Abstract
Chaperone-mediated autophagy (CMA), a subtype of autophagy, delivers select proteins into the lysosome for degradation. Defects in CMA activity have previously been linked with neurodegenerative diseases due to the accumulation of misfolded proteins, but the role of CMA in cancer is currently not well defined. In a recent study, we provide a novel mechanism by which excessive activation of CMA can be exploited as a method to eliminate cancer cells by inducing metabolic catastrophe and delineate a novel strategy to promote the degradation of HK2 (hexokinase 2) in cancer cells.
Autophagy is an important degradative mechanism, which promotes cell survival under adverse stress conditions, including in cancer cells that are responding to anticancer treatment. Accordingly, inhibition of autophagy has been proposed as a potential therapeutic strategy. However, since suppressing autophagy per se is not sufficient to induce cell death for the majority of cancer cells, identification of pathways that can sensitize these cells to autophagy inhibition might provide a strategy that can be beneficial for the treatment of this disease. As with autophagy, chaperone-mediated autophagy (CMA) involves the engagement of the lysosomal degradation machinery; however, it is a selective process that targets specific proteins but does not broadly degrade other cellular components and organelles. Chemical modulation of CMA is currently very limited; hence, prolonged starvation has been one of the best-characterized stimuli to activate CMA. There is sequential crosstalk among the different autophagy pathways and attenuation of one pathway can lead to the activation of the other. Consistently, autophagy inhibition with the small molecule spautin-1 significantly accelerates the activation of CMA under nutrient-deprived conditions. However, other mechanisms by which CMA can be activated remain to be identified.
We describe a study involving a high-throughput chemical screen to identify small molecules that could sensitize cancer cells to autophagy inhibition under normal growth conditions. Our analysis identified quizartinib (AC220), an FLT3 inhibitor, as the most effective enhancer of the original autophagy inhibitor spautin-1 and the upgraded improved derivative of spautin-1, A70. FLT3 and its cognate ligand have a well-established role as critical regulators of survival and proliferation of hematopoietic stem/progenitor cells. Interestingly, we found that AC220 provides a chemical means to selectively sensitize cancer cells to autophagy inhibition under normal growth conditions, suggesting that the FLT3 signaling pathway is a critical sensor and regulator of cellular nutritional states. Treatment with AC220 alone promotes metabolic stress by inhibiting glycolysis, but cancer cells survive due to concomitant sustained oxidative phosphorylation and a strong dependency on autophagy. Only with combinatory treatment of AC220 and spautins, is the compensatory metabolism abolished. This treatment induces a rapid collapse in cellular ATP levels and leads to cell death via metabolic catastrophe. Importantly, our study demonstrates that FLT3 may have a previously unappreciated role in regulating glucose metabolism in cancer cells beyond acute myeloid leukemia, suggesting that the FLT3 inhibitor AC220 may be applied for the treatment of non-acute myeloid leukemia-related cancers.
Since maximal CMA activation requires nutritional stress conditions, and treatment with AC220 mimics a stressful cellular state, we show that simultaneous inhibition of the FLT3 pathway and macroautophagy leads to excessive activation of CMA regardless of the cellular growth conditions. Activation of CMA induced cancer cell death through metabolic catastrophe, and through an unbiased proteomic approach our study characterized multiple metabolic proteins as targets for degradation through the CMA pathway. One important target that we identified as a novel CMA substrate in this screen is HK2, a key glycolytic enzyme. Interestingly, the CMA-targeting motif we identified on HK2 (712QRFEK716) directly mediates binding of this protein to glucose molecules. In the presence of glucose this motif is buried within the structure. However, in the absence of glucose, the CMA motif of HK2 is exposed, and accessible for recognition and binding with HSPA8/Hsc70 revealing that its degradation by CMA is regulated by the glucose availability.
HK2 performs a key function in glucose metabolism by phosphorylating glucose to generate glucose-6-phosphate, the first committed step in glycolysis. HK2 is also known as an oncogenic kinase that promotes multiple metabolic pathways that are essential to prevent metabolic stress. Its overexpression helps cancer cells maintain growth factor-independent glucose metabolism to suppress cell death after growth factor withdrawal. The ability of CMA to promote the degradation of HK2 suggests the potential utility of manipulating cellular metabolism via CMA as a means to promote cancer cell death. Molecular links between metabolic stress and cell death are poorly understood. Degradation of HK2 through the activation of the CMA pathway directly links metabolic collapse to cancer cell death. Importantly, our study suggests that degradation of metabolic proteins through the activation of the CMA pathway may provide a mechanism to promote the death of cancer cells that have acquired mutations to resist intrinsic pathways of apoptosis through metabolic catastrophe.
Metabolic reprogramming as a result of oncogenic alterations plays an important role in regulating energy and biomaterial production in cancer cells. Elevated glucose uptake in cancer cells is a common metabolic hallmark of many tumors. This dependence on glucose metabolism has been proposed as a possible anticancer therapeutic. However, most cells demonstrate metabolic adaptations in response to an altered cellular energy status. Thus, the strategy to effectively target glucose metabolism for therapeutic purposes is still being investigated. Our study supports the proposal of a model, in which the activation of CMA may be exploited as a method to effectively inhibit adapted metabolism in cancer cells through degradation of pro-oncogenic metabolic proteins, such as HK2.
We have previously identified oncogenic mutant TP53 as a CMA substrate. Thus, combined, our studies provide a new insight into why inhibition of autophagy can be beneficial for the treatment of cancers—by promoting the degradation of oncogenic HK2 and mutant TP53, which may display dominant oncogenic activities. We propose that selective activation of CMA may be considered as a novel therapeutic strategy for the removal of pathological mutant proteins involved in human cancers.
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