MUC1-C oncoprotein regulates glycolysis and pyruvate kinase M2 activity in cancer cells

Abstract

Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2). Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR)-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis.

Figure 1. MUC1-C cytoplasmic domain (MUC1-CD)-induced transformation of 3Y1 cells is associated with induction of glycolysis and PKM2 activity.

A. Structure of the MUC1-C subunit with the extracellular domain (ED), transmembrane domain (TM) and amino acid sequence of the cytoplasmic domain (MUC1-CD). The CQC motif and the EGFR phosphorylation site are highlighted. B and C. 3Y1/vector and 3Y1/MUC1-CD cells were analyzed for glucose uptake (B) and lactate production (C). The results (mean±SD of five separate experiments each performed in triplicate) are expressed as nmol/10⁶ cells. D. Lysates from rat 3Y1/vector and 3Y1/MUC1-CD cells were analyzed for PKM2 activity. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as relative PKM2 activity compared to that obtained in 3Y1/vector cells (assigned a value of 1). The student's t-test was used to determine the p-values. E and F. Lysates from 3Y1/vector and 3Y1/MUC1-CD cells were immunoblotted with the indicated antibodies. G-I. 3Y1/vector and 3Y1/MUC1-CD cells were transfected with Control siRNA or Akt siRNA pools for 72 h. Lysates from the indicated cells were immunoblotted with anti-Akt and anti-β-actin (G). The indicated cells were analyzed for glucose uptake (H, left) and lactate production (H, right). The results (mean±SD of three replicates) are expressed as nmol/10⁶ cells. Lysates from the indicated cells were also analyzed for PKM2 activity (I). The results (mean±SD of three replicates) are expressed as relative PKM2 activity compared to that obtained in 3Y1/vector cells (assigned a value of 1).

Figure 2. MUC1-C cytoplasmic domain binds to PKM2.

A. Lysates from 3Y1/MUC1-CD cells were subjected to immunoprecipitation with a control IgG or anti-PKM2. The precipitates were immunoblotted with the indicated antibodies. B. Lysates from 3Y1/vector cells were incubated with GST or GST-MUC1-CD. The adsorbates to glutathione beads were immunoblotted with anti-PKM2. Input of the GST proteins was assessed by Coomassie blue staining. C. Lysates from human ZR-75-1 breast cancer cells were immunoprecipitated with a control IgG or anti-PKM2. Precipitates were immunoblotted with the indicated antibodies. D. ZR-75-1 cell lysates were incubated with GST, GST-MUC1-CD and the indicated GST-MUC1-CD deletion mutants. The adsorbates were immunoblotted with anti-PKM2. Input of the GST proteins was assessed by Coomassie blue staining. E. PKM2 was incubated with GST, GST-MUC1-CD and the indicated GST-MUC1-CD deletion mutants. The adsorbates were immunoblotted with anti-PKM2. F. PKM2 was incubated with GST, GST-MUC1-CD and the indicated GST-MUC1-CD mutants in which Cys-1 and/or Cys-3 were substituted with Ala. The adsorbates were immunoblotted with anti-PKM2.

Figure 3. MUC1-CD binds directly to PKM2 B- and C-domains.

A. Schematic representation of the PKM2 protein with the N-terminus (N) and the A1-, B-, A2- and C-domains. Highlighted are the Cys-165 and Cys-474 residues. B and C. MUC1-CD was incubated with GST and the indicated GST-PKM2 deletion mutants. The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. D. MUC1-CD was incubated with GST, GST-PKM2(117-218) and GST-PKM2(117-218) with the indicated C152A and C165A mutations. The adsorbates were immunoblotted with anti-MUC1-C. E. MUC1-CD was incubated with GST, GST-PKM2(390-531) and GST-PKM2(390–531) with the indicated C423A, C424A and C474A mutations. The adsorbates were immunoblotted with anti-MUC1-C.

Figure 4. Phosphorylation of MUC1-CD on Tyr-46 confers binding to the PKM2 C-domain at Lys-433.

A. His-tagged MUC1-CD and MUC1-CD(Y46F) were incubated with EGFR in the absence and presence of ATP. The reaction products were analyzed by immunoblotting with anti-p-Tyr and anti-MUC1-C (left). GST and the indicated GST-PKM2 deletion mutants were incubated with EGFR-phosphorylated MUC1-CD (p-MUC1-CD) (right). The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. B. GST and GST-PKM2(390–531/C474A) were incubated with MUC1-CD or p-MUC1-CD. The adsorbates were immunoblotted with anti-MUC1-C. C. GST and GST-PKM2(390-531) were incubated with MUC1-CD(C3A) or EGFR-phosphorylated p-MUC1-CD(C3A). The adsorbates were immunoblotted with anti-MUC1-C. D. PKM2 was incubated with GST, GST-MUC1-CD(C3A) and EGFR-phosphorylated GST-p-MUC1-CD(C3A). The adsorbates were immunoblotted with anti-MUC1-C and anti-p-Tyr. E and F. GST, GST-PKM2(390-531) and GST-PKM2(390-531/K433E) were incubated with p-MUC1-CD (E) or ZR-75-1 cell lysate (F). The adsorbates were immunoblotted with anti-MUC1-C.

Figure 5. MUC1-CD CQC motif stimulates PKM2 activity.

A. Recombinant PKM2 was incubated in the absence and presence of the indicated concentrations of MUC1-CD (left) or MUC1-CD (C3A) (right) for 30 min at room temperature. The results (mean+SD of five separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control. B. PKM2 was incubated with the indicated concentrations of FBP alone, MUC1-CD alone, or FBP and MUC1-CD. The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control. C. Amino acid sequences of GO-203 and CP-2 peptides. PKM2 was incubated with 100 µM GO-203 or CP-2. The results (mean+SD of five separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control. D. PKM2 and the indicated PKM2 mutants were incubated in the absence (open bars) and presence of 100 µM GO-203 (solid bars). The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained in the absence of GO-203. E. PKM2 was incubated with the indicated concentrations of FBP alone, GO-203 alone, or FBP and GO-203. The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control.

Figure 6. PKM2 activity is inhibited by EGFR-phosphorylated MUC1-CD.

A. PKM2 was incubated with 10 µM FBP in the absence (Control) and presence of 10 µM unphosphorylated and EGFR-phosphorylated MUC1-CD, MUC1-CD(C3A) and MUC1-CD(Y46F). The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control. B. Sequences of the Tyr-46 and phospho-Tyr-46 peptides. C. PKM2 was incubated with 10 µM FBP in the absence (Control) and presence of 100 µM phospho-Tyr-46 peptide or Tyr-46 peptide. The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control. D. PKM2 was incubated in the absence (Control) and presence of 20 µM GO-203 alone, 100 µM phospho-Tyr-46 peptide alone or GO-203 with phospho-Tyr-46 peptide. The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity compared to that obtained with the control.

Figure 7. Silencing MUC1-C downregulates aerobic glycolysis in breast cancer cells.

A. Lysates from ZR-75-1 and MCF-7 cells stably expressing an empty vector or a MUC1siRNA were immunoblotted with the indicated antibodies. B. The indicated cells were analyzed for glucose uptake. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as nmol/10⁶ cells. C. The indicated cells were analyzed for lactate production. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as relative lactate production compared to that in cells expressing the empty vector (assigned a value of 1). D. The indicated cells were plated in soft agar and colonies were counted after incubation for 14 days. The results (mean±SD of two separate experiments each performed in triplicate) are expressed as the colony number relative to that obtained with ZR-75-1 or MCF-7 cells expressing an empty vector (each assigned a value of 1).

Figure 8. Effects of MUC1-C on PKM2 activity are dependent on cell context.

A and B. Lysates from the indicated ZR-75-1 (A) and MCF-7 (B) cells harvested at the indicated times after passage were analyzed for PKM2 activity (left). The results (mean+SD of three separate experiments each performed in triplicate) are expressed as relative PKM2 activity compared to that obtained in cells expressing the empty vector (assigned a value of 1). The ZR-75-1/vector (A) and MCF-7/vector (B) cell lysates were also incubated with anti-MUC1-C and the precipitates were immunoblotted with the indicated antibodies (right). C. MCF-7/vector and MCF-7/MUC1siRNA cells were left untreated and stimulated with EGF for 5 min. Lysates from MCF-7/vector cells were incubated with anti-MUC1-C and the precipitates were immunoblotted with the indicated antibodies (left). Lysates were assayed for PKM2 activity (right). The results (mean+SD of three separate experiments) are expressed as the relative PKM2 activity as compared to that in unstimulated MCF-7/vector cells.

Figure 9. Effects of MUC1 on glycolysis and PKM2 activity are attenuated by the C3A mutation.

A. Lysates from HCT116 cells stably expressing an empty vector, wild-type MUC1 or MUC1(C3A) were immunoblotted with the indicated antibodies. B. The indicated cells were analyzed for glucose uptake. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as nmol/106 cells. C. The indicated cells were analyzed for lactate production. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as relative lactate production compared to that in cells expressing the empty vector (assigned a value of 1). D. Lysates from the indicated cells were analyzed for PKM2 activity. The results (mean±SD of three separate experiments each performed in triplicate) are expressed as relative PKM2 activity compared to that obtained in cells expressing the empty vector (assigned a value of 1). E. The indicated cells were plated in soft agar and colonies were counted after incubation for 14 days. The results (mean±SD of two separate experiments each performed in triplicate) are expressed as the colony number relative to that obtained with HCT116/vector cells (assigned a value of 1).