Overall, our studies demonstrate that in the context of a potent oncogene, mutationally active Ras, autophagy both promotes adhesion-independent transformation and facilitates glycolysis. The genetic deletion or RNAi-mediated knockdown of autophagy regulators (ATGs) causes a potent decrease in anchorage-independent growth in soft agar, indicating that an intact autophagy pathway is required for robust adhesion-independent transformation by oncogenic Ras. Furthermore, autophagy inhibition during Ras transformation results in reduced proliferation and decreased glucose metabolism. The decreased rate of glycolysis found in Ras-transformed autophagy-deficient cells correlates with decreased sensitivity to declining glucose concentrations in comparison with autophagy-competent counterparts, both in terms of proliferation and adhesion-independent transformation.
In addition, our results indicate that oncogenic Ras does not suppress autophagy during ECM detachment. These data differ from previous reports in both Ras-expressing cells in vitro as well as Drosophila
development in vivo (Furuta et al.
; Berry and Baehrecke, 2007
), both of which demonstrate that Ras activation suppresses autophagy. Ras-mediated suppression of autophagy is proposed to arise secondary to the constitutive activation of the PI3K/mTOR pathway, a negative regulator of autophagy induction. However, upon ECM detachment of several K-Ras mutant cancer lines, ribosomal protein S6 phosphorylation, an established readout of mTORC1 activity, is rapidly decreased, indicating that Ras is unable to sustain mTORC1 activation in cells deprived of matrix contact. Furthermore, detached MEFs expressing H-RasV12
exhibit decreased S6 phosphorylation comparable to nontransformed controls. On the other hand, in H-RasV12
–transformed MCF10A cells, S6 phosphorylation is only slightly reduced following matrix detachment compared with nontransformed counterparts. Interestingly, although we were able to completely inhibit mTORC1 activity by treating H-RasV12
MCF10A cells with rapamycin during suspension, this did not further augment autophagy. This result indicates that the enhanced level of mTORC1 activity that persists in H-RasV12
MCF10A cells following detachment is not sufficient to suppress autophagy induction. From these results, we speculate that a partial reduction in mTORC1 activity in H-RasV12
–expressing MCF10A cells may be sufficient to promote detachment-induced autophagy. Alternatively, other mTORC1-independent pathways may promote autophagy in detached cells expressing oncogenic Ras. Importantly, our results support that oncogenic Ras activation does not inhibit detachment-induced autophagy in mammalian cells.
The mechanisms through which autophagy modulates oncogenic transformation are context dependent (Chen and Debnath, 2010
). Here we demonstrate that autophagy is required for robust Ras-driven transformation; cells deleted or depleted of multiple independent ATGs all exhibit decreased anchorage-independent transformation in soft agar. Recently, we discovered that detachment-induced autophagy protects cells from anoikis, which we proposed to facilitate oncogenic transformation (Fung et al.
). However, because the ectopic overexpression of the antiapoptotic protein Bcl-2 does not enhance soft agar growth in Ras-transformed autophagy-deficient cells, the ability of autophagy to facilitate Ras transformation cannot be completely explained by its ability to protect cells from apoptosis during ECM detachment.
These studies point to a previously unrecognized tumor-promoting function for autophagy that manifests during oncogenic Ras transformation. For example, upon matrix detachment, increased numbers of atg5−/−
cells continue to proliferate compared with atg5+/+
controls. In fact, the enhanced proliferation of autophagy-deficient cells has been proposed as a potential mechanism by which autophagy might exert tumor suppressive effects (Qu et al.
; Fimia et al.
). However, unlike nontransformed autophagy-deficient cells, atg5
genetic deletion impedes, rather than enhances, the ability of H-RasV12
–transformed MEFs to proliferate during ECM detachment. Similarly, when cultured in attached nutrient-rich conditions, Ras-transformed atg5−/−
cells exhibit a marked decrease in proliferation compared with their autophagy-competent counterparts. In addition, we have also found that MCF10A cells expressing H-RasV12
occasionally undergo growth arrest or cell death following lentiviral-driven introduction of shRNAs against ATGs; in contrast, nontransformed cells consistently remain viable and continue to proliferate upon ATG knockdown (unpublished data). These results support that autophagy competence is required for cells to proliferate and expand during oncogenic Ras transformation.
Increasing evidence indicates that stress response pathways play diverse, multifaceted roles necessary for oncogenic transformation. For example, heat shock protein 1 (HSP-1), an important mediator of the heat shock response, has been implicated as an important facilitator of Ras transformation, which correlates with its ability to modulate both proliferative capacity and glucose metabolism (Dai et al.
). Here we demonstrate that autophagy similarly supports increased glucose metabolism, suggesting a previously unrecognized mechanism by which autophagy may contribute to tumorigenesis. H-RasV12
–transformed, autophagy-competent MEFs display enhanced glucose uptake compared with their autophagy-deficient counterparts. In addition, using 13
C-NMR analysis of glucose metabolism, we observe augmented glycolytic flux in H-RasV12
–expressing autophagy-competent cells as evidenced by increased production of lactate and alanine from glucose. Notably, we have also observed reduced glucose uptake in nontransformed, autophagy-deficient cells, but unlike H-RasV12
–transformed cells, these reductions do not correlate with significant changes in lactate production or in monolayer proliferation. Increased glycolysis in tumors, first observed by Otto Warburg, is crucial to support both the increased energy and synthetic demands required for high rates of proliferation. This metabolic shift in tumor cells is coordinated by up-regulating critical components of glycolysis, resulting in enhanced glucose uptake and lactate production even in the presence of ample oxygen (Vander Heiden et al.
). It is currently unclear whether reduced autophagy specifically elicits changes in glucose metabolism or causes more global metabolic shifts during Ras transformation. We are presently evaluating whether and how other metabolic pathways are affected by the loss or reduction of autophagy.
Although glucose withdrawal and energy depletion have been shown to be potent activators of autophagy as a survival response, we have unexpectedly found that the reduction or elimination of autophagy competence can actually reduce glycolytic capacity in a Ras-transformed cell. Hence, we speculate that autophagy may promote oncogenic Ras-driven tumor growth in specific metabolic microenvironments. In support, decreasing glucose concentrations inhibits soft agar colony formation in H-RasV12–expressing wild-type cells to levels approaching that of H-RasV12 autophagy-deficient cells. In contrast, both the proliferation and adhesion-independent transformation of autophagy-deficient cells are relatively insensitive to reductions in glucose availability. These alterations in glucose metabolism in autophagy-deficient cells may similarly impact transformation by other oncogenes, such as Myc and PI3K, which orchestrate global metabolic changes that contribute to the transformed phenotype, similar to activating mutations in Ras. Thus we are presently examining the impact of autophagy inhibition on glucose metabolism and transformation driven by other oncogenes.