Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression

Abstract

The development of cancer-specific therapeutics has been limited because most healthy cells and cancer cells depend on common pathways. Pyruvate kinase (PK) exists in M1 (PKM1) and M2 (PKM2) isoforms. PKM2, whose expression in cancer cells results in aerobic glycolysis and is suggested to bestow a selective growth advantage, is a promising target. Because many oncogenes impart a common alteration in cell metabolism, inhibition of the M2 isoform might be of broad applicability. We show that several small interfering (si) RNAs designed to target mismatches between the M2 and M1 isoforms confer specific knockdown of the former, resulting in decreased viability and increased apoptosis in multiple cancer cell lines but less so in normal fibroblasts or endothelial cells. In vivo delivery of siPKM2 additionally causes substantial tumor regression of established xenografts. Our results suggest that the inherent nucleotide-level specificity of siRNA can be harnessed to develop therapeutics that target isoform-specific exons in genes exhibiting differential splicing patterns in various cell types.

Figure 1.

The screening of a tiling siRNA library reveals sequences that can discriminate between the M1 and M2 isoforms of pyruvate kinase. (A) The sequences of the M1 and M2 isoforms of pyruvate kinase are shown. Mismatches are highlighted in yellow. (B) The viability of HCT116 cells for each member of the siPKM2 library is shown relative to the no treatment control. Cells were transfected with 5 nM siRNA at 0, 48, and 96 h and were assayed after 6 d. Transfections were performed in technical quadruplicate on two independent occasions. The three most active siRNA sequences are highlighted in red. NT, no treatment.

Figure 2.

The knockdown of the M2 isoform of pyruvate kinase mRNA by si27, si155, and si156 is potent and specific. (A) HCT116 cell were transfected with 5 nM siRNA. Duplicate biological samples were collected 48 h after transfection. Quantitative real-time PCR primers were designed to amplify total PKM, PKM1 only, or PKM2 only from HCT116 total RNA. HCT116, HepG2, and SKOV3 cells were transfected with 5 nM siRNA at 0, 48, and 96 h. (B and C) Cells were assayed for cell viability (B) and apoptosis (C) after 6 d. Results are normalized relative to the no treatment control. Transfections were performed in technical triplicate on two independent occasions. NT, no treatment. siPK, siRNA targeting both M1 and M2 isoforms. siControl, siRNA targeting firefly luciferase. Error bars denote standard deviation. (D) HCT116 cells were transfected with serial dilutions of si156 at 0, 48, and 96 h. Cells were assayed for cell viability after 6 d. Results are normalized relative to the no treatment control. Transfections were performed in technical quadruplicate on two independent occasions. Error bars denote standard deviation. (E) Lysates were collected from HCT116, HepG2, and SKOV3 cells 48 h after transfection with 5 nM siRNA. Blots were probed with antibodies targeting either total PKM or PKM2. Duplicate biological samples were collected for HepG2 and SKOV3, the cell lines used in the xenograft experiment. NT, no treatment. Vinculin was used as a loading control. (F) Effect of si156 on lactate production in HCT116 cells. The assay was performed 48 h after cells were transfected with 5 nM siRNA. Results are normalized to the no treatment control, and lactate production is further normalized to cell number. The experiment was performed in biological and technical duplicate on two independent occasions. Error bars denote standard deviation.

Figure 3.

Cancer cells are addicted to metabolic reprogramming by PKM2. 10 members of the NCI-60, representing multiple types of cancer and numerous underlying genetic insults, were transfected with 5 nM siControl or si156 at 0, 48, and 96 h. Cell viability (A) and apoptosis (B) were determined after 6 d. Results are normalized to the no treatment control, and apoptosis is further normalized to cell number. Transfections were performed in technical quadruplicate on two independent occasions. NT, no treatment. Error bars denote standard deviation.

Figure 4.

si156 does not reduce the viability of normal human primary cells. (A) The knockdown of the M2 isoform of pyruvate kinase mRNA by si156 is confirmed in SF372 human adult skin fibroblasts and human umbilical vein endothelial cells (HUVECs). Primers were designed to amplify total PKM, PKM1 only, or PKM2 only. Duplicate biological samples were collected 48 h after transfection with 5 nM siRNA, and each sample was assayed in technical triplicate. (B) Lysates were collected from SF372 and HUVE cells 48 h after transfection with 5 nM siRNA. Blots were probed with antibodies targeting either total PKM or PKM2. NT, no treatment. Vinculin was used as a loading control. (C) Relative cell viability was determined after six days. SF372 and HUVE cells were transfected with 5 nM siRNA at 0, 48, and 96 h. Transfections were performed in technical quadruplicate on two independent occasions. NT, no treatment. Error bars denote standard deviation.

Figure 5.

Administration of lipidoid-formulated si156 results in a reduction in volume of established tumors. (A) HepG2. (B) SKOV3. Error bars denote SEM (n = 4). The size difference between the treatment and control groups is significant by two-tailed Student’s t test at day 20. P = 0.0005 and P = 0.0133, respectively. (C and D) TUNEL staining confirms induction of apoptosis in vivo. Sections of SKOV3 tumors from mice treated with lipidoid-formulated siControl (C) or si156 (D) are shown. Blue, DAPI; red, TUNEL. Bars, 250 µm.