Macropinocytosis: the big drinker behind cancer cell self-consumption

ABSTRACT

Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also hypoxic and rely on a number of adaptive responses, including macroautophagy/autophagy and macropinocytosis (MP), to support their bioenergetic needs. Whereas autophagy enables starved cells to recycle intracellular macromolecules via lysosomal degradation and use the liberated amino acids (AA) to fuel their metabolism, MP allows cells to take up extracellular proteins via fluid-phase endocytosis and use them as an energy source. However, how any MP-enabled organism, including the prototypical cancer cell, coordinately regulates and balances autophagy and MP is not fully understood. We recently found that inhibition of autophagy results in upregulation of MP, which enables cancer cells to overcome autophagy deficiency and continue to support their bioenergetic demands. The NFE2L2/NRF2-driven induction of MP-related genes (MRGs) is responsible for the upregulation of MP in autophagy inhibited, hypoxic, and oxidatively stressed-exposed cancer cells. Concurrent autophagy and MP blockade effectively cuts off the cancer cell’s nutrient and supplies, leading to rapid tumor regression. These findings suggest MP to be an important target in cancer treatment and that shutting off the energy spigot is a promising therapeutic strategy.

Abbreviations AA: amino acids; ADCs: autophagy deficient-cells; AI: autophagy inhibition; ALB: albumin; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; CQ: chloroquine; ECM: extracellular matrix; HCQ: hydroxychloroquine; MI: MP inhibition; MP: macropinocytosis; MRGs: MP-related genes; MRPs: MP-related proteins; PDAC: pancreatic ductal adenocarcinoma.

PDAC, the major common and aggressive pancreatic cancer type is frequently initiated by oncogenic KRAS mutations. The clinical management of PDAC has benefited minimally from traditional therapies and latest advances in targeted- and immune-therapies, underscoring the necessity to develop radically new therapeutic approaches. Autophagy is upregulated in established PDACs, where it promotes cancer cell survival. This led to the convention that autophagy inhibition (AI) should cause PDAC starvation and regression. However, most clinical attempts to target autophagy by using the lysosomal acidification inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ) have not improved overall patient survival when combined with chemotherapy. A related and greater concern regarding the clinical utility of autophagy inhibitors is our recent finding that AI activates an alternative survival pathway, MP, in which cells ingest extracellular proteins to extract nutrients [1]. MP upregulation may be a major cause of treatment resistance and failure.

While studying expression of CHUK/IKKα in human PDAC and investigating whether its downregulation disturbed autophagic flux, as previously observed in mice, we found that a subset of CHUK-low human PDAC shows defective autophagic degradation. CHUK was found to serve as a scaffold that stabilized LC3-STX17 interaction on autophagosomes and promote formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (STX17-SNAP29-VAMP8). Importantly, CHUK-low tumors overcome the autophagy defect through upregulation of MP. Previous studies had shown that MP is upregulated due to KRAS activation. However, our results showed that AI in KRAS-transformed cells results in further dramatic upregulation of MP via the well-established SQSTM1/p62-NFE2L2 axis, which we found to serve as a master regulator of genes encoding MP-related proteins (MRPs) and MP-stimulating growth factors and receptors. Through immunohistochemical analysis of human PDAC specimens, we correlated low CHUK expression with high SQSTM1, NFE2L2 and MRPs, but elevated NFE2L2 correlates even better with increased MRPs expression regardless of CHUK. Notably, forced expression of KEAP1-resistant NFE2L2^E79Q restores MP in KRAS-ablated cells. These results support a direct NFE2L2-dependent crosstalk between autophagy and MP. While NFE2L2 stimulates MP at the transcriptional level, KRAS acts post-transcriptionally by activating phosphatidylinositol 3-kinase, whose catalytic subunits are NFE2L2 inducible. Importantly, NFE2L2 serves as a convergence point for at least 3 MP-stimulating triggers: AI, hypoxia, and oxidative stress, explaining that activated NFE2L2 better correlates with MRPs expression than low CHUK in vivo. In addition, NFE2L2 ablation in autophagy-deficient cells (ADCs) dramatically inhibits MP activity and MRGs induction. These results further indicate that AI stimulates MP through NFE2L2.

ADCs are highly sensitive to MP inhibition (MI) or glutamine starvation. Addition of exogenous ALB (albumin) or culture on extracellular matrix (ECM) strongly enhances the growth of nutrient-starved ADCs, whereas the effect on the parental cells is more modest, and only ALB-supplemented or ECM-cultured ADCs are highly sensitive to MI. Importantly, MI in autophagy-compromised PDAC cells results in their killing. To investigate how MP compensates for autophagy loss, PDAC cells were cultured with [³H]proline- or [U-¹³C]glutamine-labeled ECM. Culture on ECM increases cellular ATP and AA content, as well as the NADPH:NADP ratio and bromodeoxyuridine incorporation, which are further increased in ADCs and reduced in NFE2L2-ablated or MP-blocked cells. Importantly, AI greatly increases ECM-derived isotope enrichment in glutamine-derived AA and tricarboxylic acid cycle intermediates, which is reversed by MI. In addition, we found HCQ is not a good choice to inhibit MP because low HCQ concentrations enhance MP and high concentrations are clinically unattainable due to cardiotoxicity. Although new lysosomotropic agents are being developed, we suspect that lysosome inhibitors may cause severe side effects because many vesicular trafficking pathways culminate in the lysosome. The preferred approach is to use a combination of specific autophagy and MP inhibitors. Indeed, concurrent inhibition of autophagy and MP elicit dramatic regression of transplanted and autochthonous PDACs, suggesting the therapeutic promise of such combinations.

Thus, our study highlights MP as an important target in cancer treatment and explains why autophagy inhibitors fail to starve PDAC and cannot induce its regression. Once autophagy is inhibited, cancer cells simply resort to MP to feed themselves. We expect there is more to learn about the autophagy-MP crosstalk under various physiological and pathological conditions.

Funding Statement

This work was supported by grants from the Padres Pedal the Cause/C3, the Youth Program of the National Natural Science Foundation of China 81802757 (HS) and 82002931(FY), the NIHR01CA211794 (MK), R37AI043477 (MK), P01DK098108 (MK), and the National Natural Science Foundation of China 2016YFC0905900 (BS). Additional support was provided by the UC Pancreatic Cancer Consortium to MK is the Homer T. Hirst III Professors of Oncology in Pathology.

Disclosure statement

No potential conflict of interest was reported by the author(s).