Harnessing metabolic dependencies in pancreatic cancers
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a 5-year survival rate of <10%. The tumour microenvironment (TME) of PDAC is characterized by excessive fibrosis and deposition of extracellular matrix, termed desmoplasia. This unique TME leads to high interstitial pressure, vascular collapse and low nutrient and oxygen diffusion. Together, these factors contribute to the unique biology and therapeutic resistance of this deadly tumour. To thrive in this hostile environment, PDAC cells adapt by using non-canonical metabolic pathways and rely on metabolic scavenging pathways such as autophagy and macropinocytosis. Here, we review the metabolic pathways that PDAC use to support their growth in the setting of an austere TME. Understanding how PDAC tumours rewire their metabolism and use scavenging pathways under environmental stressors might enable the identification of novel therapeutic approaches.
Autophagy is critical for cysteine metabolism in pancreatic cancer through regulation of SLC7A11
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. The elevated macroautophagy/autophagy in these tumors supports growth, promotes immune evasion, and increases therapeutic resistance. Therefore, targeting autophagy is a therapeutic strategy that is being pursued to treat PDAC patients. Whereas autophagy inhibition impairs mitochondrial metabolism in PDAC, the specific metabolite(s) that becomes limiting when autophagy is inhibited has not been identified. We report that loss of autophagy specifically results in intracellular cysteine depletion under nutrient-replete conditions. Mechanistically, we show that PDAC cells utilize the autophagy machinery to regulate the activity and localization of the cystine transporter SLC7A11 at the plasma membrane. Upon inhibition of autophagy, SLC7A11 is localized to lysosomes in an MTORC2-dependent manner. Our findings reveal a novel connection between autophagy and cysteine metabolism in pancreatic cancer.
Metabolic Codependencies in the Tumor Microenvironment
Metabolic reprogramming enables cancer cell growth, proliferation, and survival. This reprogramming is driven by the combined actions of oncogenic alterations in cancer cells and host cell factors acting on cancer cells in the tumor microenvironment. Cancer cell intrinsic mechanisms activate signal transduction components that either directly enhance metabolic enzyme activity or upregulate transcription factors that in turn increase expression of metabolic regulators. Extrinsic signaling mechanisms involve host-derived factors that further promote and amplify metabolic reprogramming in cancer cells. This review describes intrinsic and extrinsic mechanisms driving cancer metabolism in the tumor microenvironment and how such mechanisms may be targeted therapeutically.
Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism inÂ vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using inÂ vivo and inÂ vitro systems. This work revealed striking overlap of inÂ vivo metabolic dependencies with those inÂ vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define inÂ vivo metabolic dependencies and pathways that may have therapeutic utility.
Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer
Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a "hit" compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.
Harnessing metabolic dependencies in pancreatic cancers [Review]
Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive fibrosis and hypovascularization, resulting in significant intratumoral hypoxia (low oxygen) that contributes to its aggressiveness, therapeutic resistance, and high mortality. Despite oxygen being a fundamental requirement for many cellular and metabolic processes, and the severity of hypoxia in PDAC, the impact of oxygen deprivation on PDAC biology is poorly understood. Investigating how PDAC cells survive in the near absence of oxygen, we find that PDAC cell lines grow robustly in oxygen tensions down to 0.1%, maintaining mitochondrial morphology, membrane potential, and the oxidative metabolic activity required for the synthesis of key metabolites for proliferation. Disrupting electron transfer efficiency by targeting mitochondrial respiratory supercomplex assembly specifically affects hypoxic PDAC proliferation, metabolism, and inÂ vivo tumor growth. Collectively, our results identify a mechanism that enables PDAC cells to thrive in severe, oxygen-limited microenvironments.