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Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation.

Lock R, Roy S, Kenific CM, Su JS, Salas E, Ronen SM, Debnath J - Mol. Biol. Cell (2010)

Bottom Line: The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses.In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment.Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California, San Francisco Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143, USA.

ABSTRACT
The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment. Inhibiting autophagy due to the genetic deletion or RNA interference-mediated depletion of multiple autophagy regulators attenuates Ras-mediated adhesion-independent transformation and proliferation as well as reduces glycolytic capacity. Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability. Overall, increased glycolysis in autophagy-competent cells facilitates Ras-mediated adhesion-independent transformation, suggesting a unique mechanism by which autophagy may promote Ras-driven tumor growth in specific metabolic contexts.

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Effects of ATG knockdown on adhesion-independent transformation in MDA-MB-231 cells and H-RasV12 MCF10A cells. (A) MDA-MB-231 cells transduced with lentiviral vectors encoding shRNAs against the indicated ATGs (shATGs) were subjected to immunoblotting with antibodies against ATG7, ATG5 (to detect ATG12–ATG5 complex), and tubulin. (B) MDA-MB-231 cells expressing shATG7-2 or shCNT were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against LC3 and tubulin. (C) Representative images and quantification of soft agar colony formation in MDA-MB-231 cells expressing the indicated shATGs. (D) H-RasV12 MCF10A cells expressing shCNT or shATG7-2 were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against ATG7, LC3, and tubulin. (E) Representative images and quantification of soft agar colony formation in H-RasV12 MCF10A cells expressing shCNT or shATG7-2. The above results represent the mean ± SEM from three or more independent experiments. P value was calculated using Student's t test.
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Figure 4: Effects of ATG knockdown on adhesion-independent transformation in MDA-MB-231 cells and H-RasV12 MCF10A cells. (A) MDA-MB-231 cells transduced with lentiviral vectors encoding shRNAs against the indicated ATGs (shATGs) were subjected to immunoblotting with antibodies against ATG7, ATG5 (to detect ATG12–ATG5 complex), and tubulin. (B) MDA-MB-231 cells expressing shATG7-2 or shCNT were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against LC3 and tubulin. (C) Representative images and quantification of soft agar colony formation in MDA-MB-231 cells expressing the indicated shATGs. (D) H-RasV12 MCF10A cells expressing shCNT or shATG7-2 were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against ATG7, LC3, and tubulin. (E) Representative images and quantification of soft agar colony formation in H-RasV12 MCF10A cells expressing shCNT or shATG7-2. The above results represent the mean ± SEM from three or more independent experiments. P value was calculated using Student's t test.

Mentions: We next determined whether the acute reduction of autophagy in the context of preexisting oncogenic Ras activation was similarly able to inhibit adhesion-independent transformation. First, we stably expressed two independent short-hairpin RNAs (shRNAs) against ATG7 (shATG7-1 and shATG7-2) as well as a hairpin directed against ATG12 (shATG12-1) in MDA-MB-231 cells. Analysis of target protein levels by Western blot revealed high-level knockdown of ATG7 with both shATG7-1 and 2 and reduction of the ATG5–ATG12 complex in shATG12-1–expressing MDA-MB-231 cells (Figure 4A). Of these three hairpins, shATG7-2 gave the most robust reduction in autophagy, based on immunoblotting for LC3-II (unpublished data). MDA-MB-231 cells expressing this shRNA exhibited an approximately 50% decrease in both basal and detachment-induced autophagy (Figure 4B). Furthermore, the expression of all three of these shATGs in MDA-MB-231 cells resulted in a significant decrease in soft agar colony formation, ranging from approximately 50–90%, depending on the shRNA used (Figure 4C).FIGURE 4:


Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation.

Lock R, Roy S, Kenific CM, Su JS, Salas E, Ronen SM, Debnath J - Mol. Biol. Cell (2010)

Effects of ATG knockdown on adhesion-independent transformation in MDA-MB-231 cells and H-RasV12 MCF10A cells. (A) MDA-MB-231 cells transduced with lentiviral vectors encoding shRNAs against the indicated ATGs (shATGs) were subjected to immunoblotting with antibodies against ATG7, ATG5 (to detect ATG12–ATG5 complex), and tubulin. (B) MDA-MB-231 cells expressing shATG7-2 or shCNT were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against LC3 and tubulin. (C) Representative images and quantification of soft agar colony formation in MDA-MB-231 cells expressing the indicated shATGs. (D) H-RasV12 MCF10A cells expressing shCNT or shATG7-2 were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against ATG7, LC3, and tubulin. (E) Representative images and quantification of soft agar colony formation in H-RasV12 MCF10A cells expressing shCNT or shATG7-2. The above results represent the mean ± SEM from three or more independent experiments. P value was calculated using Student's t test.
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Figure 4: Effects of ATG knockdown on adhesion-independent transformation in MDA-MB-231 cells and H-RasV12 MCF10A cells. (A) MDA-MB-231 cells transduced with lentiviral vectors encoding shRNAs against the indicated ATGs (shATGs) were subjected to immunoblotting with antibodies against ATG7, ATG5 (to detect ATG12–ATG5 complex), and tubulin. (B) MDA-MB-231 cells expressing shATG7-2 or shCNT were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against LC3 and tubulin. (C) Representative images and quantification of soft agar colony formation in MDA-MB-231 cells expressing the indicated shATGs. (D) H-RasV12 MCF10A cells expressing shCNT or shATG7-2 were grown attached (A) or suspended (susp) for the indicated times in the presence or absence of E64d and pepstatin A (E/P) and subjected to immunoblotting with antibodies against ATG7, LC3, and tubulin. (E) Representative images and quantification of soft agar colony formation in H-RasV12 MCF10A cells expressing shCNT or shATG7-2. The above results represent the mean ± SEM from three or more independent experiments. P value was calculated using Student's t test.
Mentions: We next determined whether the acute reduction of autophagy in the context of preexisting oncogenic Ras activation was similarly able to inhibit adhesion-independent transformation. First, we stably expressed two independent short-hairpin RNAs (shRNAs) against ATG7 (shATG7-1 and shATG7-2) as well as a hairpin directed against ATG12 (shATG12-1) in MDA-MB-231 cells. Analysis of target protein levels by Western blot revealed high-level knockdown of ATG7 with both shATG7-1 and 2 and reduction of the ATG5–ATG12 complex in shATG12-1–expressing MDA-MB-231 cells (Figure 4A). Of these three hairpins, shATG7-2 gave the most robust reduction in autophagy, based on immunoblotting for LC3-II (unpublished data). MDA-MB-231 cells expressing this shRNA exhibited an approximately 50% decrease in both basal and detachment-induced autophagy (Figure 4B). Furthermore, the expression of all three of these shATGs in MDA-MB-231 cells resulted in a significant decrease in soft agar colony formation, ranging from approximately 50–90%, depending on the shRNA used (Figure 4C).FIGURE 4:

Bottom Line: The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses.In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment.Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California, San Francisco Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143, USA.

ABSTRACT
The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment. Inhibiting autophagy due to the genetic deletion or RNA interference-mediated depletion of multiple autophagy regulators attenuates Ras-mediated adhesion-independent transformation and proliferation as well as reduces glycolytic capacity. Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability. Overall, increased glycolysis in autophagy-competent cells facilitates Ras-mediated adhesion-independent transformation, suggesting a unique mechanism by which autophagy may promote Ras-driven tumor growth in specific metabolic contexts.

Show MeSH
Related in: MedlinePlus