We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically. As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. .

While cancer cell proliferation depends on access to extracellular nutrients, inadequate tumour perfusion means that glucose, amino acids and lipids are often in short supply. To overcome this obstacle to growth, cancer cells utilize multiple scavenging strategies, obtaining macromolecules from the microenvironment and breaking them down in the lysosome to produce substrates for ATP generation and anabolism. Recent studies have revealed four scavenging pathways that support cancer cell proliferation in low-nutrient environments: scavenging of extracellular matrix proteins via integrins, receptor-mediated albumin uptake and catabolism, macropinocytic consumption of multiple components of the tumour microenvironment and the engulfment and degradation of entire live cells via entosis. New evidence suggests that blocking these pathways alone or in combination could provide substantial benefits to patients with incurable solid tumours. Both US Food and Drug Administration (FDA)-approved drugs and several agents in preclinical or clinical development shut down individual or multiple scavenging pathways. These therapies may increase the extent and durability of tumour growth inhibition and/or prevent the development of resistance when used in combination with existing treatments. This Review summarizes the evidence suggesting that scavenging pathways drive tumour growth, highlights recent advances that define the oncogenic signal transduction pathways that regulate scavenging and considers the benefits and detriments of therapeutic strategies targeting scavenging that are currently under development.

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The constitutive anabolism of cancer cells supports proliferation but also addicts tumor cells to a steady influx of exogenous nutrients. Limiting access to metabolic substrates could be an effective and selective means to block cancer growth. In this review, we define the pathways by which cancer cells acquire the raw materials for anabolism, highlight the actionable proteins in each pathway, and discuss the status of therapeutic interventions that disrupt nutrient acquisition. Critical open questions to be answered before apical metabolic inhibitors can be successfully and safely deployed in the clinic are also outlined. In summary, recent studies provide strong support that substrate limitation is a powerful therapeutic strategy to effectively, and safely, starve cancer cells to death.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. However, the actual mechanism of action giving rise to these phenotypes remains unclear. Here, dynamic phosphoproteomics was used to further understand how the activity of PP2A is affected following cell treatment with SH-BC-893. These analyses combined with functional assays identified the differential regulation of Akt and Gsk3b by SH-BC-893 and C2-ceramide as responsible for the vacuolation of cells by SH-BC-893 but not C2-ceramide.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Our recent work demonstrates that inactivating mutations in phosphatase and tensin homolog () are sufficient to drive macropinocytosis in the context of AMP-activated protein kinase (AMPK) activation. Given that blocking macropinocytosis limits PTEN-deficient prostate tumor growth, AMPK or phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitors could have therapeutic value in castration-resistant prostate cancer patients, particularly when used in combination with standard of care therapies. ATG5: autophagy related 5; NHE: Na(+)/H(+) exchanger; PAK1: p21-activated kinase 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP: phosphatidylinositol (3,4,5)-trisphosphate; PIP: phosphatidylinositol (4,5)-bisphosphate; RAC1: Rac family small GTPase 1.