Effect of TIPE on melanoma cell glycolysis.

A, B. Transcriptomics analysis by unsupervised hierarchical clustering and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that TIPE increased glycolysis and HIF-1α pathways. C. GSEA analysis of glycolysis showed that TIPE enhanced glycolysis compared to the control. D. Untargeted metabolomics analysis indicated that interfering with TIPE decreased the glycolysis pathway. E-G. TIPE decreases ATPase activity and ATP content and increases lactate levels. H, I. Overexpression of TIPE promotes glycolysis and glycolytic capacity according to ECAR analysis. J, K. Interfering with TIPE decreased glycolysis and glycolytic capacity using ECAR analysis. L. TIPE significantly activated HRE activity. *P<0.05; **P<0.01; ***P<0.001. The data represent the means ± SEM of 3 replicates.

TIPE interacts with PKM2 to govern its nuclear import in a dimeric form dependent manner.

A. Co-IP/MS analysis demonstrated that PKM2 interacted with TIPE in A375 cells. The results indicated that there were 5 peptides of PKM2 were detected to interact with TIPE (below), and one of which was shown at the upper. B, C. Immunoprecipitation (IP) and western blot analysis of the exogenous TIPE/PKM2 proteins interaction in the HEK-293T cells co-transfected with Flag-tagged PKM2 and HA-tagged TIPE. D, E. IP and western blot analysis of the endogenous TIPE/PKM2 proteins interaction in the A375 cells. F. GST-pull down assay analysis of TIPE/PKM2 proteins interaction using purified GST-tagged PKM2 and TIPE-HA. G. TIPE (rabbit source, TIPER) endogenously interacted with PKM2(mouse source, PKM2M) in G361 cells by using the Doulink assay. The results suggested that TIPE interacted with PKM2 to form a red color complex. The red color signal is generated only when the two proteins are too close to interaction. H, I. IP and western blot analysis of HA-tagged TIPE and Flag-tagged PKM2 fragment protein interaction in HEK-293T cells. J. IP and western blot analysis of the Flag-tagged PKM2 and HA-tagged TIPE fragment protein interaction in HEK-293T cells. K. Interfering TIPE decreased PKM2 dimeric formation but increased tetramer formation, as analyzed by BN-PAGE with β-actin as a loading control. L. Overexpression of TIPE increased PKM2 dimeric formation and decreased tetramer formation. M. Western blot showed that TIPE promoted PKM2 translocation into the nucleus. N. TIPE enhanced PKM2 translocation into the nucleus, and this phenomenon was diminished by the administration of TEPP-46 (100μM). *P<0.05; **P<0.01. The data represent the means ± SEM of 3 replicates.

TIPE promotes HIF-1α transcription in a PKM2-dependent manner.

A. Proposed molecular mechanism by which dimeric PKM2 regulates cell proliferation and glycolysis by modulating HIF-1α activity. B. TIPE, especially when combined with PKM2, boosts relative HRE luciferase activity, as examined by luciferase reporter assay. C. TIPE promoted HRE activity in a dose- and PKM2-dependent manner. D, E. TIPE increases HIF-1α targeted genes, including LDHA and GLUT1, in a dose- and PKM2-dependent manner. F, G. TIPE promoted endogenous interaction between PKM2 and HIF-1α in melanoma cells using a Doulink assay. Interference of TIPE in A375 cells promoted the interaction between PKM2 and HIF-1α(upper) compared to that overexpression of TIPE in G361 cells decreased their interaction (lower). The density of the red color signaling means the interactive strength between PKM2 and HIF-1α affected by TIPE. H. TIPE enhanced the PKM2/HIF1a interaction in the nucleus. I. TIPE increased the exogenous interaction between PKM2 and HIF-1α in a dose-dependent manner in HEK-293T cells. J. TCGA dataset revealed that TIPE has a positive relationship with hypoxia score in melanoma. K. Higher expression of TIPE is associated with a relatively higher hypoxia score in melanoma. *P<0.05; **P<0.01; ***P<0.001. The data represent the means ± SEM of 3 replicates.

TIPE increases HIF-1α activity depending on PKM2 Ser37 phosphorylation.

A. TIPE enhanced PKM2 Ser37 phosphorylation, but not that of Tyr105. B. PKM2 Ser37, but not Tyr105, increased its interaction with TIPE. C. PKM2 Ser37 mutation (S37A) hampered its interaction with HIF-1α promoted by TIPE. D. TIPE elevated PKM2 Ser37 phosphorylation in an ERK-dependent manner. E, F. TIPE enhanced PKM2 binding to the HRE for LDHA and GLUT1 in a dimeric form dependent manner. G. PKM2 Ser37 mutation (S37A) inhibited the HRE activity induced by TIPE. H, I. PKM2 Ser37 mutation (S37A) decreased the expression of LDHA and GLUT1 that promoted by TIPE. **P<0.01; ***P<0.001. The data represent the means ± SEM of 3 replicates.

TIPE promotes melanoma cells proliferation and glycolysis depending on PKM2 dimerization.

A. Pyridoxine (which facilitates PKM2 dimerization) treatment reversed the inhibition of cell proliferation that induced via interfering TIPE. B. Administration of TEPP-46 rescued TIPE overexpression induced cell proliferation. C, D. TIPE promoted melanoma clone formation in a dimeric PKM2-dependent manner. E-G. In vivo experiments showed that TIPE promoted melanoma proliferation via the dimerization of PKM2. H-J. Activation of PKM2 dimerization reversed the inhibition of glycolysis and glycolytic capacity after TIPE interfering. In contrast, inhibition of PKM2 dimerization altered this phenomenon (K-M). N. Pyridoxine increased the cell proliferation that was inhibited by TIPE-sh. It was rescued by inhibition of HIF-1α. O. TEPP-46 inhibited the TIPE-induced cell proliferation, and was rescued by overexpression of HIF-1α. P. Pyridoxine enhanced the Warburg effect that was suppressed by TIPE-sh, and it was diminished via administration of PX-478(a HIF-1α inhibitor). Q. TEPP-46 can suppression of the Warberg effect that was increased by overexpression of TIPE, this phenomenon was diminished by overexpression of HIF-1α. *P<0.05; ***P<0.001. The data represent the means ± SEM of 3 replicates.

TIPE fostered PKM2 dimerization increases melanoma stem-like phenomenon.

A, B. TIPE increased the cancer stem-like phenotype markers, including NANOG, NOTCH, OCT3/4, SOX2, BMI-1, NES and SOX10, measured by qPCR. C-F. The effects of TIPE on the stemness of melanoma cells are shown by in vitro cell migration (C), sphere formation (D), and chemoresistance (E, F). G. Limiting dilution xenograft formation of A375 cells with TIPE interference. Nude mice were subcutaneously injection of indicated cells. After about 60 days later, the mice were sacrificed and the confidence intervals (CIs) for 1 / (stem cell frequency) was calculated. H, I. TIPE increased the expression of CD44+ cells in melanoma. J, K. The effects of TIPE on the stemness of melanoma cells following the administration of TEPP-46, and this phenomenon was reversed by overexpression of HIF-1α, as evidenced by sphere formation (J) and CD44+ cell population (K). *P<0.05; **P<0.01; ***P<0.001. The data represent the means ± SEM of 3 replicates.

TIPE positively corelated with cancer stem cell markers and the levels of p-PKM2(Ser37).

A, B. Higher expression of TIPE was observed in melanoma tumor tissues than in the control, as evidenced by immunohistochemistry. C, D. The expression of TIPE correlated well with p-PKM2(Ser37) in melanoma tumor tissues. E. Similarly, a good correlation was observed between TIPE, p-PKM2(Ser37), LDH, and CD44 in mouse xenografts. F-I. In addition, the expression of TIPE was positively correlated with CSCs markers, including BMI1, NANOG, NOTCH1, and OCT3/4(POU5F1) in TCGA dataset. J. A brief model depicting the functional impact of TIPE on metabolic reprogramming in melanoma. ***P<0.001. The data represent the means ± SEM of 3 replicates.1