Lapatinib Inhibits Breast Cancer Cell Proliferation by Influencing PKM2 Expression

Abstract

Pyruvate kinase type M2, which is expressed in multiple tumor cell types and plays a key role in aerobic glycolysis, also has nonglycolytic functions and can regulate transcription and cell proliferation. The results of this study show that epidermal growth factor receptor activation induces pyruvate kinase type M2 nuclear translocation. To further determine the relationship between pyruvate kinase type M2 and epidermal growth factor receptor, we analyzed pathological data from mammary glands and performed epidermal growth factor receptor/human epidermal growth factor receptor 2 knockdown to reveal that pyruvate kinase type M2 is associated with epidermal growth factor receptor and human epidermal growth factor receptor 2. Lapatinib is a small molecule epidermal growth factor receptor tyrosine kinase inhibitor that can inhibit epidermal growth factor receptor and human epidermal growth factor receptor 2, though its effect on pyruvate kinase type M2 remains elusive. Accordingly, we performed Western blotting and reverse transcription polymerase chain reaction and analyzed pathological data from mammary glands, with results suggesting that lapatinib inhibits pyruvate kinase type M2 expression. We further found that the antitumor drug lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, as based on Cell Counting Kit-8 analyses and pyruvate kinase type M2 overexpression experiments. Signal transducer and activator of transcription 3, which is a transcription factor-associated cell proliferation and the only transcription factor that interacts with pyruvate kinase type M2, we performed pyruvate kinase type M2 knockdown experiments in Human breast cancer cells MDA-MB-231 and Human breast cancer cells SK-BR-3 cell lines and examined the effect on levels of Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3. The results indicate that pyruvate kinase type M2 regulates Signal transducer and activator of transcription 3 and phospho-Stat3 (Tyr705) expression. Together with previous reports, our findings show that lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, which results in a reduction in both Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3.

Keywords: breast cancer; cell proliferation; isoenzyme; lapatinib; pyruvate kinase.

Figure 1.

Pyruvate kinase type M2 is highly expressed in breast cancer tissues. A, Immunohistochemical staining with an anti-PKM2 antibody was performed on breast cancer tissues and adjacent normal tissues. (a), (c), and (e) Positive staining of PKM2 in tumor tissues (at ×400). (b), (d), and (f) Negative results for PKM2 in normal tissues (at ×400). (g) Negative control, with the primary antibody against PKM2 omitted and replaced with preimmune serum (at ×400). B, Western blot of breast cancer tissues and adjacent normal tissues was performed with an anti-PKM2 antibody. β-Actin was used as a loading control. PKM2 denotes pyruvate kinase type M2.

Figure 2.

Expression of EGFR, HER2, HER3 and PKM2 in breast cancer cell lines. Inhibition of PKM2 expression by siEGFR in MDA-MB-231 cells, inhibition of PKM2 expression by siHER2 in SK-BR-3 cells, and determination of the working concentrations of lapatinib and EGF. A, Epidermal growth factor receptor was expressed in MDA-MB-231 cells, whereas HER2 and HER3 were not. HER2 and HER3 were expressed in SK-BR-3 cells, whereas EGFR was not. Human epidermal growth factor receptor 3 was expressed in MCF-7 cells, whereas EGFR and HER2 were not. Three cell lines expressed PKM2. The experiments were repeated 3 times. B, Epidermal growth factor receptor knockdown analyses were performed in MDA-MB-231 cells. Cells were transfected with 20 μM siEGFR for 48 hours and lysed for Western blotting. We successfully achieved EGFR gene silencing with Stealth RNAi siRNA oligoribonucleotide duplexes of EGFR. Knocking down EGFR resulted in a reduction in PKM2 expression compared to control groups. WT represents the wild-type control group. Scr represents the negative control group. The experiments were repeated 3 times. C, Human epidermal growth factor receptor 2 knockdown analyses were performed in SK-BR-3 cells. Cells were transfected with 20 μM siHER2 for 48 hours and lysed for Western blotting. We successfully achieved HER2 gene silencing with Stealth RNAi siRNA oligoribonucleotide duplexes of HER2. Knocking down HER2 resulted in a reduction in PKM2 expression compared to control groups. WT represents the wild-type control group. Scr represents the negative control group. The experiments were repeated 3 times. D-F, The working concentration of lapatinib was 1.0 μM. Expression of PKM2 was significantly reduced with 1.0 μM lapatinib treatment in MDA-MB-231 (D) and SK-BR-3 (E) cells compared with lower concentrations of 0 μM and 0.5 μM; expression was not notably changed compared with 2.0 μM lapatinib treatment. In MCF-7 cells, the effect of Lapatinib on the expression of PKM2 was not obvious (F). All cells were treated for 48 hours. The experiments were repeated 3 times. G, The working concentration of EGF was 10 ng/mL. Expression of PKM2 was significantly increased with 10 ng/mL EGF treatment of MDA-MB-231 cells compared to lower concentrations of 0 and 5 ng/mL, and there was no marked change compared to higher concentrations of 20, 50, and 100 ng/mL. All cells were treated for 48 hours. The experiments were repeated 3 times. H, The working concentration of EGF was 50 ng/mL. Expression of PKM2 was significantly increased with 50 ng/mL EGF treatment of SK-BR-3 compared to lower concentrations of 0, 5, 10, and 20 ng/mL, and there was no obvious change compared to higher concentration of 100 ng/mL. All cells were treated for 48 hours. The experiments were repeated 3 times. I, In MCF-7 cells, there was a trend of increasing the protein levels of PKM2 in MCF-7 cells induced with EGF. All cells were treated for 48 hours. The experiments were repeated 3 times. EGFR indicates epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; PKM2, pyruvate kinase type M2; siRNA, small inhibitory RNA; siEGFR, small inhibitory EGFR; siHER2, small inhibitory HER2; MDA-MB-231, Human breast cancer cells MDA-MB-231; MCF-7, Michigan Cancer Foundation-7; SK-BR-3, Human breast cancer cells SK-BR-3.

Figure 3.

Lapatinib inhibits PKM2 expression in breast cancer cell lines. A, Lapatinib inhibits PKM2 and PKM2 (Tyr (P)-105) expression in MDA-MB-231 cells. In DMSO treatment groups, PKM2 expression was increased after stimulation with EGF compared to controls, and PKM2 expression after EGF stimulation for 48 hours was more increased than after 24 hours. PKM2 (Tyr(P)-105) expression was increased after stimulation with EGF compared to controls (left). In lapatinib treatment groups, PKM2 and PKM2 (Tyr(P)-105) expression was not notably changed after stimulation with EGF compared to controls (right). The PKM2 expression in DMSO treatment groups increased more than in lapatinib treatment groups with EGF stimulation for the same time period. The experiments were repeated 3 times. B, Lapatinib inhibits PKM2 and PKM2 (Tyr (P)-105) expression in SK-BR-3 cells. In DMSO treatment groups, PKM2 expression was increased after stimulation with EGF for 48 hours compared to controls. The PKM2 (Tyr(P)-105) expression was increased after stimulation with EGF compared to controls. The PKM2 (Tyr(P)-105) expression with EGF stimulation for 48 hours was increased more than after 24 hours (left). In lapatinib treatment groups, PKM2 and PKM2 (Tyr(P)-105) expression were not obviously changed after stimulation with EGF compared to controls (right). PKM2 expression in DMSO treatment groups was increased more than in lapatinib treatment groups with EGF stimulation for the same time period. The experiments were repeated 3 times. C, Lapatinib inhibits mRNA levels of PKM2 in MDA-MB-231. In DMSO treatment groups, PKM2 expression was significantly increased after stimulation with EGF compared to controls (P < .05). In lapatinib treatment groups, PKM2 expression was not markedly changed after stimulation with EGF compared to controls. The PKM2 expression in DMSO treatment groups was increased more than in lapatinib treatment groups with EGF stimulation for the same time period (P < .05). The experiments were performed in triplicate. Data are presented as means (SD). D, Lapatinib inhibited mRNA levels of PKM2 in SK-BR-3 cells. In DMSO treatment groups, PKM2 expression was significantly increased after stimulation with EGF compared to controls (P < .05). In lapatinib treatment groups, PKM2 expression was not obviously changed after stimulation with EGF compared to controls. PKM2 expression in DMSO treatment groups was increased more than in lapatinib treatment groups with EGF stimulation for the same time period (P < .05). The experiments were performed in triplicate. Data are presented as means (SD). DMSO denotes dimethyl sulfoxide; EGF, epidermal growth factor; mRNA, messenger RNA; PKM2, pyruvate kinase type M2; SD, standard deviation; MDA-MB-231, Human breast cancer cells MDA-MB-231; SK-BR-3, Human breast cancer cells SK-BR-3.

Figure 4.

Lapatinib inhibits breast cancer cell proliferation by influencing PKM2 expression. A, Lapatinib inhibits MDA-MB-231 cell proliferation. MDA-MB-231 cells were treated with lapatinib and DMSO. The results showed that cells proliferated at a significantly slower rate in the lapatinib treatment group compared with DMSO treatment and untreated groups (P < .05). There were no obvious changes in the DMSO treatment group compared to the untreated group in terms of cell proliferation. Both DMSO treatment groups and untreated groups served as control groups. Data represent the means (SD) of 6 independent experiments. B, Lapatinib inhibits SK-BR-3 cell proliferation. SK-BR-3 cells were treated with lapatinib and DMSO. The results showed that cells proliferated at a significantly slower rate in the lapatinib treatment group compared to DMSO treatment and untreated groups (P < .05). There were no obvious changes in cell proliferation in the DMSO treatment group relative to the untreated group. Dimethyl sulfoxide treatment groups and untreated groups served as control groups. Data represent the means (SD) of 6 independent experiments. C and E, The pcDNA3.1-PKM2 recombinant plasmid was successfully transfected into MDA-MB-231 and SK-BR-3 cell lines. WT represents the wild-type group. Vec represents the negative control group. The experiments were repeated 3 times. D and F, Lapatinib inhibits breast cancer cell proliferation by influencing PKM2 expression. The PKM2 sequences were transfected into MDA-MB-231 and SK-BR-3 cell lines under treatment with lapatinib for overexpression. The CCK-8 results show that cells proliferated at a significantly faster rate in transfected groups compared to control groups (P < .05), and there was no obvious change between negative control groups and wild-type groups. WT represents the wild-type group. Vec represents the negative control group. Data represent the means (SD) of 6 independent experiments. CCK-8 denotes Cell Counting Kit-8; DMSO, dimethyl sulfoxide; PKM2, pyruvate kinase type M2; SD, standard deviation; MDA-MB-231, Human breast cancer cells MDA-MB-231; SK-BR-3, Human breast cancer cells SK-BR-3.

Figure 5.

Inhibition of PKM2 by lapatinib reduces levels of Stat3 and phosphorylated Stat3. A, Knockdown of PKM2 results in reduced Stat3 and Stat3 (Tyr(P)-705) expression in MDA-MB-231 cells. Cells were transfected with 20 μM siPKM2 for 48 hours and lysed for Western blotting. Knockdown of PKM2 resulted in decreased Stat3 and Stat3 (Tyr(P)-705) expression compared to WT and negative control groups. The experiments were repeated 3 times. B, Knockdown of PKM2 results in reduced Stat3 and Stat3 (Tyr(P)-705) expression in SK-BR-3 cells. Cells were transfected with 20 μM siPKM2 for 48 hours and lysed for Western blotting. Knockdown of PKM2 resulted in decreased Stat3 and Stat3 (Tyr(P)-705) expression compared to WT and negative control groups. The experiments were repeated 3 times. C and D, Inhibition of PKM2 by lapatinib reduces levels of Stat3 and phosphorylated Stat3. MDA-MB-231 and SK-BR-3 cell lines were treated with lapatinib. The results showed that PKM2 expression was significantly reduced in lapatinib treatment groups compared to control groups; Stat3 and Stat3 (Tyr(P)-705) expression was also significantly reduced after treatment with lapatinib compared to control groups in both cell lines. The experiments were repeated 3 times. PKM2 denotes pyruvate kinase type M2; Stat3, Signal transducer and activator of transcription 3; Stat3(Tyr(P)-705), phospho-Stat3 (Tyr705); MDA-MB-231, Human breast cancer cells MDA-MB-231; SK-BR-3, Human breast cancer cells SK-BR-3; WT, Wild type.

Figure 6.

Lapatinib inhibits breast cancer cell proliferation by influencing PKM2 expression, which reduces levels of Stat3 and phosphorylated Stat3. Downregulation of PKM2 expression by lapatinib-mediated suppression of EGFR and HER2 reduces Stat3 and pStat3 expression, which leads to a lower level of gene transcription and inhibition of tumor cell proliferation. EGFR denotes epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; PKM2, pyruvate kinase type M2; Stat3, Signal transducer and activator of transcription 3; pStat3, Phosphorylated Stat3.