Dichotomous role of the human mitochondrial Na+/Ca2+/Li+ exchanger NCLX in colorectal cancer growth and metastasis

  1. Trayambak Pathak
  2. Maxime Gueguinou
  3. Vonn Walter
  4. Celine Delierneux
  5. Martin T Johnson
  6. Xuexin Zhang
  7. Ping Xin
  8. Ryan E Yoast
  9. Scott M Emrich
  10. Gregory S Yochum
  11. Israel Sekler
  12. Walter A Koltun
  13. Donald L Gill
  14. Nadine Hempel
  15. Mohamed Trebak  Is a corresponding author
  1. Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, United States
  2. Department of Public Health Sciences, The Pennsylvania State University College of Medicine, United States
  3. Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, United States
  4. Penn State Cancer Institute. The Pennsylvania State University College of Medicine, United States
  5. Department of Surgery, Division of Colon and Rectal Surgery, The Pennsylvania State University College of Medicine, United States
  6. Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel
  7. Department of Pharmacology, The Pennsylvania State University College of Medicine, United States
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
The expression of NCLX, a mtCa2+ extrusion mediator in CRC cells, is decreased in CRC tumor samples from human patients.

(A) TCGA data analysis showing SLC8B1 mRNA levels in tumor tissues and adjacent normal tissues of COADREAD (colon and rectal adenocarcinoma) patients. Each data point represents an individual …

Figure 1—figure supplement 1
Loss of NCLX reduces tumor number and size in the colitis-associated cancer model.

(A) TCGA data analysis showing SLC8B1 mRNA levels in tumors of male and female COADREAD patients. (B, C) TCGA data analysis showing a comparison of SLC8B1 mRNA levels in COAD patients based on age (B

Figure 2 with 1 supplement
Loss of NCLX has a dichotomous role in tumor growth and metastasis in vivo.

(A) Schematic representation of CRISPR-generated HCT116 NCLX KO #33 cells. Luciferase-tagged control HCT116 and HCT116 NCLX KO #33 cells were injected at 5 × 105 cells/mice into spleens of male …

Figure 2—figure supplement 1
Xenografts of HCT116 cells and HCT116 NCLX KO #33 cells in NOD/SCID.

Bioluminescence images of male NOD/SCID mice injected with luciferase-tagged HCT116 cells or HCT116 NCLX KO #33 cells.

Figure 3 with 1 supplement
Loss of NCLX inhibits proliferation but enhances migration and invasion of CRC cells.

(A, B) CyQUANT proliferation assays of HCT116 cells (A), DLD1 cells (B), and their respective NCLX KO clones. (C, D) Representative bright-field images of in vitro wound healing migration assay (C), …

Figure 3—figure supplement 1
Deletion of NCLX results in reduced proliferation and increased migration and invasion.

(A–D) CRISPR/Cas9 knockout strategy of the SLC8B1 gene in NCLX KO clones #33 (A), #37 (B), and #59 (C) with g1 and g2 representing the cut site and red box non-translated region. Location of primers …

Figure 4 with 2 supplements
Abrogation of NCLX function in CRC cells causes mitochondrial damage, and enhances mitochondrial ROS.

(A, B) Flow cytometric analysis of mitochondrial membrane potential using the dye TMRE in HCT116 cells (A) and DLD1 cells (B) and their respective NCLX KO clones. Each data point represents one …

Figure 4—figure supplement 1
Loss of NCLX in CRC cells inhibits mtCa2+ extrusion.

(A) Representative images of HCT116 cells and HCT116 NCLX KO cells loaded with the mitochondrial Ca2+ dye Rhod-2 AM. Cells were stimulated with 300 µM ATP in 5 mM extracellular Ca2+ at time 0, and …

Figure 4—figure supplement 2
Loss of NCLX causes mitochondrial damage.

(A–C) Representative transmission electron micrographs (TEM) of mitochondria in HCT116, HCT116 NCLX KO #37 cells, HCT116 NCLX KO #59 cells (A), DLD1 cells, DLD1 NCLX KO#06 cells (B) with red arrows …

Figure 5 with 1 supplement
Loss of NCLX leads to pro-metastatic transcriptional reprogramming.

(A) Volcano plot of differentially expressed genes between HCT116 cells and HCT116 NCLX KO #33 cells showing Log2 fold change vs. false discovery rate. The thresholds in the figure correspond to a …

Figure 5—figure supplement 1
Abrogation of NCLX function leads to transcriptional reprogramming.

(A) Heat map of expression of glycolytic genes that are significantly different between HCT116 cells and HCT116 NCLX KO #33 cells. (B, C) Gene set enrichment analysis (GSEA), and of HCT116 cells, …

Figure 6 with 1 supplement
NCLX deficiency stabilizes HIF1α and regulates migration in a mtROS-dependent manner.

(A–F) Western blot of HCT116 cells and clones of HCT116 NCLX KO cells (A), DLD1 cells and clones of DLD1 NCLX KO cells (C) and HCT116 cells infected with either scramble shRNA or three combinations …

Figure 6—figure supplement 1
NCLX KO CRC cells show chemoresistance to 5-FU.

(A) The proliferation of HCT116 cells and HCT116 NCLX KO #33 cells in the presence of 0 µM, 1 µM, 10 µM, and 25 µM 5-FU (n = 3). (B) Measurement of IC50 of 5-FU for HCT116 and HCT116 NCLX KO #33 …

Figure 7 with 1 supplement
Glycolysis is critical for migration of NCLX-deficient colorectal cancer cells.

(A) RT-qPCR analysis of glycolytic gene expression in HCT116 cells and three clones of HCT116 NCLX KO cells. RT-qPCR results are plotted as 2-ΔCt relative to tubulin and normalized to control. (B, C)…

Figure 7—figure supplement 1
Loss of NCLX enhances glycolysis and diminishes mitochondrial respiration.

(A–D) Western blots for HK2, ALDOA, LDHA, with GAPDH as a loading control from HCT116, HCT116 NCLX KO #37, #59 (A), DLD1, clones of DLD1 NCLX KO cells (C), and quantification of band intensity …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)SLC8B1/NCLXGenBankGene ID: 80024
Gene Mus musculusSlc8b1/NCLXGenBankGene ID: 170756
Genetic reagent Mus musculusNOD.CB17-Prkdcscid/JJackson LaboratoryStock No: 010636, RRID:IMSR_JAX:010636Male
Cell line (Homo-sapiens)HCT116 (colon, epithelial)ATCCATCC# CCL-247Male
Cell line (Homo-sapiens)HT29 (colon, epithelial)ATCCATCC# HTB-38Female
Cell line (Homo-sapiens)DLD1 (colon, epithelial)ATCCATCC# CCL-221Male
Cell line (Homo-sapiens)HCT116 NCLX KO (colon, epithelial)This paperNCLX Knockout clones of HCT116 cells were generated by the Trebak lab using CRISPR/Cas9 and are available upon request
Cell line (Homo-sapiens)DLD1 NCLX KO (colon, epithelial)This paperNCLX Knockout clones of DLD1 cells were generated in the Trebak lab using CRISPR/Cas9 and are available upon request
Cell line (Homo-sapiens)HCT116 shNCLX (colon, epithelial)This paperHCT116 cells with stable shRNA-mediated knockdown of NCLX (shNCLX) were generated by the Trebak Lab using shRNA sequences (listed below in this table) cloned in the lentiviral vector pLKO. These plasmids are available upon request
Cell line (Homo-sapiens)DLD1 shNCLX (colon, epithelial)This paperDLD1 cells with stable shRNA-mediated knockdown of NCLX (shNCLX) were generated by the Trebak Lab using shRNA sequences (listed below in this table) cloned in the lentiviral vector pLKO. These plasmids are available upon request
Antibodyanti-Hif1α (Rabbit polyclonal)Cell Signaling TechnologyCat# 14179s,
RRID:AB_2622225
WB (1:500)
Antibodyanti- ALDOA (Rabbit polyclonal)Cell Signaling TechnologyCat# 8060S,
RRID:AB_2797635
WB (1:1000)
Antibodyanti- HK2 (Rabbit polyclonal)Cell Signaling TechnologyCat# 2867S, RRID:AB_2232946WB (1:1000)
Antibodyanti- LDHA (Rabbit polyclonal)Cell Signaling TechnologyCat# 2012S,
RRID:AB_2137173
WB (1:1000)
Antibodyanti- MMP1 (Rabbit polyclonal)AbcamCat# ab38929, RRID:AB_776395WB (1:1000)
Antibodyanti- MMP2 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-13594, RRID:AB_627956WB (1:500)
Antibodyanti- MMP9 (Rabbit polyclonal)AbcamCat# ab73734, RRID:AB_1860201WB (1:1000)
Antibodyanti- LC3B (Rabbit polyclonal)AbcamCat# ab51520, RRID:AB_881429WB (1:1000)
Antibodyanti- OXPHOS (Mouse monoclonal)AbcamCat# ab110413, RRID:AB_2629281WB (1:5000)
Antibodyanti- GAPDH (Mouse monoclonal)Millipore SigmaCat# MAB374, RRID:AB_2107445WB (1:10000)
Antibodyanti- HSC70 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-24, RRID:AB_627760WB (1:5000)
Antibodyanti- p62 (Rabbit polyclonal)AbcamCat# ab109012, RRID:AB_2810880WB (1:1000)
Antibodyanti- cleaved caspase-3 (Rabbit polyclonal)Cell Signaling TechnologyCat# 9661S, RRID:AB_2341188WB (1:1000)
IF (1:500)
Antibodyanti- pAMPK (Rabbit polyclonal)Cell Signaling TechnologyCat# 2535S, RRID:AB_331250WB (1:1000)
Antibodyanti- AMPK (Rabbit polyclonal)Cell Signaling TechnologyCat# 5831S, RRID:AB_10622186WB (1:1000)
Antibodyanti- pS6K (Rabbit polyclonal)Cell Signaling TechnologyCat# 9234S, RRID:AB_2269803WB (1:1000)
Antibodyanti- S6K (Rabbit polyclonal)Cell Signaling TechnologyCat# 2708S, RRID:AB_390722WB (1:1000)
Sequence-based reagentSLC7A11_FThis paperRT-PCR primersAGGGTCACCTTCCAGAAATC
Sequence-based reagentSLC7A11_RThis paperRT-PCR primersGAAGATAAATCAGCCCAGCA
Sequence-based reagentGCLM _FThis paperRT-PCR primersCATTTACAGCCTTACTGGGAGG
Sequence-based reagentGCLM _RThis paperRT-PCR primersATGCAGTCAAATCTGGTGGCA
Sequence-based reagentFOXO3_FThis paperRT-PCR primersCGGACAAACGGCTCACTCT
Sequence-based reagentFOXO3_RThis paperRT-PCR primersGGACCCGCATGAATCGACTAT
Sequence-based reagentNANOG _FThis paperRT-PCR primersTTTGTGGGCCTGAAGAAAACT
Sequence-based reagentNANOG _RThis paperRT-PCR primersAGGGCTGTCCTGAATAAGCAG
Sequence-based reagentOCT4_FThis paperRT-PCR primersTTCAGCCAAACGACCATCTG
Sequence-based reagentOCT4_RThis paperRT-PCR primersCACGAGGGTTTCTGCTTTGC
Sequence-based reagentSOX2_FThis paperRT-PCR primersGCCGAGTGGAAACTTTTGTCG
Sequence-based reagentSOX2_RThis paperRT-PCR primersGGCAGCGTGTACTTATCCTTCT
Sequence-based reagentGLUT1_FThis paperRT-PCR primersTATCGTCAACACGGCCTTCACT
Sequence-based reagentGLUT1_RThis paperRT-PCR primersAACAGCTCCTCGGGTGTCTTAT
Sequence-based reagentHK2_FThis paperRT-PCR primersGCCATCCTGCAACACTTAGGG
Sequence-based reagentHK2_RThis paperRT-PCR primersGTGAGGATGTAGCTTGTAGAGGGT
Sequence-based reagentGPI_FThis paperRT-PCR primersTGTGTTCACCAAGCTCACAC
Sequence-based reagentGPI_RThis paperRT-PCR primersGTAGAAGCGTCGTGAGAGGT
Sequence-based reagentALDOA_FThis paperRT-PCR primersAGGCCATGCTTGCACTCAG
Sequence-based reagentALDOA_RThis paperRT-PCR primersAGGGCCCAGGGCTTCAG
Sequence-based reagentENO1_FThis paperRT-PCR primersGACTTGGCTGGCAACTCTG
Sequence-based reagentENO1_RThis paperRT-PCR primersGGTCATCGGGAGACTTGAAG
Sequence-based reagentLDHA_FThis paperRT-PCR primersGGTTGGTGCTGTTGGCATGG
Sequence-based reagentLDHA_RThis paperRT-PCR primersTGCCCCAGCCGTGATAATGA
Sequence-based reagentMMP1_FThis paperRT-PCR primersATGCTGAAACCCTGAAGGTG
Sequence-based reagentMMP1_RThis paperRT-PCR primersGAGCATCCCCTCCAATACCT
Sequence-based reagentMMP2_FThis paperRT-PCR primersACCAGCTGGCCTAGTGATGATG
Sequence-based reagentMMP2_RThis paperRT-PCR primersGGCTTCCGCATGGTCTCGATG
Sequence-based reagentMMP9_FThis paperRT-PCR primersACGCACGACGTCTTCCAGTA
Sequence-based reagentMMP9_RThis paperRT-PCR primersCCACCTGGTTCAACTCACTCC
Sequence-based reagentqNCLX_1_FThis paperRT-PCR primersGCGTGCTGGTTACCACAGT
Sequence-based reagentqNCLX_1_RThis paperRT-PCR primersCCACGGAAGAGCATGAGGAA
Sequence-based reagentqNCLX_2_FThis paperRT-PCR primersCCGGCAGAAGGCTGAATCTG
Sequence-based reagentqNCLX_2_RThis paperRT-PCR primersACCTTGCGGCAGTCTACCAC
Sequence-based reagentGAPDH_FThis paperRT-PCR primersCCCTTCATTGACCTCAACTACA
Sequence-based reagentGAPDH_RThis paperRT-PCR primersATGACAAGCTTCCCGTTCTC
Sequence-based reagentNONO_FThis paperRT-PCR primersTCCGAGGAGATACCAGTCGG
Sequence-based reagentNONO_RThis paperRT-PCR primersCCTGGGCCTCTCAACTTCGAT
Sequence-based reagentTubulin_FThis paperRT-PCR primersAGTCCAAGCTGGAGTTCTCTAT
Sequence-based reagentTubulin_RThis paperRT-PCR primersCAATCAGAGTGCTCCAGGGT
Sequence-based reagentG6PD_FThis paperRT-PCR primersCGAGGCCGTCACCAAGAAC
Sequence-based reagentG6PD_RThis paperRT-PCR primersGTAGTGGTCGATGCGGTAGA
Sequence-based reagentPGD_FThis paperRT-PCR primersATGGCCCAAGCTGACATCG
Sequence-based reagentPGD_RThis paperRT-PCR primersAAAGCCGTGGTCATTCATGTT
Sequence-based reagentTKT_FThis paperRT-PCR primersTCCACACCATGCGCTACAAG
Sequence-based reagentTKT_RThis paperRT-PCR primersCAAGTCGGAGCTGATCTTCCT
Sequence-based reagentg1This paperGuide RNA sequences (Figure 2A and Figure 3—figure supplement 1A)GCGCAGATTCAGCCTTCTGC
Sequence-based reagentg2This paperGuide RNA sequences (Figure 3—figure supplement 1B and C)GGGATACTCACGTCTACCAC
Sequence-based reagentg3This paperGuide RNA sequences (Figure 3—figure supplement 1E)GTAGACGTGAGTATCCCGGT
Sequence-based reagentg4This paperGuide RNA sequences (Figure 3—figure supplement 1E)ACCCACACCAGCAGTCCGTC
Sequence-based reagentshRNA (shNCLX#2)This paperFigure 3—figure supplement 1IGCCTTCTTGCTGTCATGCAAT
Sequence-based reagentshRNA (shNCLX#3)This paperFigure 3—figure supplement 1IGCTCCTCTTCTACCTGAACTT
Sequence-based reagentsiRNA (siNCLX)This paperFigure 4—figure supplement 1MAACGGCCCCUCAACUGUCUT
Sequence-based reagentNCLX_1This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)GCCAGCATTTGTGTCCATTT
Sequence-based reagentNCLX_2This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)AATTCGTCTCGGCCACTTAC
Sequence-based reagentNCLX_3This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)ACTTAGCACATCGCCACCTG
Sequence-based reagentNCLX_4This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)CTGATCTGCACGCTGAATGG
Sequence-based reagentNCLX_5This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)GAGGTACACAGCAGTTCT CCC
Sequence-based reagentNCLX_6This paperPCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F)CAGCTGGTGCCCTCAAACAC
Sequence-based reagentPX75 NCLX test FThis paperPCR primers for screening genomic DNA of NCLX KO HCT116 cellsGTTGTTGAGACAGAGTCTTGC TTC
Sequence-based reagentPX76 NCLX test RThis paperPCR primers for screening genomic DNA of NCLX KO HCT116 cellsTCCAGCGAGACTGTGCAGAA
Sequence-based reagentpx77This paperPCR primers for genotyping NCLX -/- miceTACAGTCTGGCTCGTTCC CT
Sequence-based reagentpx78This paperPCR primers for genotyping NCLX -/- miceCGGTCCCAGACGCCG T
Sequence-based reagentpx79This paperPCR primers for genotyping NCLX -/- miceCGCTGGGGTCCATCT TTG AT
Sequence-based reagentpx80This paperPCR primers for genotyping NCLX -/- miceTGGGTCTCCGGTCCCAGT A
Commercial assay or kitcDNA Reverse Transcription KitApplied biosystemsCat# 4368814
Commercial assay or kitSeahorse XFp Mito Fuel Flex Test KitAgilent TechnologiesCat# 103270-100
Commercial assay or kitSeahorse XFp Glycolysis Stress Test KitAgilent TechnologiesCat# 103017-100
Commercial assay or kitSeahorse XFp Cell Mito Stress Test KitAgilent TechnologiesCat# 103010-100
Commercial assay or kitBCA assay kitThermo Fisher ScientificCat# A53225
Commercial assay or kitTMREThermo Fisher ScientificCat# T669
Commercial assay or kitCyQUANTThermo Fisher ScientificCat# C35006
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Chemical compound, drugMOPS SDS running bufferThermo Fisher ScientificCat# NP0001
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Chemical compound, drugCaCl2Fisher ScientificCat# C614
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Chemical compound, drugLDS sample bufferThermo Fisher ScientificCat# NP0007
Chemical compound, drugNuPAGE Bis-Tris precast gelsThermo Fisher ScientificCat# NP0321
Chemical compound, drugPolyvinylidene difluoride membraneLi-Core BiosciencesCat# 88518
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Additional files

Supplementary file 1

Genomic sequencing of NCLX KO clones of HCT116 and DLD1 cells.

This file shows the genome sequencing of NCLX KO clones #33, #37, and #59 of HCT116 and #06, #24, and #32 of DLD1 cells. For genomic sequencing, PCR was performed to amplify specific parts of the NCLX gene using primers listed in the key resources table. The primers used for cloning genomic DNA from HCT116 cells were PX75 NCLX test F and PX76 NCLX test R. The primers used for cloning genomic DNA from DLD1 cells were NCLX_4 and NCLX_5. PCR products were cloned using StrataClone Blunt PCR Cloning Kit and sent to Genewiz for sanger sequencing. The genome sequencing data for the HCT116 NCLX KO clones confirm the introduction of STOP codons in coding sequences of HCT116 NCLX KO #33 at predicted positions 181-183 bp and 184-186 bp, in HCT116 NCLX KO #37 at 16-18 bp and 46-48 bp, and in HCT116 NCLX KO #59 at 31-33 bp, and 52-54 bp. Furthermore, the genomic sequencing of NCLX KO clones of DLD1 cells showed that the middle ~32 kb portion of the NCLX gene was deleted in all clones. Therefore, providing decisive evidence that these cells are indeed NCLX KO.

https://cdn.elifesciences.org/articles/59686/elife-59686-supp1-v4.docx
Source data 1

Source data RNAseq_HCT116_HCT116 NCLX KO.

https://cdn.elifesciences.org/articles/59686/elife-59686-data1-v4.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/59686/elife-59686-transrepform-v4.docx

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