Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

Abstract

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV⁺ than HPV^- cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV⁺ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.

Keywords: HPV16 E7; ML265; PKM2; cervical cancer; non-pyruvate kinase function.

Figure 1

PKM2 is upregulated and has a prognostic value in cervical cancer. (A) TPM values of PKM2 mRNA in cervical cancer tissues (n = 304) and normal cervical tissues (n = 11) are shown in the log scale. * p = 1.3 × 10⁻⁶ (Welch’s unequal variances t-test). (B) Cervical cancer tissues shown in A were grouped based on the HPV status. The level of PKM2 mRNA was 2.1-fold higher in HPV⁺ cervical cancer (n = 169) than HPV^– cervical cancer (n = 9). * p = 0.05 (Welch’s unequal variances t-test). (C) Overall survival was worse in cervical cancer patients with high levels of PKM2 (red, n = 76) than those with low levels of PKM2 (blue, n = 75).

Figure 2

HPV16 E7 upregulates and interacts with PKM2 in cervical cancer cells. (A) HPV16 E7 interacts with PKM2 in SiHa cells. SiHa cell extracts (0.5 mg) were subject to co-immunoprecipitation using an anti-HPV16 E7 antibody. Normal IgG was used as a negative control. The input lane represents 50 μg of cell extracts. (B) GST proteins fused to HPV18 E7 (GST-18E7) and HPV45 E7 (GST-45E7) were incubated with 293T cell extracts overexpressing HA-tagged PKM2 (HA-PKM2). GST was used as a negative control. PKM2 was detected by western blot using an anti-HA antibody. GST fusion proteins were visualized by Coomassie blue staining. Intervening lanes were deleted and indicated by vertical lines. (C) PKM2 levels were higher in HPV⁺ than in HPV⁻ cervical cancer cells. Cell extracts were subject to western blot. Intervening lanes were deleted and indicated by vertical lines. (D) The levels of PKM2 mRNA was higher in SiHa cells than C33A cells. Total RNA was subject to semi-quantitative RT-PCR. The number of PCR cycle was 25 for PKM2 and 22 for GAPDH. (E) HPV16 E7 increased the level of PKM2. Cells were transfected with an empty (mock) or HPV16 E7-expressing plasmid. Cell extracts were subject to western blot. Actin was used as a loading control.

Figure 3

HPV16 E7-induced proliferation of cervical cancer cells depend on PKM2. (A) C33A cells stably expressing HPV16 E7 or empty vector were generated using retroviral vectors. Cell extracts were analyzed with western blot. (B) Cells described in A were seeded in 24-well plates (20,000 cells/well) and counted after 5 days. Results from five independent experiments are shown as mean ± S.E.M. V, vector. * p = 0.005 (two-sided Student’s t-test). (C) C33A-E7 cells were transduced with retrovirus expressing scrambled (SC) shRNA or PKM2 shRNA. Total cell extracts were subject to western blot. (D) Cells described in C were subject to cell counting assay, as described in B. Results from three independent experiments are shown as mean ± S.E.M. * p = 0.01 (two-sided Student’s t-test).

Figure 4

PKM2 is required for proliferation of cervical cancer cells. (A) SiHa cells were transiently transfected with scrambled (SC) shRNA or PKM2 shRNA vector. Total cell extracts were analyzed with western blot. (B) SiHa cells were transiently transfected as described in A and photographed 5 days later. Note that cells transfected with an PKM2 shRNA plasmid were sub-confluent. Scale bar, 100 µm. (C) SiHa cells were transiently transfected as described in A and counted 5 days later. Data are presented as mean ± S.E.M. (n = 3). * p = 0.01 (two-sided Student’s t-test).

Figure 5

ML265 inhibits cell cycle progression and colony formation in cervical cancer cells. (A) SiHa cells were seeded in 24-well plates (20,000/well) and treated with vehicle (veh) and 40 μm of ML265 (ML) for 5 days. The left panel shows photographed cells (scale bar, 100 µm). Red arrows point to cells growing on top of other cells. The right panel shows cell counting results as mean ± S.E.M. (n = 3). * p = 0.03 (two-sided Student’s t-test). (B) SiHa cells were seeded in 6-well plates (100 cells/well) and treated with 40 μm of ML265 for 2 weeks. Cells were stained with crystal violet and photographed (left panel). Quantified results (right panel) are shown as mean ± S.E.M. (n = 3). * p = 0.04 (two-sided Student’s t-test). (C) SiHa cells were treated with ML265 (40 µm) for 24 h. Cell cycle was analyzed with flow cytometry. Shown are representative results from two independent experiments with triplicate samples (p < 0.05, Student’s paired t-test). At least 10,000 cells per sample were analyzed. (D) SiHa cells were treated with ML265 (40 µm) for 24 h and stained with Hoechst 33342 to visualize DNA (blue). Random fields of view were photographed, and mitotic cells were quantified. The left panel shows the representative images of each group (scale bar, 50 µm). Cells in anaphase and telophase are circled, and each circle is counted as one cell. More than 1,000 cells per sample were analyzed. Quantification results are shown in the right panel as mean ± S.E.M (n = 3). * p = 0.003 (two-sided Student’s t-test).

Figure 6

ML265 decreases the phosphorylation of PKM2 at the Y105 position. (A) SiHa cells were treated with ML265 (40 µm) or vehicle (veh) for 24 h and then treated with paraformaldehyde for cross-linking. Total cell extracts were analyzed with western blot. GAPDH was used as a loading control. Note that ML265 decreased monomer and increased tetramer. (B) SiHa cells were treated with vehicle and ML265 for 24 h. Cells were nearly confluent at the endpoint. The cytoplasmic fraction (C) and the nuclear fraction (N) were analyzed with western blot. GAPDH and lamin A/C were used as a cytoplasmic and nuclear fraction marker, respectively. (C) SiHa cells were treated with ML265 for 24 h. Total cell extracts were subject to western blot analysis. (D) Total cell extracts from C33A-vector and C33A-E7 cells described in Figure 3A were analyzed with western blot. Note that both pY105-PKM2 and total PKM2 were increased in C33A-E7 cells compared to the control cells.