TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1α axis in melanoma
- Center of Translational Medicine, Zibo Central Hospital Affiliated to Binzhou Medical University, China
- Shandong First Medical University, China
- The Second Medical College, Xinjiang Medical University, China
- School of Laboratory Medicine, Xinxiang Medical University, China
Figures
Figure 1 with 4 supplements
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 extracellular acidification rate (ECAR) analysis. (J, K) Interfering with TIPE decreased glycolysis and glycolytic capacity using ECAR analysis. (L) TIPE significantly activated hypoxia response element (HRE) activity. *p < 0.05; **p < 0.01; ***p < 0.001. The data represent the means ± SEM of three replicates.
Figure 1—figure supplement 1
Verification of TIPE expression in different melanoma cell lines and the effects of TIPE interference and overexpression.
(a) Western blot validation of TIPE in melanoma cell lines. (b) Western blot and qPCR analysis of TIPE expression after TIPE interference in A375 cells. (c) Western blot and qPCR analysis of TIPE expression after overexpression of TIPE in G361 cells ***P<0.001.
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Figure 1—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing original western blots for Figure 1—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
TIPE promotes melanoma cell proliferation in vitro and in vivo.
(a) CCK8 assay showed that interfering TIPE (TIPE-sh) decreased the cells proliferation in A375 cells compared to control (Ctrl-sh). (b) Overexpression of TIPE (TIPE) elevated G361 cell proliferation compared to control (Ctrl). (c, d) Fluorescence-activated cell sorting (FACS) analysis of the effects of TIPE on cell cycle. (e) Effects of TIPE on colony formation in A375 and G361 cells. (f–h) Effect of TIPE on tumor formation in a nude mouse xenograft model. Nude mice were subcutaneously injection of cells containing Ctrl-sh and TIPE-sh, respectively. The tumor volume was measured every 7 days for five times until sacrifice. Representative images of tumors from the TIPE-sh and control groups (Ctrl-sh), n = 4 for each group. (i) Interfering TIPE decreases the expression of Ki67 in nude mice, measured by immunohistochemistry. (j) TCGA dataset shows a positive relation between the mRNA expression of TIPE and Ki67 in melanoma patients. *p < 0.05; ***p < 0.001.
Figure 1—figure supplement 3
Volcano plots and heatmaps generated following TIPE interference by using untargeted metabolomics.
Principal component analysis (PCA) and volcano plots of the samples from TIPE interference group vs. control in the negative mode (a, b) or positive mode (c, d) by using untargeted metabolomics. (e) Heatmap indicated that the glycolysis pathway including pyruvate and lactic acid is decreased after TIPE interference.
Figure 1—figure supplement 4
TIPE enhances the expression of HIF-1α mRNA and protein.
(a) TIPE upregulated HIF-1α mRNA expression using a qPCR assay. (b) Western blot analysis showed that TIPE increased HIF-1α expression. (c) TCGA dataset indicated that TIPE has a positive correlation with HIF-1α ***P<0.001.
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Figure 1—figure supplement 4—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig1-figsupp4-data1-v1.zip
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Figure 1—figure supplement 4—source data 2
PDF file containing original western blots for Figure 1—figure supplement 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig1-figsupp4-data2-v1.zip
Figure 2 with 2 supplements
TIPE interacts with PKM2 to govern its nuclear import in a dimeric form-dependent manner.
(A) Co-immunoprecipitation mass spectrometry (Co-IP/MS) analysis demonstrated that PKM2 interacted with TIPE in A375 cells. The results indicated that there were five 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 (100 μM). *p < 0.05; **p < 0.01. The data represent the means ± SEM of three replicates.
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Figure 2—source data 1
Original files for western blot analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing original western blots for Figure 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-data2-v1.zip
Figure 2—figure supplement 1
TIPE engages in an interaction with PKM2.
(a, b) Molecular docking with a superposition of the three structures showed binding domain of TIPE and PKM2 by using PyMol 2.2.0 (S = −246.61). (c) Western blot analysis by using the TIPE antibody to bait its potential partner prior to the ‘IN-GEL DIGESTION’ step. Before the ‘nano-HPLC-MS/MS ANALYSIS’ step, the quality control including false discovery rate (d), score distribution (e), peptides feature detection (f), verifiable de novo sequences (g), and quality accuracy control (h) were performed.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
TIPE has no effect on the expression levels of PKM2.
(a, b) TIPE did not impact the expression levels of PKM2. (c) TCGA dataset showed that there was no significant correlation between TIPE and PKM2.
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Figure 2—figure supplement 2—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
PDF file containing original western blots for Figure 2—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig2-figsupp2-data2-v1.zip
Figure 3
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 hypoxia response element (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 SLC2A1, 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 three replicates *p<0.05.
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Figure 3—source data 1
Original files for western blot analysis displayed in Figure 3.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing original western blots for Figure 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig3-data2-v1.zip
Figure 4
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 hypoxia response element (HRE) for LDHA and SLC2A1 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 SLC2A1 that promoted by TIPE. **p < 0.01; ***p < 0.001. The data represent the means ± SEM of three replicates.
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing original western blots for Figure 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/92741/elife-92741-fig4-data2-v1.zip
Figure 5
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 three replicates.
Figure 6
TIPE fostered PKM2 dimerization increases melanoma stem-like phenomenon.
(A, B) TIPE increased the cancer stem-like phenotype markers, including NANOG, NOTCH, POU5F1, SOX2, BMI-1, NES, and SOX10, measured by qPCR. 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) were 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 three replicates.
Figure 7 with 1 supplement
TIPE positively corelated with cancer stem cell (CSC) 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 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 three replicates "*p<0.05.
Figure 7—figure supplement 1
TCGA dataset showed a paradoxical observation that elevated TIPE expression is associated with a favorable prognosis in melanoma patients.
Author response image 1
Additional files
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Supplementary file 1
Supplementary tables.
(a) The top candidate interacting proteins of TIPE identified by mass spectrometry. (b) Genes upregulated in response to low-oxygen levels (hypoxia) in melanoma. (c) Limiting dilution data. (d) Confidence intervals for 1/(stem cell frequency). (e) Primer or siRNA sequences. (f) Experimental materials. (g) The clinicopathological characteristics of 48 melanoma specimens.
- https://cdn.elifesciences.org/articles/92741/elife-92741-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92741/elife-92741-mdarchecklist1-v1.docx
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TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1α axis in melanoma
eLife 13:RP92741.
https://doi.org/10.7554/eLife.92741.4
