Giuliani, Charlett (2022) Investigation of the Role of Autophagy in Breast Cancer Progression. PhD thesis, Victoria University.

Abstract

Autophagy is a degradation process that removes proteins and organelles and culminates at the lysosome to degrade its “cargo”. The three types of autophagy are macro, micro and chaperone-mediated autophagy. Macroautophagy is an indiscriminate degradation pathway that degrades defunct organelles and protein aggregates into recyclable components for reuse in metabolism and replenishes amino acid reserves. Its activity is shown to be minimal at basal levels within the cell and more commonly associated with an intracellular stressors such as nutrient deprivation, hypoxia and amino acid deficits. However, it also responds to extracellular insults such as chemotherapeutics and radiation. It was generalised as a survival mechanism that maintains cellular homeostasis by removing cellular debris that could lead to genotoxicity and or cell death. Therefore, it was surprising that this protective mechanism would be upregulated in the context of cancer. It has been agreed that its role is contradictory and has also been shown to be context-specific. The current dogma suggests macroautophagy prevents carcinogenesis in the non-transformed cell, but once the cell has transformed, it provides support and even facilitates chemoresistance. The exact mechanisms of this activity have been examined in this study in relation to mutant RAS. Initial gene array data collected from MCF10A transformed with HRASV12 resulted in the upregulation of approximately 60 macroautophagy-related genes. Macroautophagy was examined in a panel of breast cell lines with different RAS expressions such as mutant, null and activated wild type. This panel included normal mammary epithelial cell MCF10A with ectopic expression of HRASV12, RAS mutant breast cancer cell lines HS578T and MDA-MB-231 and a model of RAS activation in MDA-MB-468 breast cancer cell lines through EGF treatment. It was found that the expression of the crucial macroautophagy marker LC3-II was increased at basal levels for all transformed cell lines, and with extracellular stress induction, there was an increase in the LC3-II marker as expected except in those breast cancer cell lines with mutant RAS where expression did not alter. Further functional analysis of autophagic flux determined that autophagic activity was present at the basal level. When the RAS mutant breast cancer cell lines underwent autophagic induction, this activity was suppressed, suggesting that in the context of these RAS perturbations, mutant cell lines cannot respond to “normal” induction ques to activate autophagy. This reliance on autophagy in these more aggressive cell lines was reflected in the patient, demonstrating that high expression of the LC3 isoform LC3B correlates explicitly to poor prognosis. Interestingly this was in contrast to the LC3A and LC3C isoforms, with their increased expression correlating to a better prognosis, suggesting that the LC3B isoform is associated with a pathologic form of autophagy. Several autophagic inhibitors were utilised to examine the importance of autophagy in cancer survival and growth to extrapolate the significance of this role of macroautophagy in further supporting mutant RAS. Given that macroautophagy is a stepwise process, it has numerous points where it can “potentially” be inhibited, and this is still an area of ongoing development. Two stages of the process that can be inhibited are at the initial stages through the inhibition of PI3KC3/VPS34, of which three drugs were examined, and the final stage of the autophagic process, which was inhibited at the lysosome using the VTPase inhibitor Bafilomycin A1. All transformed cell lines were sensitive to growth inhibition and cell viability compared to the control non-transformed mammary epithelial cell line with high sensitivity to 3Ma-IV of the VPS34 inhibitors and an even higher sensitivity to Bafilomycin A1. These results highlight the importance of autophagy to oncogenic cell lines and the significance of the lysosome. The lysosome, as previously mentioned, is a hub for all three autophagic processes, so the scope of the study was broadened to include chaperone-mediated autophagy, which has known crosstalk with macroautophagy. Chaperoned-mediated autophagy (CMA) differs from macroautophagy because it discriminates and targets specific proteins for degradation. The key effector of chaperone-mediated autophagy is Lamp2A, which resides on the lysosome. This study has shown that Lamp2A is expressed in all RAS mutant breast cell lines but demonstrated a clear trend based on molecular subtypes, which was observed to be significantly upregulated in the triple-negative subtypes. The biological relevance of this increase in chaperone-mediated autophagy was examined with the development of a doxycycline inducible-shRNA against Lamp2A specifically. The functional studies examined the viability and metastatic potential in three oncogenic triple-negative subtypes and one normal. The results demonstrated that chaperone-mediated autophagy activity has a pivotal role in the three oncogenic triple-negative subtypes cell lines compared to one normal, with all biological aspects significantly impacted by the knockdown of Lamp2A. This research has demonstrated the importance of chaperone-mediated autophagy and postulates that it targets specific substrates, directly regulating the cancer cell's ability to proliferate, migrate, invade and colonise. In summary, this research has demonstrated that macroautophagy supports RAS activity in the context of mutation and exposes itself as a viable biomarker for combination treatment with autophagic inhibitors. In concert with these findings, chaperone-mediated autophagy has also revealed itself to be a fundamental regulator in the metastatic phenotype. There are no viable treatment options to inhibit chaperone-mediated autophagy, but the development of these is warranted. Chaperone-mediated autophagy has also revealed itself as a significant potential treatment target not only for breast cancer but also for metastatic disease.

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