trials.
We propose a preliminary trial in humans with melanoma (a Phase O trial) to see if administering hydroxychloroquine (HCQ), a FDA approved drug with little to no side effects, can block the process of autophagy, a survival mode for several different types of tumor cells. We will use a method called electron microscopy to study if HCQ is inhibiting autophagy in melanoma tumor tissue and blood and skin samples, but since this method is expensive and laborious we will also examine four proteins -- Beclin1, LC3, p62 and GRp170 -- which detect autophagy inhibition in cell lines, animal models, and some preserved human tissue samples to demonstrate that these simpler tests may also detect inhibition of autophagy in human beings. Our hope is that we will be able to use these results to better understand and detect autophagy, making it easier to develop future drugs against this pathway in cancer.
