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. 2014 Jun 20;9(6):1340-50.
doi: 10.1021/cb5001907. Epub 2014 Apr 28.

Inositol Phosphate Recycling Regulates Glycolytic and Lipid Metabolism That Drives Cancer Aggressiveness

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Free PMC article

Inositol Phosphate Recycling Regulates Glycolytic and Lipid Metabolism That Drives Cancer Aggressiveness

Daniel I Benjamin et al. ACS Chem Biol. .
Free PMC article

Abstract

Cancer cells possess fundamentally altered metabolism that supports their pathogenic features, which includes a heightened reliance on aerobic glycolysis to provide precursors for synthesis of biomass. We show here that inositol polyphosphate phosphatase 1 (INPP1) is highly expressed in aggressive human cancer cells and primary high-grade human tumors. Inactivation of INPP1 leads to a reduction in glycolytic intermediates that feed into the synthesis of the oncogenic signaling lipid lysophosphatidic acid (LPA), which in turn impairs LPA signaling and further attenuates glycolytic metabolism in a feed-forward mechanism to impair cancer cell motility, invasiveness, and tumorigenicity. Taken together these findings reveal a novel mode of glycolytic control in cancer cells that can serve to promote key oncogenic lipid signaling pathways that drive cancer pathogenicity.

Figures

Figure 1
Figure 1
INPP1 is highly expressed in aggressive cancer cells and primary tumors. (A–C) INPP1 gene (A) and protein (B) expression and INPP1 activity (C) across aggressive ovarian, melanoma, breast, and prostate cancer cells (SKOV3, C8161, 231MFP, and PC3) compared to their less aggressive counterparts (OVCAR3, MUM2C, MCF7, and LNCaP) as measured by quantitative PCR (qPCR) (A), Western blotting (B), and inositol-1,4-bisphosphate phosphatase activity measuring inositol phosphate product formation by LC–MS (C). (D) INPP1 enzyme activity (for ovarian tumors) and mRNA expression (for melanoma and breast tumors) in high-grade compared to low-grade primary human ovarian tumors or melanoma or breast tumors compared to normal tissue. *p < 0.05. Data are presented as mean ± SEM; n = 3–5/group for panels A–C and n = 3–39 for panel D.
Figure 2
Figure 2
INPP1 inactivation leads to impairments in cancer pathogenicity. (A) INPP1 was knocked down using both a short-hairpin RNA (shRNA) oligonucleotide (shINPP1) as well an small-interfering RNA (siRNA) oligonucleotide (siINPP1), resulting in >70% reduction in both INPP1 expression and activity in C8161 and SKOV3 cells compared to their respective sh and siControl cells. (B,C) shINPP1 and siINPP1 cells show decreased migration (B) and invasion (C) compared to shControl and siControl cells in both SKOV3 and C8161 cells. Migration and invasion assays were performed by transferring cancer cells to serum-free media for 4 h prior to seeding 50,000 cells into inserts with 8 μm pore size containing membranes coated with collagen (10 μg/mL) or BioCoat Matrigel, respectively. C8161 and SKOV3 migration times were 5 and 8 h, respectively. Migrated or invaded cells refer to average numbers ± SEM per four fields counted at 400 X magnification. (D) shINPP1 cells show impaired tumor growth in SCID mice compared to shControl cells. A total of 2 ×106 C8161 or SKOV3 cells/100 μL were injected subcutaneously into the flank, and tumor growth was measured using calipers. Significance is presented as *p < 0.05 compared to shControl or siControl. Data are presented as mean ± SEM; n = 3 or 4/group for panels A–C and n = 5 or 6/group for panel D.
Figure 3
Figure 3
Metabolomic profiling links INPP1 to glycolysis and lipid metabolism. (A) Metabolomic analyses of cancer cell steady-state metabolomes with impaired INPP1 activity compared to control cells. The volcano plot shows all ions that were detected by targeted or untargeted metabolomic profiling of shControl and shINPP1 SKOV3 cells. Gray points show the ions and metabolites that were not significantly altered between shControl and shINPP1 cells. The red and blue points to the right of the dotted black line are metabolites that were significantly (p < 0.05) and commonly elevated or lowered, respectively, across C8161 and SKOV3 sh and siINPP1 compared to their respective sh and siControl cells. C16:0, C18:0, C18:1 refer to acyl chain length:unsaturation on LPA. C16:0e LPAe refers to the ether lipid counterpart of LPA (LPA-ether). All targeted data are in Supplementary Table S1. (B,C) Levels of metabolites that were altered upon INPP1 knockdown in SKOV3 (B) and C8161 (C) cells, quantified by SRM. Data are presented as means ± SEM of n = 4 or 5/group with significance expressed as *p < 0.05 for INPP1 knockdown compared to control.
Figure 4
Figure 4
INPP1 modulates glycolytic and glucose-derived LPA metabolism. (A) Media glucose and lactate levels at 0, 8, 16, and 24 h in siControl and siINPP1 cells, measured by glucose assay kit and SRM-based LC–MS/MS, respectively. (B) Steady-state isotopic [13C] incorporation into glycolytic intermediates from treatment of siControl and siINPP1 SKOV3 cells with either 10 mM concentration of nonisotopic glucose or 10 mM concentration of isotopic [U-13C]glucose for 24 h in otherwise glucose-free RPMI1640 media. Confirmation that we are measuring isotopic glycolytic intermediates at steady state is provided in Supplementary Figure S4. Full isotopomer distribution of metabolites is shown in Supplementary Figure S5. (C) Relative gene expression by qPCR of glucose transporters and glycolytic enzymes in SKOV3 and C8161 cells of siControl (black) compared with siINPP1 (blue) cells. (D) Phenotypic and metabolic effects of 2-deoxyglucose (2-DG) in SKOV3 cells. Treatment of SKOV3 cells with 2-DG (in water, 5 mM, 24 h) impairs SKOV3 cell migration (right panel) and lowers post-PGI glycolytic intermediates and LPA levels (left panel). (E) GLUT4 overexpression partially rescues migratory deficits conferred by INPP1 knockdown in SKOV3 cells. qPCR of GLUT4 expression is shown in the left panel, and migration data are shown in the right panel. Data are presented as means ± SEM of n = 3–5/group with significance expressed as *p < 0.05 compared to siControl or control cells and #p < 0.05 comparing siINPP1+GLUT4 to siINPP1 groups.
Figure 5
Figure 5
LPA modulates the migratory defects and glycolytic impairments conferred by INPP1 knockdown. (A,B) The migratory impairment in shINPP1 SKOV3 ovarian (A) and C8161 melanoma (B) cells is fully rescued upon treating cells with low concentrations of LPA (100 nM). Treatment with DMSO or LPA (100 nM) was initiated concurrently with the seeding of cells for assessment of cancer cell migration (24 h). (C) Reduced [13C] incorporation into glycolytic intermediates from labeling siINPP1 cells with [13C]glucose (24 h) is rescued upon treatment of cells with LPA (1 μM). Treatment with LPA was initiated 2 days after transfection of siINPP1 oligonucleotides and 24 h prior to labeling of cells with either [12C] or [13C]glucose (10 mM, 24 h). (D) Isotopic incorporation into glycolytic intermediates is reduced upon treating SKOV3 cells with the LPA antagonist Ki16425 (10 μM). The antagonist or DMSO was pretreated with SKOV3 cells 24 h prior to seeding of cells for labeling with [12C] or [13C]glucose (10 mM, 24 h). For panels C and D, isotopic incorporation of [13C]glucose into glycolytic intermediates and glycerol-3-phosphate were quantified by SRM-based LC–MS/MS. (E) The reduction in GLUT1 and HK2 expression conferred by INPP1 knockdown is partially to fully rescued by LPA (1 μM). (F) Model depicting the metabolic role of INPP1 in controlling glycolytic metabolism and LPA signaling. Data are average ± SEM, n = 3–5/group. Significance is expressed in panels A, B, and E as *p < 0.05 comparing shControl to all other groups and #p < 0.05 comparing the LPA-treated shINPP1 to DMSO-treated shINPP1 groups. Significance in panel C is expressed as *p < 0.05 comparing siINPP1 with siControl groups and #p < 0.05 comparing LPA-treated siINPP1 with DMSO-treated siINPP1 groups. Significance in panel D is expressed as *p < 0.05 comparing Ki16425-treated and DMSO-treated groups.
Figure 6
Figure 6
INPP1 knockdown affects the Hippo transducer YAP. (A) INPP1 knockdown increases phosphorylated YAP (p-YAP) protein levels compared to siControl cells in SKOV3 cells grown in serum-free media for 24 h by Western blotting. This increase in p-YAP is partially rescued upon addition of LPA (100 nM, 24 h). (B) YAP was knocked down by >75% in SKOV3 cells using two independent si oligonucleotides, and YAP knockdown was confirmed by qPCR. After 48 h of transfection with siControl or siYAP oligonucleotides, media was replaced, and media glucose and lactic acid levels were measured after 24 h by glucose assay kit and SRM-based LC–MS/MS, respectively. Data are represented as n = 3–5/group. Significance expressed as *p < 0.05 compared to siControl, #p < 0.05 comparing siINPP1+LPA to siINPP1.

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