Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism

Abstract

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer.

FIGURE 1.

Knockdown, proliferation, and viability of Ras-3T3 cells depleted of several glycolytic enzymes. A, relative knockdown of glycolytic enzymes in Ras-3T3 cells. Cell extracts harvested 4 days after transfection were assayed for the indicated enzyme activity with and without substrate, and the no substrate control was subtracted before calculation of units/mg protein in each lysate. Relative specific activity was normalized to the specific activity of mock-transfected cells for each enzyme. Mock transfection (black) and siRNA to the indicated enzyme (white) are plotted. Experiments were done ≥3 times, and error bars are expressed as S.E. Mock transfection values used for normalization were 0.027 ± 0.003 (aldolase), 0.23 ± 0.04 (GAPDH), 1.3 ± 0.17 (triose-phosphate isomerase; TIM), and 0.12 ± 0.02 (enolase) units/mg. B, cells were plated at 2.5 × 10⁴/35 mm dish, and at the indicated time points after transfection, the number of cells for each treatment was determined using a hemocytometer and plotted for mock-transfected cells (●), and cells were transfected with aldolase siRNA (○), GAPDH siRNA (■), triose-phosphate isomerase siRNA (□), or enolase siRNA (×). Error bars represent 1 S.D. C, mock (♦) and aldolase siRNA-transfected (♢) cells were stained with crystal violet each day after transfection and plotted versus time. Values are normalized to 1 day post-transfection values for each treatment. Error bars are represented as S.D. D, mock (♦) and aldolase siRNA-transfected (♢ cells were assayed for proliferation using MTS each day after transfection. The absorbance reading at 490 nm for each treatment each day is plotted versus time. Error bars are represented as S.D. E, cells treated as in B were subjected to trypan-blue exclusion assay. Live cells were counted, normalized to total number of cells and plotted as fraction of viable cells for each treatment. Symbols and error bars are as described in B.

FIGURE 2.

Effect of aldolase depletion on proliferation in a panel of transformed cell lines. A, the indicated cell lines were transfected with siRNAs to aldolase and were monitored for aldolase activity as described in Fig. 1A. Relative activity at 4 days after transfection is plotted for mock transfection (black) and siRNA to aldolase (white) treatments. Experiments were done ≥3 times, and error bars are expressed as S.E. (B–D). For the indicated cell lines (top left of each panel), crystal violet staining determined the fold change in cell number for mock-transfected (●) and aldolase siRNA-transfected (○) cells as a function of time after transfection. Values at 1 day were used for normalization, and error bars are represented as 1 S.D.

FIGURE 3.

Rescue of 9L cells with exogenously expressed aldolase. A, anti-Myc immunoblot showing expression of Myc-tagged rabbit aldolase A in MycAld cells, but not in MSCV cells. Arrow indicates the position of 43 kDa, the expected size of the Myc-tagged aldolase. B, immunoblot showing Myc-tagged rabbit aldolase A expression in lysates following transfection of mock-treated and aldolase-knockdown (siAldolase) MycAld-9L cells. Cells were harvested 4 days after transfection, protein extracted, and assayed for MycAldolase via immunoblot. Actin was blotted as a loading control. C, MSCV-9L cells transfected with siRNAs to mouse aldolase A were monitored for knockdown by aldolase activity assay as described in Fig. 1A. Mock-transfected (0.02 ± 0.0014 units/mg) (black) and aldolase siRNA-transfected (white) values are shown. Error bars are represented as S.E. D, the number of cells/day were measured and plotted as described in Fig. 2 for mock (black) or siRNA to aldolase (open) transfected MSCV-9L (●, ○) and MycAld-9L (♦,♢) cells, respectively. Error bars are represented as 1 S.D. E, soft agar colony assay for parental 9L, MSCV-9L, or MycAld-9L mock-transfected (black) or aldolase-siRNA transfected (white) cells. After 14 days in soft agar, colonies were counted. Error bars represent 1 S.D. Statistical significance determined by t test is represented as ** (p < 0.005), triple asterisks (p < 0.001), or not significant (N.S., p > 0.05). IB, immunoblot.

FIGURE 4.

Proliferation of transformed versus non-transformed cells derived from the same parental cell line. Growth rates were calculated from the slope of a log plot of cell number (determined by crystal violet) versus days after transfection for mock-transfected (black) or aldolase siRNA-transfected (white) cells. A, NIH-3T3 and Ras-3T3 cells; B, Rat1 and DN-p53 Rat1 cells. Error bars are represented as S.E. Statistical significance as determined by t test is represented as *, p < 0.05; **, p < 0.005; or not significant (N.S.) p > 0.05).

FIGURE 5.

Glycolytic flux and intracellular ATP concentrations in aldolase siRNA-transfected Ras-3T3 cells. A, glycolytic flux was measured in NIH-3T3 (black) and Ras-3T3 (white) cells that were untreated, mock-transfected, or aldolase siRNA (siAld)-transfected using the rate of lactate production per cell. Values were normalized to NIH-3T3 mock-transfected treatment values (average 1.84 ± 0.17 × 10⁻⁷ μmol/h·cell). Error bars are represented as S.E. No significant differences were seen among any of the treatments within one cell type. B, intracellular [ATP]/cell was measured using a bioluminescence assay in NIH-3T3 (black) or Ras-3T3 (white) cell lysates after treatment as described in A. Values are normalized to NIH-3T3 mock-transfected treatment values (average value 0.1 fmol/cell). Error bars are represented as S.E. No significant differences were seen among any of the treatments within one cell type.

FIGURE 6.

Catalytically dead D33S-aldolase rescues proliferation defect. A, immunoblot of MSCV-9L and MycAld-9L cells that were mock- or aldolase siRNA-transfected (Mock or siAld). Cells were harvested 4 days post transfection, and protein was extracted and assessed for MycAldolase expression via immunoblot (IB; top row). Actin was blotted as a loading control (bottom row). B, comparison of growth rates for MSCV-, MycAld-, and MycD33S-9L cells that were mock- (black) or aldolase siRNA (white)-transfected. Cells were stained with crystal violet each day after transfection. Rates were determined and compared by plotting ln A₅₇₀ versus time. Each treatment was performed in triplicate, and error bars are expressed as 1 S.D. *, p < 0.05; **, p < 0.005; ***, p < 0.001, or not significant (N.S.), p > 0.05.

FIGURE 7.

Increased multinucleation in aldolase siRNA-transfected Ras-3T3 cells. A–F, representative images of mock-transfected (A–C) and aldolase siRNA-transfected (D–F) Ras-3T3 cells stained for nuclei (DAPI) (A and D), counterstained with Alexa Fluor 488 phalloidin (B and E), or the merged images (C and F) are depicted. An arrow indicates the presence of multiple nuclei in one cell. G, quantification of the percentage of multinucleated cells and percentage of cells with >2 nuclei for mock-transfected (black) or aldolase siRNA-transfected (white) Ras-3T3 cells. The experiment was performed ≥4 times, and at least 50 cells were counted for each treatment. Error bars are represented as S.E., and significance is represented as double asterisks (p < 0.005) or triple asterisks (p < 0.001).

FIGURE 8.

Comparison of the effects of glycolytic enzyme depletion on proliferation in transformed cell lines. Relative protein expression (white) and relative rates of proliferation (black) for RNAi-treated cells for each enzyme were normalized to the controls for protein expression or cell proliferation, respectively. Boldface type indicates enzymes investigated in this study. Enzymes are shown in the order of the reactions they catalyze in the glycolytic pathway (although PFK-2 is not directly involved in glycolytic reactions, it is a positive regulator of PFK-1, and its knockdown is functionally equivalent to inhibition of PFK-1; thus, it is depicted in parentheses): no knockdown (KD; no RNAi), hexokinase (HK), phosphoglucoisomerase (PGI) (7), phosphofructokinase-1 (PFK-1) (6), phosphofructokinase-2 (PFK-2) (5), aldolase A (Ald-A), triose-phosphate isomerase, GAPDH, phosphoglycerate kinase (PGK) (4), phosphoglycerate mutase (PGM), enolase-1 (Eno-1), PK-M2 (9), lactate dehydrogenase-A (LDH-A; 10). ND indicates no report of this enzyme affecting growth of cells in culture. All data are for cells grown under normoxic conditions with the exception of PFK-1, which was measured under hypoxic conditions and is indicated by an asterisk.