Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Suppressing Pyrimidine Synthesis in Malignant Gliomas


By: Carol A. Rouzer, VICB Communications
Published:  February 26, 2015



An inhibitor targeting phospholipase D suppresses pyrimidine biosynthesis, leading to reduced nucleotide levels in the cells.


A hallmark of cancer cells is their excessive use of glucose via the glycolytic pathway. Originally, many investigators thought that the cells were using glycolysis to fulfill their energy needs, but we now know that most cancer cells obtain their energy primarily through the process of oxidative phosphorylation. Instead, recent evidence suggests that rapidly growing and dividing cancer cells require compounds produced in the glycolytic pathway for the biosynthesis of other cellular components such as lipids, amino acids, and nucleotides. Dysfunctional regulation of signaling pathways is frequently responsible for the abnormal metabolism seen in cancer cells, leading to the hypothesis that drugs targeting these pathways might induce growth suppression or death by disrupting cellular metabolism. So far, however, most attempts to block key metabolic signaling pathways have been thwarted by unacceptable side effects. Now, Vanderbilt Institute of Chemical Biology members Alex Brown and Craig Lindsley report a new approach to metabolic disruption in glioma cells using inhibitors of phospholipase D (PLD) [T. P. Mathews, et al. (2015) ACS Chem. Biol., published online February 3, doi:10.1021/cb500772c].


PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PtdOH), an important lipid signaling molecule (Figure 1). Prior research in the Brown laboratory had shown that PtdOH generated primarily by the PLD2 isoform plays a role in activating the protein kinase Akt in glioma cells under nutrient-poor conditions (serum starvation, Figure 2). Akt activates many pathways that promote cell growth and survival. In particular, it promotes glycolysis through stimulation of glucose transport and the enzyme hexokinase. These findings led the Brown lab investigators to explore other metabolic responses to inhibition of PLD using isoform-selective inhibitors developed in collaboration with the Lindsley laboratory.




Figure 1. Reaction catalyzed by PLD. The substrate is phosphatidylcholine and the products are phosphatidic acid and choline. Structures of the substrates and products may vary depending on the fatty acids in the lipids.


The Brown lab investigators began their studies with metabolomics analyses of various cancer cell lines incubated under normal growth conditions in the presence or absence of PLD1- or PLD2-selective inhibitors. The results quickly pointed to a decrease in deoxyribonucleotide triphosphate (dNTP) levels in glioma cells treated with the PLD1-selective inhibitor. Other cancer cell lines did not respond to the PLD1 inhibitor in this way. Further studies with serum-starved cells demonstrated a similar decrease in dNTP levels, but in this case, both the PLD1- and PLD2-selective inhibitors were effective. Under these conditions, an inhibitor of Akt also reduced dNTP levels. This finding, along with the observation that glioma cells expressing a constitutively active, myristoylated form of Akt (Myr-Akt) were insensitive to the PLD inhibitors but sensitive to Akt inhibition, suggested that the dNTP suppressive effects of the inhibitors under serum-starvation were due to inhibition of Akt activation. This finding was consistent with prior results from the lab (Figure 2).



Figure 2.  Role of PLD2 in the activation of the PI3K/Akt pathway. In this example, a receptor tyrosine kinase (RTK) binds its ligand (L), leading to activation of the receptor. Receptor activation leads to autophosphorylation at key tyrosine residues. PI3K binds to the RTK at one of these phosphotyrosines, resulting in its activation in proximity of the membrane. It now can convert its substrate PIP2 (magenta hexagons) to PIP3 (green octagons). Akt binds to PIP3 in the membrane through its plextrin homology domain. Under conditions of serum-starvation, this binding is facilitated by PtdOH (blue starbursts) produced from PC (red spheres) by PLD2. PDK1 also binds to PIP3, and phosphorylates Akt at threonine-308. Other kinases, including mTORC2, phosphorylate Akt at serine-473. Phosphorylated Akt is now activated. PTEN converts PIP3 back to PIP2 as the first step to reverse the signaling process. Based on this model, inhibition of PLD2 will result in reduced Akt activity.

To better understand the effects of PLD1 inhibition on dNTP levels under normal growth conditions, the Brown lab investigators evaluated the effects of direct Akt inhibition on dNTP levels in this setting. They discovered that, while Akt inhibition could mimic the effects of the PLD1 inhibitor on dNTP levels in wild-type glioma cells, it was ineffective in cells expressing Myr-Akt. This result suggested that PLD1 inhibition must be able to suppress dNTPs through a pathway that does not require Akt. A clue to the identity of this pathway came from studies using an inhibitor of mTOR, a kinase complex downstream of Akt. mTOR inhibitors reduced dNTP levels in both wild-type and Myr-Akt-expressing glioma cells, suggesting a direct link between PLD1 and mTOR that bypasses Akt.


To test the hypothesis that mTOR regulates dNTP levels through a PLD-regulated mechanism in glioma cells, the investigators used a phosphoproteomics screen to detect changes in the pattern of phosphorylated proteins elicited by PLD1 inhibition. For these studies, they preincubated control cells in standard culture medium and cells to be treated with the PLD1 inhibitor in medium containing arginine and lysine labeled with nonradioactive “heavy” isotopes. After the cells had incorporated the labeled amino acids into their proteins, the investigators treated them with the PLD1 inhibitor. They then combined the lysates from the “light” control cells and “heavy” PLD1 inhibitor-treated cells and subjected them to proteomics analysis directed toward the identification of phosphorylated peptides. The results demonstrated that treatment with the PLD1 inhibitor suppressed the phosphorylation of multiple targets of the mTOR signaling pathway. Among these were 4E-BP1 and EIF3A, modulators of protein translation, ULK1, a regulator of autophagy, RAPTOR a regulator of the mTORC1 complex, and CAD, an enzyme complex that catalyzes the initial steps of pyrimidine biosynthesis (Figure 3). These findings clearly supported a direct link between PLD1 and mTOR activation.


Figure 3. Proposed mechanism for the activation of mTOR by PLD1. Studies suggest that mTOR is directly activated by PtdOH produced by PLD. Activation of mTOR leads to increased phosphorylation and activation of its targets. Particularly relevant to this work is the activation of CAD, which catalyzes the first steps in pyrimidine biosynthesis. The data suggest that the isoform involved in mTOR activation in glioma cells is PLD1. The form of mTOR involved shown here is mTORC1, due to the proteomics data suggesting that RAPTOR phosphorylation is decreased in the presence of PLD1 inhibition. RAPTOR is a part of the mTORC1 complex.



Further experiments using immunoblotting with an antibody directed against a phosphopeptide of 4E-BP1 confirmed that PLD1 inhibition reduced phosphorylation at that site in both parental and Myr-Akt-expressing glioma cells. Of even greater interest was the finding that PLD1, Akt, and mTOR inhibitors reduced the activity of CAD as indicated by the reduction in the levels of one of its products, carbamoyl aspartate, in parental glioma cells. Myr-Akt-expressing cells behaved similarly with the exception that they were unresponsive to Akt inhibition, again suggesting a direct link between PLD1 and mTOR without a requirement for Akt. The effects of PLD1 inhibition on carbamoyl aspartate levels suggested that modulation of CAD activity contributed to the reduction in dNTP levels observed in the presence of the inhibitor. This conclusion was further supported by the finding that overexpression of CAD reversed the effects of PLD1 inhibition on dNTP levels in parental glioma cells. Finally, to further confirm that the results observed were due to inhibition of PLD1, the investigators showed that all of the same effects could be reproduced by raloxifene, a structurally distinct PLD inhibitor.


Together, the results demonstrate that PLD1 regulates a fundamental metabolic process, dNTP biosynthesis, through modulation of mTOR activity in glioma cells. The importance of mTOR dysregulation in multiple forms of cancer is well recognized; however, attempts to suppress cancer cell growth by direct blockade of this pathway have been met with limited success due to unwanted side effects. The toxicity of PLD inhibitors in vivo is remarkably low, suggesting that PLD inhibition may be a more successful approach to suppress the abnormal metabolic functions observed in malignant gliomas. The applicability of this approach to other forms of cancer is unclear. However, glioma is a difficult to treat, highly deadly form of brain cancer, so progress in the therapy of this disease would be a great step forward.





The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center

  • Areas of Interest