KO of 5-InsP 7 kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype

Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):11968-11973. doi: 10.1073/pnas.1702370114. Epub 2017 Oct 25.

Abstract

The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.

Keywords: bioenergetics; inositol pyrophosphates; signaling.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Colonic Neoplasms / metabolism
  • Cyclin-Dependent Kinase Inhibitor p21 / biosynthesis*
  • G1 Phase Cell Cycle Checkpoints / genetics
  • Gene Knockout Techniques
  • Glycolysis / genetics
  • Glycolysis / physiology
  • HCT116 Cells
  • HEK293 Cells
  • Humans
  • Inositol Phosphates / metabolism*
  • Mitochondria / metabolism
  • Phosphotransferases (Phosphate Group Acceptor) / genetics*
  • Phosphotransferases (Phosphate Group Acceptor) / metabolism
  • Signal Transduction
  • Tumor Suppressor Protein p53 / biosynthesis*

Substances

  • Cyclin-Dependent Kinase Inhibitor p21
  • Inositol Phosphates
  • Tumor Suppressor Protein p53
  • 1-diphosphoinositol pentakisphosphate
  • Adenosine Triphosphate
  • Phosphotransferases (Phosphate Group Acceptor)
  • diphosphoinositol pentakisphosphate kinase
  • PPIP5K1 protein, human
  • inositol hexakisphosphate kinase