to survive, the cells must communicate with their environment. This communication occurs either through activation of cell surface receptors by their extracellular ligands, or through endocytosis, macropinocytosis, and phagocytosis. Whereas endocytosis ensures nutrient uptake, cell surface receptor function, and cell migration in all cell types, macropinocytosis and phagocytosis are essential in specialized immune cells for antigen presentation and activation of the immune system. In addition to these physiological roles of endocytosis, cancer cells often display elevated levels of macropinocytosis, driven by oncogenes like Src and Ras that are components of the signaling cascades downstream of growth factor receptors (4).
Endosome and macropinosome maturation into lysosomes requires several derivatives of phosphatidylinositol, among which phosphatidylinositol trisphosphate (PtdIns3P), is produced by the class III phosphoinositide 3-kinase, Vps34, and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] is synthetized from PtdIns3P by the phosphoinositide kinase PIKfyve. Thus, Vps34 and PIKfyve cooperate to produce PtdIns(3,5)P2. Inactivation of Vps34 or PIKfyve generates a drop in the cellular level of PtdIns(3,5)P2, and an arrest of endosomes and macropinosome maturation at a late stage, before fusion with the lysosomes, leading to an accumulation of enlarged cytosolic vesicles, frequently named as “aberrant vacuolation” (9). Aberrant vacuolation might result not only from Vps34 or PIKfyve deficiency, but also from an abnormal increase of endocytic activity that might saturate the capacity of endosome-lysosome fusion machinery, the capacity of endosomal recycling, or both. Such a hyperstimulation of macropinocytosis has been observed in human glioblastoma cells overexpressing the constitutively active form of the oncoprotein H-Ras (G12V), a situation in which, unexpectedly, cellular viability was strongly affected (4). The cellular non-apoptotic death induced by H-RAS (G12V) overexpression is termed “methuosis,” from the Greek “methuo” (i.e., heavy drinker), and is preceded by an intracellular accumulation of large aberrant vacuoles derived from macropinosomes. Vulnerability of some cancer cells to this type of cell death triggered research to find drugs that can interfere with endocytic vesicle maturation, particularly for cancer cells resistant to apoptosis-mediated death. Two classes of small compounds, indole-based chalcones (6, 7) and vacquinols (3), were found to induce non-apoptotic cell death of glioblastoma tumors. For both compounds, the cancer cell death has been preceded by massive accumulation of intracellular vacuoles, but the molecular targets of these drugs are still unknown.
The identification of the targets of vacquinols and chalcones, as well as the alternative methods to manipulate the endocytic pathways of cancer cells, will strongly benefit from a better characterization of molecular mechanisms leading to aberrant vacuolation, a prerequisite for tumor death by methuosis.
In this issue of American Journal of Physiology-Cell Physiology, Compton et al. (1) provide new insights into the mechanisms involved in accumulation of large cytosolic vacuoles that affect cellular viability. The authors inactivate Vps34 or PIKfyve using pharmacological or genetic approaches. As expected, inactivation of Vps34 or PIKfyve induced a depletion of intracellular levels of PtdIns(3,5)P2 and the accumulation of aberrant large vacuoles in the cytosol. This effect relied on the activation of the small GTPase Rab5 and of the vacuolar H+ ATPase. Requirement of Rab5 GTPase activity, as demonstrated by expression of dominant-negative mutant of Rab5, the GFP-Rab5S34N mutant protein, which rescued the aberrant vacuolation phenotype, even if Vps34 or PIKfyve were inactivated. These data indicated that the GTP-GDP Rab5 cycle is essential for endosome fusion and growth of intracellular large vacuoles that resemble early steps in endosome maturation. The implication of vacuolar H+ ATPase was demonstrated by a pharmacological approach, using the macrolide antibiotic bafilomycin A1. Bafilomycin A1 treatment rescued and prevented the formation of cytosolic vacuoles due to Vps34 or PIKfyve inhibition. Nevertheless, this protective effect of bafilomycin A1 was probably independent of the alkalinization of endosomal pathways by the drug, as suggested by the lack of effect of NH4Cl or chloroquine, two compounds that increase the endocytic pH but did not rescue the vacuolation phenotype induced by Vps34 or PIKfyve inactivation. These results suggested that the H+ V-ATPase display pH-independent activities through a mechanism that remains to be identified. Alternatively, bafilomycin A1 may also impact on the aberrant vacuoles formation via inhibition of heterotypic and/or homotypic endosome fusion (2) or via secondary targets of the compound, such as the recently identified protein SERCA, a Ca2+ pump (5).
The work by Compton et al. brings new insights into the central role of Rab5 and Bafilomycin A1 targets in non-apoptotic cell death caused by aberrant vacuolation. These results provide a better understanding of the basic mechanisms leading to aberrant vacuolation, a prerequisite for tumor death by methuosis that may give clues to potential new opportunities for cancer therapy (4), as already described in the context of aggressive tumors such as glioblastoma (3). While the molecular targets of vacquinol remains to be identified, Compton et al. results further strengthen the importance of Vps34 and PIKfyve kinases in aberrant vacuolation, and suggest that the development of specific inhibitors of these kinases could represent an interesting anti-tumor strategy (8). In addition, the discovery that Rab5 is required for vacuolation opens new avenues for the use of a combination of kinase inhibitors with Rab5 modulators. This might be especially useful if the tumor cells downregulate Rab5 activity in response to Vps43 or PIKfyve inhibition. Probably the most enigmatic result presented by Compton et al. is the inhibition of vacuolation by bafilomycin A1. Finding the mechanism by which bafilomycin A1 inhibits vacuolation could lead to the identification of a new potential therapeutic target for tumor control.
No conflicts of interest, financial or otherwise, are declared by the authors.
L.S. drafted manuscript; L.S. and S.L. edited and revised manuscript; S.L. approved final version of manuscript.
- Copyright © 2016 the American Physiological Society