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Sources of Funding: National Institutes of Health (CA134514 and CA130908 to H.H.), the Department of Defense (W81XWH-09-1-622 and W81XWH-14-1-0486 to H.H.), National Natural Science Foundation of China (81101931 to C.W.P) and Shanghai Municipal Commission of Health and Family Planning (201640041 to C.W.P).

Introduction

Elevated glucose uptake and lactate production in the availability of oxygen, a phenomenon called the Warburg effect, is important for cancer cell growth. Phosphorylation of Pyruvate kinase isozyme M2 (PKM2) by ERK promotes its nuclear localization and the Warburg effect. O-class forkhead factor 1 (FOXO1) acts as a tumor suppressor in the nucleus. FOXO1 can be phosphorylated by activated AKT and exported to cytoplasm, thereby losing its tumor suppressor function. Here, we revealed that AKT-phosphorylated FOXO1 inhibits IQGAP1-augmented activation of ERK. We also investigated whether AKT-phosphorylated FOXO1 inhibits PKM2 nuclear localization and Warburg effect in prostate cancer.

Methods

We used co-IP to determine protein interaction, Western-blot to detect the protein level, RT-PCR to detect gene expression level, immunocytochemistry to detect PKM2 cellular localization, immunohistochemistry to analysize FOXO1 and phosphor-ERK level in human prostate cancer using tissue microarray (TMA). Glucose or lactate levels were determined using a glucose assay kit or lactate assay kit. IQBP was a small phosphor-mimicking peptide derived from FOXO1. PC-3-Luc cells were injected into NSG mice to generate xenograft model.

Results

We identified the scaffold protein IQGAP1 as a binding partner of FOXO1 (Fig. a). We demonstrated that activated AKT is important for FOXO1-IQGAP1 interaction (Fig. b and c), and AKT-phosphorylated FOXO1 at serine-319 is critical for FOXO1 binding to IQGAP1 (Fig. d). Phosphorylated FOXO1 inhibits IQGAP1-augmented phosphorylation of ERK(Fig. e-g). We also found that there is an inverse correlation between FOXO1 and phosphorylated ERK1/2 in human prostate cancer tissues (Fig. h and i). Phosphorylated FOXO1 (cytoplasmic) inhibits PKM2 nuclear localization (Fig. j) and knockdown of FOXO1 promotes PKM2 nuclear localization (Fig. k). We further demonstrated that phosphorylated FOXO1 (cytoplasmic) inhibits expression of PKM2 target gene and induces glucose uptake and lactate production (Fig. l and m). Finally, we demonstrated that IQBP overcomes docetaxel (DTX)-induced chemoresistance through inhibiting Warburg effect in vivo (Fig. n and o).

Conclusions

AKT-phosphorylated FOXO1 inhibits PKM2 nuclear localization and Warburg effect in prostate cancer.

Funding

National Institutes of Health (CA134514 and CA130908 to H.H.), the Department of Defense (W81XWH-09-1-622 and W81XWH-14-1-0486 to H.H.), National Natural Science Foundation of China (81101931 to C.W.P) and Shanghai Municipal Commission of Health and Family Planning (201640041 to C.W.P).