Glucose is an energy metabolite necessary for cellular survival and growth whether or not the cell is part of a tumor. Not only do cancer cells switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect) to gain more glucose, a hallmark of cancer, but they also increase the amount of glucose taken up from the surrounding extracellular space. However, the lack of glucose can have a negative effect on cells, causing them to become apoptotic in the absence of this metabolite. Cancer cells have methods to get around the requirement for glucose, including upregulating glucose transporters to improve access to the energy metabolite. In this Redox Biology article, researchers describe how activating androgen receptor in response to a lack of glucose affects the amount of GLUT1 expressed on prostate cancer cells, making the cells resistant to glucose deprivation.
To set the stage, two prostate cancer cell lines, LNCaP, an androgen-sensitive cell line, and LNCaP-R, an androgen-insensitive cell line, were deprived of glucose. Both cell lines showed signs of cell death, but LNCaP-R cells died in greater numbers. To probe how LNCaP cells died, several inhibitors (a pan-caspase inhibitor, two necroptosis inhibitors and a ferroptosis inhibitor) were added but did not change the way the cells died. However, an autophagy inhibitor enhanced cell death, suggesting the cells were necrotic not apoptotic. Teasing apart if the necrosis of LNCaP cells was due to glucose availability or merely disrupted glycolysis, the glucose analog 2DG was added to the medium with glucose. The cells survived when treated with 2DG, suggesting it was the absence of glucose that induced necrosis. When LNCaP cells were cultivated in medium that replaced glucose with mannose or fructose, the cells survived, another point in favor of sugar depletion causing cell death.
Examining Glucose Uptake
With the prostate cancer cells surviving when glucose was in the medium, glucose uptake was likely the method used for cell survival. Gonzalez-Menendez et al. added 2DG to the glucose-free medium used for LNCaP cells and found that the cells accumulated 2DG. GLUT1 is the major glucose transporter in prostate cells so the amount of GLUT1 was evaluated in both prostate cancer cell lines. LNCaP increased production of GLUT1 while LNCaP-R downregulated the transporter. However, both cell lines increased the amount of surface GLUT1. With a higher level of GLUT1 expressed in the androgen-sensitive cell line, researchers hypothesized that the presence of androgens was responsible for the changes in the expression levels of the transporter. AMP-activated protein kinase (AMPK) regulates GLUT1 levels and membrane location, and is activated by androgens. When AMPK phosphorylation was investigated in LNCaP and LNCaP-R cells that were deprived of glucose, the androgen-sensitive LNCaP cells had greater levels of phosphoAMPK while there was no significant change in LNCaP-R cells.
Because GLUT1 was more highly expressed in glucose-deprived cells with access to androgens, does glucose affect androgen receptor (AR) levels? In medium without glucose, AR increased in the nucleus; nuclear translocation and AR activity was associated with prostate-specific antigen (PSA). When GLUT1 and AR protein abundance were studied during the cell cycle of LNCaP cells, the increase in AR correlated to GLUT1 during the same phases of the cell cycle.
Testing the Hallmarks of Necrosis
Cells undergoing necrosis are known to produce less ATP and more free radicals. Did this hold true for glucose-deprived prostate cancer cells? The ratio of ATP to AMP was reduced in LNCaP cells and the amount of H2O2 and mitochondrial superoxide increased after removing glucose from the medium. When treated with antioxidants, glucose-deprived LNCaP cells survived in the presence of catalase or N-acetyl-L-cysteine, suggesting oxidative stress occurred when glucose was removed. To probe the relationship between GLUT1 and free radicals, H2O2 was added to LNCaP cells and GLUT1 levels, AR translocation and phosphoAMPK were measured. The amount of GLUT1 increased under these conditions as did translocation of AR and phosphorylation of AMPK. From these results, Gonzalez-Menendez et al. hypothesized that the increase in H2O2 after glucose withdrawal may be responsible for activating AR and thus, increasing GLUT1 expression.
Exploring the Role of GLUT1
To investigate the role of GLUT1 in cell death after glucose removal, GLUT1 was overexpressed in LNCaP cells. Comparing LNCaP cells to those overexpressing GLUT1, fewer GLUT1-overexpressing cells died after glucose removal and H2O2 treatment was less toxic. To differentiate whether this GLUT1 protection was mediated by an extracellular or intracellular signal, a specific inhibitor of GLUT1, phloretin, was added to block the transporter. Under glucose deprivation, cell death increased in both LNCaP and GLUT1-overexpressing LNCaP cells. Furthermore, if GLUT1-overexpressing cells were grown under low serum conditions and glucose removed, there was no protective effect of GLUT1, suggesting that serum is needed for preventing cell death under glucose deprivation.
While GLUT1 overexpression protects LNCaP cells from oxidative stress, what role does GLUT1 have in antioxidant pathways? Protein levels of five cellular antioxidants were measured in LNCaP and LNCaP-GLUT1 overexpressing cells. When glucose was removed, superoxide dismutase 2 (SOD2) levels decreased in LNCaP cells but recovered to control levels in GLUT1-overexpressing cells. Glutathione peroxidase 1 expression and catalase activity increased in both cell types. In GLUT1-overexpressing cells, the GSH/GSSG ratio increased after removing glucose, suggesting that the cells had a greater reducing ability because of GLUT1.
Probing Androgen-Insensitive Cancer Cells
While researchers mostly focused on studying androgen-sensitive prostate cancer cells and the role GLUT1 plays in cell death protection, they discovered GLUT1 also benefits androgen-insensitive prostate cancer cells. When deprived of glucose, PC-3 cells, prostate cancer cells that do not produce AR or respond to androgens, died. Like with LNCaP cells, PC-3 cells treated with glucose analog 2DG did not change the levels of cell death, but supplementing glucose-free medium with other sugars (mannose or fructose) did increase cell survival. However, GLUT1 levels remained the same whether or not glucose was present in the medium, another result that suggests AR needs to be active to increase GLUT1 levels in glucose-deprived cells.
Repeating the GLUT1 overexpression in PC-3 cells paralleled the results in LNCaP cells. That is, cell death did not occur when deprived of glucose or treated with H2O2. Similar changes in cellular antioxidant levels were seen in PC-3 cells grown without glucose except for catalase, which decreased under glucose deprivation. Finally, like GLUT1-overexpression LNCaP cells, PC-3 cells over expressing GLUT1 altered the GSH/GSSG ratio, indicating that these androgen-insensitive cells resisted oxidative stress induced by glucose deprivation.
This research probed the relationship of androgen response to glucose deprivation in prostate cancer cell lines. Gonzalez-Menendez et al. discovered that androgen-sensitive cells increased GLUT1 when there was no glucose in the medium, allowing the cells to avoid cell necrosis and resist oxidative stress. While the amount of GLUT1 did not change for androgen-insensitive cells, these cells were able to survive glucose deprivation by resistance oxidative stress. These differences in androgen sensitivity offer insight into the changes in prostate tumors that are in early stages or those that become more aggressive in later stages.
Gonzalez-Menendez, P., Hevia, D., Alonso-Arias, R., Alvarez-Artime, A., Rodriguez-Garcia, A., Kinet, S., Gonzalez-Pola, I., Taylor, N., Mayo, J.C. and Sainz, R.M. (2018) GLUT1 protects prostate cancer cells from glucose deprivation-induced oxidative stress. Redox Biol. 17, 112–127. doi: 10.1016/j.redox.2018.03.017