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. 2022 Sep 22;20(9):e3001753.
doi: 10.1371/journal.pbio.3001753. eCollection 2022 Sep.

Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function

Neal K Bennett  1 , Hiroki J Nakaoka  2 , Danny Laurent  2 , Ross A Okimoto  3 , Yoshitaka Sei  1 , Andrew E Horvai  4 , Trever G Bivona  3 , Johanna Ten Hoeve  5 , Thomas G Graeber  5 , Ken Nakamura  1   6   7 , Jean L Nakamura  2
Affiliations

Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function

Neal K Bennett et al. PLoS Biol. .

Abstract

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Human lung cancer cells demonstrate differential requirements for mitochondrial and respiratory genes when grown in vivo in flank or orthotopic lung models.
HCC827 and H1975 human EGFR-mutant lung cancer cells were transduced with the mini-CRISPRi library and then injected into either the subcutaneous space in flanks (HCC827) or the left lower lung lobes (H1975) of nude mice and grown for 28 days. DNA from each tumor was sequenced and read counts for each sgRNA quantified. The read count for each sgRNA was normalized to the sum of reads for negative control sgRNAs and the ratio of each sgRNA’s frequency in the tumor model relative to its frequency in vitro (immediately preinjection). (A) Read count frequency in the HCC827 cell model. Two independent replicate experiments were performed, with n = 6 for each experiment (for n = 12 total). Each dot represents a single sgRNA and indicates its average normalized representation (computed from 2 independent replicate experiments), organized and displayed as control sgRNAs and sgRNAs targeting ATP-modulating genes classified as glycolytic or mito-respiratory (termed Mito-Resp). Mito-Resp sgRNAs were among the most severely depleted in vivo (HSD17β10, TMEM261, MALSU1, c14orf2) (mean and SEM shown) (mean % representation of nontargeting, glycolytic and mito-respiratory sgRNAs as groups are 99.9%, 62.6%, and 42.2%, respectively. One-way ANOVA of all 3 groups of sgRNAs demonstrate p-value < 0.0001, with Tukey’s multiple comparisons test between glycolytic and control p < 0.02, mito-resp and control p < 0.0001, and between glycolytic and mito-resp ns). (B) H1975 cells grown orthotopically in the lungs of nude mice (n = 6) were dissected and analyzed using the same approach as for flank tumors (A) (mean % representation of nontargeting, glycolytic, and mito-respiratory sgRNAs are 85.4%, 61.6%, and 40.9%, respectively. One-way ANOVA of all 3 groups of sgRNAs demonstrate p-value = 0.006, with Tukey’s multiple comparisons test between glycolytic and control ns, mito-resp and control p-0.005, and between glycolytic and mito-resp ns). (C) Bioluminescent imaging of tumor growth and metastasis in the H1975 model (2 representative mice shown), demonstrating tumor progression in the primary site and metastatic spread to mediastinum and contralateral lung by day 28. In addition, an extrathoracic distant metastasis (left femoral metastasis) developed (blue signal in the leg of the animal on the right). All metastases were confirmed at necropsy and analyzed by sequencing. (D) Mean % representation of nontargeting, glycolytic, and mito-respiratory sgRNAs in H1975 metastases (7 individual metastases from 4 mice, normalized to the matched primary tumor) are plotted on the y-axis (mean and SEM shown, mean % representation of nontargeting, glycolytic, and mito-respiratory sgRNAs are 183%, 192%, and 177%, respectively, one-way ANOVA of all 3 groups of sgRNAs with Tukey’s multiple comparisons test demonstrate ns). (E-G) Validation of individual sgRNAs indicates that silencing mito-translational genes suppresses in vivo tumor growth. sgRNAs targeting top mito-respiratory hits identified from the mini-CRISPRi library screen—c14orf2, MALSU1, and TMEM261—were transduced into HCC827 cells, cells were selected with antibiotics, then injected into the flanks of nude mice. Tumors were allowed to grow for 28 days. (E) qPCR analysis of transduced HCC827 cells assessing level of silencing achieved with single sgRNAs (mean and SEM shown, t test **p < 0.01). (F, G) Tumor weight (F) and tumor volume (G) of HCC827 cells expressing each of the sgRNAs, mean and SEM shown (Student t test, *p < 0.05, **p < 0.01, ***p < 0.001). Underlying data can be found in S1 Data.
Fig 2
Fig 2. Transcriptome profiling of human lung cancer cells expressing sgRNAs against mito-respiratory hits reveals overexpression of glycolytic pathway genes in vitro and repression in vivo.
RNA-Seq was performed on HCC827 cells expressing individual sgRNA against top hits c14orf2, MALSU1, TMEM261 (or control sgRNA) (n = 4 samples per cell line) to determine changes in gene expression in vitro and in vivo. (A) Averaged expression for each mito-respiratory sgRNA was compared to control cells, fold-change, indicated by log2(FC) plotted on the x-axis, and log2(p-value) on the y-axis. SgRNA-targeted genes are indicated by green triangles, with each cell line demonstrating the expected reduced expression of its targeted gene (upper left quadrant). Glycolytic genes were the most overexpressed genes overall, with ENO2, PGK1, and HK2 being the most overexpressed genes across all 3 cell lines. (B) Gene set enrichment analysis identifies glycolysis and mitochondria-associated pathways and ontologies as enriched in all 3 cell lines. Significance was set at p < 0.05. (C) Volcano plots shown for each of the single sgRNA cell lines. Expected sgRNA-mediated silencing was observed in all 3 cell lines (blue dot). (D) HCC827 cells transduced with either control sgRNA or MALSU1 sgRNA were injected into the flanks of nude mice, then grown for 28 days, after which tumors were removed and analyzed by RNA-Seq. Pathway analysis was performed comparing the MALSU1 sgRNA cells to control sgRNA cells grown in vivo (blue) or in vitro (red), and displayed in a bubble plot indicating normalized enrichment score (NES) and log10 p-value for significantly altered pathways. Control sgRNA tumors n = 4, sgMALSU1 tumors n = 2. Underlying data can be found in S1 Data.
Fig 3
Fig 3. Mito-respiratory hits have distinct metabolic signatures that differ between in vitro and in vivo contexts.
HCC827 cells expressing individual CRISPRi sgRNA (control, or mito-respiratory hits c14orf2, MALSU1,TMEM261 silencing confirmation shown in Fig 2A) were grown in basal media or media with 10 mM 2DG (n = 4 per group, individual data points shown as grey dots) with either [U-13C]glucose or [U-13C]glutamine for 18 hours. Cells were collected, metabolites extracted and analyzed by mass spectrometry. (A) Estimated metabolic flux ratios for cells expressing sgRNAs targeting c14orf2, MALSU1, or TMEM261. Using the measured % of total all labeled and unlabeled glycolytic [[U-13C]glucose→F16BP indicates % total unlabeled (0 carbon) or completely labeled (6 carbon)] and respiratory metabolite values [[U-13C]glutamine→glutamate analysis indicates total unlabeled (0 carbon labeled) on the left and fully labeled (all 5 carbons labeled)] (shown in Panels A and B of Fig F in S1 File), the estimated metabolic flux ratios of glycolytic flux (6C/unlabeled) to the respiratory flux (5C/unlabeled) are shown for 3 mito-respiratory hits (TMEM261, c14orf2, and MALSU1). The ratios distinguish the glycolysis-shifted metabolism apparent in the 3 cell lines in which mito-respiratory hits are silenced. (n = 4 replicates per sample, 1-way ANOVA, Dunnett’s multiple comparisons test, *p < 0.05, ***p < 0.001). (B, C) PCA was applied to the fractional contribution values of the metabolomics data for HCC827 cells expressing individual CRISPRi sgRNA (control, or mito-respiratory hits c14orf2, MALSU1, or TMEM261). 13C glucose-derived labeling of cells grown under either control or 2DG media (D) was compared in (B) control or oligomycin in (C). The absolute change in PC1 and PC2 values of the fractional contribution analysis for each cell line comparing control to 2DG growth (ΔPC1 = PC1control − PC12DG, ΔPC2 = PC2control − PC22DG) shown in (B) or control to oligomycin shown in (C) are plotted (n = 4 replicates). (D, E) HCC827 cells expressing either control or MALSU1 sgRNA were injected into the flanks of nude mice. After 28 days of growth, mice were injected with 13C glucose to label tumor metabolites, after which tumors were collected and metabolites analyzed. (D) PCA was performed using the fractional labeling values of glucose-derived metabolites comparing MALSU1-deficient and control HCC827 cells grown in vitro or in vivo. (E) PCA was performed on the amount labeled of glucose-derived metabolites in MALSU1-deficient and control HCC827 cells grown in vitro or in vivo. Underlying data can be found in S1 Data. c14orf2, chromosome 14 open reading frame 2; F16BP, fructose 1,6-bisphosphate; MALSU1, mitochondrial assembly of ribosomal large subunit 1; PCA, principal component analysis.
Fig 4
Fig 4. Mito-respiratory gene silencing alters metabolic network structures and in vivo tumor growth.
(A-D) Pathway analysis of HCC827 cells expressing individual CRISPRi sgRNA (control or mito-respiratory hits c14orf2, MALSU1, or TMEM261) grown in vitro (A-C) or HCC827 cells expressing an individual CRISPRi sgRNA against MALSU1 grown in vivo as flank tumor (D) was performed by integrating transcriptome and metabolomics data and plotted on a KEGG graph depicting pathway components for glycolysis/gluconeogenesis. (E) H1975 cells transduced with either control sgRNA or individual sgRNAs against c14orf2, MALSU1, or TMEM261 were analyzed using Seahorse. OCR shown, compilation of 3 independent experiments, each analyzing n = 4 per cell line, normalized to cell numbers. Mean shown, 2-way ANOVA, **p < 0.01, ***p < 0.001. (F) OCR and ECAR measurements for cells grown under basal or forced glycolysis (oligomycin). Compilation of 3 independent experiments, each analyzing n = 4 replicates per cell line and normalized to cell numbers. (G) ATP levels were measured in different substrate conditions (basal, respiratory (10 mM 2DG), glycolytic (5 μM oligomycin) or depletion (no glucose or pyruvate)) mean and SEM shown, 2-way ANOVA, ***p < 0.001). (H, I) NAD+/NADH ratios and NAD+/NADH pool size. Shown are compilation of 2 independent experiments **p < 0.01, ***p < 0.001. (J) MitoSox assay measuring mitochondrial superoxide. Compilation of 3 independent experiments, each analyzing n = 4 replicates per cell line. Two-way ANOVA, **p < 0.01. (K) Cells expressing either control sgRNA or MALSU1 sgRNA were injected orthotopically into the left lungs of mice, then mice were imaged using BLI 21 days postinjection. (L) Day 21 radiance within the left 1/3, central 1/3, and right 1/3 chest regions in mice injected with control sgRNA tumor cells. N = 4 mice, mean shown. (M) Day 21 radiance within left 1/3, central 1/3, and right 1/3, chest regions in mice injected with MALSU1 sgRNA tumor cells. N = 6 mice, mean shown. (N) Central 1/3 and contralateral right 1/3 lung radiance each normalized to the matched left 1/3 radiance for control and MALSU1 mice, mean shown, t test *p < 0.05. (O) Day 21postinjection radiance within the left 1/3 chest in mice injected with either control or TMEM261-silenced tumor cells (n = 4 mice for control, n = 5 mice for TMEM261, mean shown Mann–Whitney test, *p < 0.05). (P) Day 28 postinjection central 1/3 and contralateral right 1/3 lung radiance, each reading normalized to the matched left 1/3 radiance for control and TMEM261 cells (n = 4 mice and n = 5 mice, respectively) mean shown, Mann–Whitney test, ns. (Q) HE-stained sections of right lungs from mice injected with either control sgRNA tumor cells or TMEM261 sgRNA tumor cells showing micrometastatic tumor deposits. Red arrows indicate intraparenchymal micrometastases; black arrows indicate perivascular metastases. (R) Systems-level testing implicates context-specific growth effects of mitochondrial/respiratory function and ATP levels. ATP-modulating CRISPRi hits grown in lung cancer cells produce specific energy substrate-driven growth effects in vitro and in vivo. In vitro growth correlates with glycolytic ATP, while in vivo primary growth in the subcutaneous and orthotopic lung setting correlate with mitochondrial-derived ATP. Silencing discrete mito-respiratory genes also impacts the growth of regional metastases, which are distinguishable from the primary tumor. Underlying data can be found in S1A–S1D and S2E and S2F and S3G–S3J Data. BLI, bioluminescence imaging; ECAR, extracellular acidification rate; HE, hematoxylin–eosin; OCR, oxygen consumption rate.
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