PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis
Alexander Nguyen, … , Elisa de Stanchina, Sohail F. Tavazoie
Alexander Nguyen, … , Elisa de Stanchina, Sohail F. Tavazoie
Published January 19, 2016
Citation Information: J Clin Invest. 2016;126(2):681-694. https://doi.org/10.1172/JCI83587.
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Research Article Oncology

PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis

Abstract

Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.

Authors

Alexander Nguyen, Jia Min Loo, Rohit Mital, Ethan M. Weinberg, Fung Ying Man, Zhaoshi Zeng, Philip B. Paty, Leonard Saltz, Yelena Y. Janjigian, Elisa de Stanchina, Sohail F. Tavazoie

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Figure 6

PKLR negatively regulates PKM2 pyruvate kinase activity.

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PKLR negatively regulates PKM2 pyruvate kinase activity. (A) Cell lysate...
(A) Cell lysates from LS-LVM3b cells with indicated overexpression constructs were subjected to immunoprecipitation using anti-FLAG beads followed by LC-MS/MS. Protein peptide-spectrum matches (PSMs), as compared with label-free quantification (LFQ) of protein enrichment (relative to empty vector immunoprecipitation), are shown. (B) Cell lysates from LS-LVM3b cells were subjected to immunoprecipitation using anti-FLAG beads followed by Western blot analysis. The experiment was conducted at least 3 times. (C) Cell lysates from LS174T cells were subjected to immunoprecipitation using anti-HA beads followed by Western blot analysis. The experiment was conducted at least 3 times. (D) Sequentially immunoprecipitated PKM2 complexes from transfected HEK293T cells (n = 3) were assayed for pyruvate kinase activity. Fusion protein expression was confirmed by Western blotting. The experiment was conducted twice. *P < 0.05, 2-sided t test. (E) Immunoprecipitated PKM2 complexes from LS174T cells (n = 3), subjected to 16 hours under high cell density and hypoxia, were assayed for pyruvate kinase activity. Activity was normalized to immunoprecipitated HA-PKM2, as measured by quantitative Western blotting. The experiment was conducted 3 times. ***P < 0.001, 2-sided t test. (F and G) Pyruvate kinase activity was measured from (F) LS174T and (G) SW620 cell lysates after 24 hours under hypoxic, cell-dense conditions. Activity was normalized to live cells by cell count. Data shown are from 3 biological replicates and 2 independent experiments. **P < 0.01, 1-sided t test between indicated sample and shControl. (H) Glucose uptake rates and (I) lactate excretion rates were determined from conditioned media of LS174T cells grown in high cell density and hypoxia for 24 hours. Data shown are from 3 biological replicates. *P < 0.05, 1-sided t test between indicated sample and shControl.