Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment

1. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, and Brugge JS

2. Excess proliferation displaces cells from their niches autophagy Vargo-Gogola T, Nat Rev Cancer (2007). Determine the basis for the observed metabolic defect in matrix deprived cells and provide evidence that an oncogene can rescue this defect.

3. EGFR expression is reduced after detachment in MCF10A cells and overexpression can block anoikis EGFR levels decrease upon matrix detachment and forced expression rescues downstream signaling and allows cell survival Reginato MJ, et al Nat Cell Biol (2003)

4. Glucose metabolism yields more than just energy +

5. Fatty acid oxidation (FAO) • I’m not fat I just have a lot of potential energy! • Each cycle trims 2 C from the chain and produces 1 acetyl-CoA, 1 FADH2, and 1 NADH ATP ATP ATP Adapted from Mira A (2009)

6. The Take-Home Messages • Detachment from ECM causes an ATP deficiency that occurs as a result of reduced glucose uptake • This metabolic deficiency can be rescued by overexpression of the oncogene ErbB2, via constitutive signaling through EGFR and PI3K, and it depends on flux through the PPP • Antioxidants can reverse the metabolic defect, independent of glucose uptake, by increasing flux through the PPP • Antioxidants can enhance the transforming activity of oncogenic cells.

7. MCF10A cells that are in contact with ECM Glucose EGFR ECM PI3K Glucose Glycolysis, TCA, ETC Fermentation to lactate ATP PPP ATP NADPH NADPH NADPH ATP FAO ROS

8. MCF10A cells that are matrix deprived Glucose X PI3K Glucose Glycolysis, TCA, ETC Fermentation to lactate PPP ATP NADPH Bim FAO ROS

9. Expression of ErbB2 removes the need for ECM signaling ErbB2 Glucose PI3K Glucose Glycolysis, TCA, ETC Fermentation to lactate ATP PPP ATP NADPH NADPH NADPH ATP FAO ROS

10. Figure 1. Loss of matrix attachment causes reduction in cellular ATP that is rescued by ERBB2 • Depriving MCF10A cells of matrix leads to a drop in ATP levels that is not seen in cells that overexpress ErbB2.

11. Supplementary Figures 4 & 5. Drop in ATP after detachment is not affected by inhibition of apoptosis 24 hrs 48 hrs Inhibition of apoptosis does not affect the drop in ATP after detachment ATP has dropped by 24 hours of matrix detatchment but cells are not apoptotic

12. Supplementary Figure 6. Drop in ATP after detachment is not affected by inhibition of apoptosis or autophagy Beclin-1 and ATG5 are key autophagy proteins Inhibition of autophagy does not affect drop in ATP Metabolic defect at 24 hours in detached MCF10A cells that is not a result of apoptosis or autophagy

13. EGFR expression is reduced after detachment in MCF10A cells and overexpression can block anoikis EGFR levels decrease upon matrix detachment and forced expression rescues downstream signaling and allows cell survival Reginato MJ, et al Nat Cell Biol (2003)

14. Figure 1. Loss of matrix attachment causes reduction in cellular ATP that is rescued by ERBB2 • EGFR is markedly stabilized in cells overexpressing ErbB2 • Targeted knockdown of EGFR in ErbB2-MCF10A cells leads to a drop in ATP following matrix deprivation • EGFR expression allows maintenance of ERK signaling and an enhancement of PI3K signaling

15. Figure 1. Loss of matrix attachment causes reduction in cellular ATP that is rescued by ERBB2 through PI3K pathway activation • Sustained ATP is lost after inhibition of the PI3K pathway but not the ERK pathway • Constitutive activation of PI3K or AKT allows a sustained level of ATP

16. Figure 1. Loss of matrix attachment causes reduction in cellular ATP that is rescued by ERBB2 through PI3K pathway activation • Sustained ATP is lost after inhibition of the PI3K pathway but not the ERK pathway • Constitutive activation of PI3K or AKT allows a sustained level of ATP ErbB2 sustains ATP levels by preventing downregulation of EGFR, which maintains PI3K pathway signaling.

17. Figure 2. Matrix detachment causes a reduction in glucose uptake The increase in glucose uptake seen in ErbB2-MCF10As is important for sustaining ATP levels after detachment and is dependent upon PI3K signaling

18. Figure 2. Matrix detachment causes a reduction in glucose uptake • Addition of pyruvate can rescue the drop in ATP in detached MCF10A cells. • Pyruvate rescue is not seen when the mitochondria is inhibited. • Addition of a toxic glucose analog (2DG) does not allow sustained levels of ATP in detached ErbB2-MCF10As The increase in glucose uptake seen in ErbB2-MCF10As is important for the elevated level of ATP in these cells after detachment.

19. Pentose phosphate pathway is an alternative mode of glucose oxidation • Major products are NADPH and ribose-5-phosphate • Controlled by glucose 6 phosphate dehydrogenase (G6PD) G6PD

20. Figure 2. Matrix detachment causes a reduction in glucose uptake and ERBB2 rescue of this defect is dependent on PPP flux ErbB2-MCF10A • ROS is elevated and GSH drops in detached cells • Not seen in ErbB2-MCF10A cells • Antioxidant NAC rescues GSH reduction in MCF10A cells MCF10A

21. Figure 2. Matrix detachment causes a reduction in glucose uptake and ERBB2 rescue of this defect is dependent on PPP flux ErbB2-MCF10A DHEA & 6AN • ATP levels drop and ROS levels increase in ErbB2-MCF10A cells following PPP inhibition

22. Pentose phosphate pathway is an alternative mode of glucose oxidation • Major products are NADPH and ribose-5-phosphate • Controlled by glucose 6 phosphate dehydrogenase (G6PD) G6PD

23. Figure 2. Matrix detachment causes a reduction in glucose uptake and ERBB2 rescue of this defect is dependent on PPP flux ErbB2-MCF10A • G6PD levels increase after matrix detachment • Knockdown increases ROS and does not allow sustained ATP in ErbB2-MCF10A cells Suggests that sustained ATP levels in ErbB2-MCF10A cells is dependent on flux through the PPP

24. Figure 3. Antioxidants rescue low ATP levels in detached cells MCF10A • Cells treated with the antioxidants NAC and Trolox do not experience an ATP drop upon detachment • Rescue is independent of changes in glucose uptake

25. Figure 3. Antioxidants rescue low ATP levels in detached cells by permitting fatty acid oxidation • MCF10A detachment causes a drop in FAO that can be reversed by Trolox treatment • Antioxidant rescue of FAO is reversed by inhibiting fatty acid transport into the mitochondria

26. Figure 3. Antioxidants rescue low ATP levels in detached cells by permitting fatty acid oxidation • Restoration of NADPH to detached MCF10A cells rescues their metabolic defects • Blocking FAO in detached ErbB2-MCF10A cells reduces ATP significantly implicating this pathway in the maintenance of ATP in these cells.

27. Figure 4. Analysis of antioxidant effects on acinar morphogenesis and colony formation in soft agar day 7 • Visualization of NAD(P)H and ROS demonstrates the metabolic dichotomy between matrix-attached and matrix-starved cells in a more physiologic setting.

28. Figure 4. Analysis of antioxidant effects on acinar morphogenesis and colony formation in soft agar • Treatment of acini formed in 3D culture with Trolox diminishes the metabolic dichotomy between the matrix-attached and matrix-starved cells ROS is contributing to the observed metabolic dichotomy

29. Figure 4. Analysis of antioxidant effects on acinar morphogenesis and colony formation in soft agar • Neutralization of ROS can reduce luminal clearing alone or in combination with an apoptotic block. Matrix-independent survival requires circumventing both metabolic defects and apoptosis

30. Figure 4. Analysis of antioxidant effects on acinar morphogenesis and colony formation in soft agar • Neutralization of ROS can reduce luminal clearing alone or in combination with an apoptotic block. Can antioxidants promote the transforming activity of mammary epithelial cells?

31. Figure 4. Analysis of antioxidant effects on acinar morphogenesis and colony formation in soft agar ErbB2-MCF10A BT-474 • Colony formation was increased by treatment with Trolox in E7/BCL2-MCF10As, ErbB2-MCF10As, and and ErbB2-expressing breast cancer cell line

33. MCF10A cells that are in contact with ECM Glucose EGFR ECM PI3K Glucose Glycolysis, TCA, ETC Fermentation to lactate ATP PPP ATP NADPH NADPH NADPH ATP FAO ROS

34. Expression of ErbB2 removes the need for ECM signaling ErbB2 Glucose PI3K Glucose Glycolysis, TCA, ETC Fermentation to lactate ATP PPP ATP NADPH NADPH NADPH ATP FAO ROS Antioxidants

35. The Take-Home Messages • Detachment from ECM causes an ATP deficiency that occurs as a result of reduced glucose uptake • This metabolic deficiency can be rescued by overexpression of the oncogene ErbB2, via constitutive signaling through EGFR and PI3K, and it depends on flux through the PPP • Antioxidants can reverse the metabolic defect, independent of glucose uptake, by increasing flux through the PPP • Antioxidants can enhance the transforming activity of oncogenic cells.

36. Final thoughts and possible therapeutic implications • Provides and explanation of how tumor cells can adapt to glucose deprivation • Are antioxidants more harmful than previously thought? • Prevent DNA damage vs. promote anchorage-independent survival • Does this lend support to the relevance of PI3K inhibition?