HER2-driven mammary tumorigenesis enhances bioenergetics despite reductions in mitochondrial content
- Department of Human Health Sciences, University of Guelph, Canada
- Centre for Metabolism, Obesity and Diabetes Research, McMaster University, Canada
- Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Canada
- Department of Biomedical Sciences, University of Guelph, Canada
- Department of Biochemistry, McGill University, Rosalind and Morris Goodman Cancer Research, Canada
- Department of Molecular and Cellular Biology, University of Guelph, Canada
- Department of Cancer Biology, Medical Center Boulevard, Wake Forest University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Experimental design and canonical HER2 signaling.
(A) Schematic of mammary tumors from the mammary fat pad of female MMTV-neundl-YD5 mice and normal (benign) mammary tissue, representative images of tumor morphology (tumor capsule, peripheral tumor tissue, and necrotic core), tissue processing workflow for transcriptomics, proteomics, western blotting, mitochondrial respiration, ROS emission, and histology (transmission electron microscopy (TEM) and immunofluorescence (IF)). (B) Schematic of classical signaling pathways (Ras/MAPK and PI3K/AKT/mTOR) downstream of HER2 receptor stimulation in the YD5 model. (C) Her2 gene expression (transcripts per million, TPM). (D) Gene expression for canonical HER2 signaling. (E) Absolute abundance of HER2 protein detected by quantitative label-free proteomics (n.d. = HER2 not detected in benign). (F) Representative western blots of proteins involved in canonical HER2 signaling. Quantified western blots (% of benign) for (G) total protein content and (H) phosphorylated targets normalized to the total protein content of each target. p-values in panels D and E listed as adjusted p-values from transcriptomic (differentially expressed gene, DEG) data (p-adj. <0.1 considered statistically significant). In panels G, H data is presented as means ± SD and statistical significance was determined using an unpaired, two-tailed Student’s t-test (*p < 0.05, n = 5–6 biological replicates per group/protein target). Abbrev. AKT – protein kinase B; eEf2 – eukaryotic elongation factor 2; eIF4E – eukaryotic translation initiation factor 4E; ERK – extracellular signal-regulated kinase; GRB2 – growth factor receptor-bound protein-2; MEK – mitogen-activated protein kinase kinase; mTORC1/2 – mechanistic target of rapamycin complex 1 and 2; PDK – 3-phosphoinositide-dependent protein kinase-1; PI3K – phosphoinositide 3-kinase; PIP2/3 – phosphatidylinositol 4,5-bisphosphate; PIP3 – phosphatidylinositol 3,4,5-triphosphate; p70-S6K1 – ribosomal protein S6 kinase beta-1; RAF – rapidly accelerated fibrosarcoma; RAS – rate sarcoma virus; SHC – Src homolog and collagen homolog; 4EBP1 – eukaryotic translation initiation factor 4E-binding protein 1.
-
Figure 1—source data 1
PDF file containing original western blots for Figure 1F with annotated bands and molecular weight markers.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig1-data1-v1.zip
-
Figure 1—source data 2
Original TIF files for western blot images in Figure 1F–H.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig1-data2-v1.zip
Figure 1—figure supplement 1
Absolute phosphorylated protein targets downstream of HER2 activation.
(A) Quantified western blots for phosphorylated canonical HER2 signaling proteins (n = 5–6 biological replicates/group). Phosphorylation sites are indicated for each target. (B) Representative blot for each phosphorylated target. Data are presented as mean ± SD and analyzed with an unpaired, two-tailed Student’s t-test (*p < 0.05).
-
Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1A, B with annotated bands and molecular weight markers.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig1-figsupp1-data1-v1.zip
-
Figure 1—figure supplement 1—source data 2
Original TIF files for western blot images in Figure 1—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig1-figsupp1-data2-v1.zip
Figure 2
Assessment of HER2-driven mammary tumor and benign mammary tissue transcriptomes.
(A) Principal component analysis (PCA) of benign mammary tissue and mammary tumors (n = 6 biological replicates/group). (B) Volcano plot showing differentially expressed genes (DEGs) identified between mammary tissue and mammary tumors. (C) Heatmap of top 3000 DEGs, (D) top 10 upregulated pathways (Reactome 2022) and (E) gene ontologies (GO Biological Process 2023) in tumors compared to benign mammary tissue, (F) top 10 downregulated pathways (Reactome 2022) and (G) gene ontologies (GO Biological Process 2023) in tumors compared to benign mammary tissue. Panels D, E assess the top 3000 upregulated DEGs (log2fc >1), panels F, G assess the top 3000 downregulated DEGs (log2fc <1). An adjusted p-value of <0.1 was considered statistically significant across all analyses. The Enrichr open access analysis web tool was used to generate panels D–G which were sorted by p-value.
Figure 3 with 1 supplement
Assessment of HER2-driven mammary tumor and benign mammary tissue proteomes.
(A) Venn diagram showing all detected proteins. Of the 1848 overlapping targets, 420 were significantly different based on a p-adj. <0.1 and 411 of those proteins were downregulated in tumors. (B) Principal component analysis (PCA) of benign mammary samples and mammary tumors. (C) Volcano plot showing proteins identified between mammary tumors and benign mammary tissue, those in blue (downregulated) or red (upregulated) were significantly different (n = 420 proteins, p-adj. <0.1). (D) Heatmap of 420 differentially expressed proteins in benign and tumor samples. (E) Top 10 downregulated pathways (Reactome 2022) and (F) gene ontologies (GO Biological Process, 2023), (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of downregulated proteins. The Enrichr open access analysis web tool was used to generate panels E, F, which were sorted by p-value.
Figure 3—figure supplement 1
Unique HER2-driven tumor and benign mammary tissue proteins.
(A) Venn diagram depicting 741 and 600 unique proteins identified in benign mammary tissue and HER2-driven tumors, respectively, (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of all detected unique benign mammary tissue proteins, (C) top 10 pathways (Reactome 2022), (D) top 10 gene ontologies (GO Biological Process) in benign mammary tissue, (E) KEGG analysis of unique HER2-driven tumor proteins, (F) top 10 pathways (Reactome 2022), and (G) top 10 gene ontologies (GO Biological Process) in HER2-driven tumors. KEGG, pathway, and GO analyses were conducted on all mammary and tumor proteins detected by quantitative label-free proteomics. The Enrichr open access analysis web tool was used to generate panels C, D, F, and G which were sorted by p-value.
Figure 4
Overlapping genes and proteins between HER2-driven mammary tumor transcriptome and proteome.
Correlation of log2FC for overlapping targets between the HER2-driven tumor and benign mammary tissue transcriptomes and proteomes grouped by up/downregulation (A), and (B) protein localization (mitochondrial vs other). (C) Venn diagram showing 144 overlapping downregulated targets between the assessed HER2-driven tumor and benign mammary tissue transcriptomes and proteomes. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for common downregulated targets. Top 10 downregulated (E) pathways (Reactome 2022) and (F) gene ontologies (GO Biological Process 2023). An adjusted p-value of <0.1 was utilized for all analyses. The Enrichr open access analysis web tool was used to generate panels E, F, which were sorted by p-value.
Figure 5 with 2 supplements
Assessment of the mitochondrial transcriptome and proteome.
(A) Venn diagram showing mitochondrial proteins detected in the current dataset when cross-referenced with the Mouse MitoCarta 3.0 database. (B) Heatmap of 125 mitochondrial proteins identified in benign mammary tissue and HER2-driven tumors (n = 5 biological replicates/group). (C) Absolute abundance of all mitochondrial proteins detected in benign and tumor tissues. (D) Gene expression (detected by transcriptomics; transcripts per million) of all detected OXPHOS subunits separated per respiratory complex; black and white bars represent subunits with no significant differences between groups. Heatmaps of (E) Complex I, (F) Complex II, (G) Complex III, (H) Complex IV, (I) Complex V (ATP synthase), and (J) tricarboxylic acid (TCA) cycle transcriptomes. All proteomic analyses (panels A–C) were conducted on n = 5 biological replicates/group. All transcriptomic analyses (panels D–J) were conducted on n = 6 biological replicates/group. Panels B, C were analyzed using an unpaired two-tailed Student’s t-test (*p < 0.05). In panel D, * and colored (blue and green bars) represent significant differences between benign and tumor samples based on an adjusted p-value of <0.1. OXPHOS complex schematics in panel D were obtained from the open access RCSB Protein Data Bank (Complex I = TW1V, Complex II = 2A06, Complex III = 3SFD, Complex IV = 6NMP, Complex V = 5FIL). Bars in panel D are listed in the following order: Complex I: Ndufa1, Ndufa10, Ndufa11, Ndufa12, Ndufa13, Ndufa2, Ndufa3, Ndufa5, Ndufa6, Ndufa7, Ndufa8, Ndufa9, Ndufab1, Ndufb10, Ndufb11, Ndufb2, Ndufb3, Ndufb4, Ndufb5, Ndufb6, Ndufb7, Ndufb8, Ndufb9, Ndufc1, Ndufc2, Ndufs1, Ndufs2, Ndufs3, Ndufs4, Ndufs5, Ndufs6, Ndufs7, Ndufs8, Ndufv1, Ndufv2, Ndufv3; Complex II: Sdha, Sdhb, Sdhc, Sdhd; Complex III: Cyc1, Uqcr10, Uqcr11, Uqcrb, Uqcrc1, Uqcrc2, Uqcrfs1, Uqcrh, Uqcrq; Complex IV: Cox4i1, Cox4i2, Cox5a, Cox5b, Cox6a1, Cox6b1, Cox6b2, Cox6c, Cox7a1, Cox7a2, Cox7a2l, Cox7b, Cox7c, Cox8a, Cox8b, Ndufa4; Complex V: Atp5a1, Atp5b, Atp5c1, Atp5d, Atp5e, Atp5g1, Atp5g2, Atp5g3, Atp5h, Atp5j, Atp5j2, Atp5k, Atp5l, Atp5md, Atp5mpl, Atp5o, Atp5pb, Atpif1, Dmac2l.
Figure 5—figure supplement 1
Mitochondrial enrichment in benign mammary tissue and HER2-driven mammary tumors.
(A) Total and mitochondrial (Mouse MitoCarta 3.0) absolute protein abundance assessed by quantitative label-free proteomics. (B) Mitochondrial enrichment factor (MEF) calculated as the ratio of all detected MitoCarta proteins relative to the underlying total proteome per sample (n=5 biological replicates/group). Statistical analysis was conducted using an unpaired two-tailed Student’s t-test (*p < 0.05). In panel A, *# denotes significant differences between both total and MitoCarta proteomes between groups.
Figure 5—figure supplement 2
Proteomic analysis of detected OXPHOS subunits.
Heatmaps showing subunits detected for (A) Complex I, (B) Complex II, (C) Complex III, (D) Complex IV, and (E) Complex V (ATP synthase) using quantified label-free proteomics. (F) OXPHOS Complex I–V absolute abundance as a percentage of the mitochondrial proteome (n = 5 biological replicates/group). (G) OXPHOS Complex I–V absolute abundance as a percentage of the total detected proteome (n = 5 biological replicates/group). (H) Representative and quantified western blots for Complexes II and IV expressed as a percentage of benign. Data in panels F, G, H are presented as means ± SD and analyzed with an unpaired, two-tailed Student’s t-test (*p < 0.05).
-
Figure 5—figure supplement 2—source data 1
PDF file containing original western blots for Figure 5—figure supplement 2H with annotated bands and molecular weight markers.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig5-figsupp2-data1-v1.zip
-
Figure 5—figure supplement 2—source data 2
Original TIF files of western blot images in Figure 5—figure supplement 2H.
- https://cdn.elifesciences.org/articles/104079/elife-104079-fig5-figsupp2-data2-v1.zip
Figure 6
Cell-type-specific OXPHOS mRNA analysis in benign and tumor samples.
(A) OXPHOS expression per cell type from a publicly available single-cell RNA sequencing dataset (Wu et al., 2021) shown as a dot plot for each respiratory complex (Complexes I–V) subunit that was detected. Dot size indicates the percentage of cells of a given major type expressing each subunit gene (% cells expressing). Dot color represents gene expression scaled relative to each gene’s expression distribution across all major cell types. (B) Percentage of cells expressing OXPHOS subunits, arranged in ascending order per cell type for Complexes I–V. (C) Representative immunofluorescence images of benign mammary tissue and tumor samples stained for vimentin, cytokeratin, CD68, CD31, CD4, and CD138. (D) Quantification of cell-type proportion (% immunopositive area) in benign and tumor samples. (E) OXPHOS mRNA Index calculated for Complexes I–V. , where CT represents cell type. (F) Total OXPHOS mRNA Score for each cell type. , where i represents mitochondrial respiratory complexes.
Figure 7 with 1 supplement
Assessment of lipid and carbohydrate-linked mitochondrial respiration and OXPHOS coupling in benign mammary tissue and HER2-driven mammary tumors.
(A) Schematic depicting methods of normalization for respiration rates (per wet weight, total protein, mitochondrial protein/enrichment). (B) Complex I- and II-supported respiration normalized to tissue wet weight, (C) lipid-supported respiration normalized to tissue wet weight, (D) Complex I- and II-supported respiration normalized to total protein content, (E) lipid-supported respiration normalized to total protein content, and (F) Complex I- and II-supported respiration normalized to mitochondrial protein (mitochondrial enrichment factor, MEF). (G) Lipid-supported respiration normalized to mitochondrial protein. All individual values represent biological replicates (n = 9–22 biological replicates/group depending on the protocol). Data are presented as means ± SD, statistical analyses were conducted using an unpaired, two-tailed Student’s t-test (*p < 0.05). Abbrev: E = Endogenous, PM = pyruvate (5 mM), malate (2 mM), D = ADP (5 mM), G = glutamate (10 mM), S = succinate (10 mM), PC = palmitoyl-carnitine (20 µM), CI + CII = maximal CI + CII-supported respiration (PMDGS).
Figure 7—figure supplement 1
Correlation of tumor size and volume with maximal carbohydrate and lipid-supported respiration rates.
(A) Tumor wet weight (whole tumor post excision). (B) Tumor volume calculated as a modified ellipse (V = 1/2(length*width2)). (C–F) Endogenous (no substrate, CI + CII protocol), Endogenous (no substrate, lipid protocol), State 3 (5 mM pyruvate, 2 mM malate, 5 mM ADP), and maximal lipid-supported (2 mM malate, 5 mM ADP, 20 μM palmitoyl-carnitine) respiration rates correlated with tissue wet weight. (G–J) Same respiration data as (C–F) but correlated with tumor volume. The Pearson correlation coefficient (R) values are listed for each correlation.
Figure 8 with 1 supplement
Lipid and carbohydrate linked respiration and lipid metabolism transcriptome and proteome.
(A) Rates of carbohydrate (pyruvate, malate, ADP) and lipid (palmitoyl-carnitine, malate, ADP) supported respiration in tumors and benign mammary tissue. (B) Gene expression (transcripts per million) of mitochondrial pyruvate carrier and carnitine palmitoyltransferase isoforms mediating mitochondrial pyruvate and long chain fatty acid transport, respectively. (C) Network interaction showing downregulated (unsupervised analysis) gene clustering in the HER2-driven tumor transcriptome. (D) Heatmap of differentially expressed genes involved in lipid oxidation. (E) Heatmap of differentially expressed proteins involved in lipid metabolism. (F) Representative trace of protocol used to assess mitochondrial coupling. Following maximal rates of Complex I- and II-supported respiration, 1 µM oligomycin (OMY) was added to inhibit Complex V (ATP synthase), followed by 2.5 µM antimycin A (AMA) and 1 µM rotenone (ROT) to inhibit complexes III and I, respectively. (G) Group data for experiment depicted in (F) depicted as relative inhibition from maximal Complex I- and II-supported respiration (PMDGS) in each group. In panels A, B, F, and G data are presented as means ± SD and statistical analyses were conducted using an unpaired, two-tailed Student’s t-test (*p < 0.05).
Figure 8—figure supplement 1
Reactive oxygen species emission in benign mammary tissue and HER2-driven mammary tumors.
(A) Schematic depicting major sites of superoxide (O2*−) emission from respiratory Complexes I and III. Superoxide dismutase 2 (SOD2) subsequently catalyzes the dismutation of O2*− into hydrogen peroxide (H2O2), which can be measured fluorometrically. (B) H2O2 emission per wet tissue weight. (C) H2O2 emission per total protein. (D) H2O2 emission per mitochondrial protein (MEF, mitochondrial enrichment factor). Statistical analyses were conducted using an unpaired, two-tailed Student’s t-test (*p < 0.05).
Figure 9
Mitochondrial morphology and assessment of mitochondrial fusion, mitochondrial fission, and mitophagy transcriptomes and proteomes in benign mammary tissue and HER2-driven tumors.
(A) Transmission electron microscopy (TEM) images of tumor and benign samples showing punctate (tumor) versus elongated (benign) mitochondria. (B) Gene expression (detected by transcriptomics; transcripts per million) of fusion, fission, and mitophagy related genes separated per process filtered by MitoCarta, colored bars and * represent genes with an adjusted p-value of <0.1. Black and white bars represent genes with no significant differences between groups. (C) Heatmap of fusion transcriptome. (D) Heatmap of fusion proteome. (E) Heatmap of fission transcriptome. (F) Heatmap of fission proteome. (G) Heatmap of mitophagy transcriptome. (H) Heatmap of mitophagy proteome. TEM images are from independent tumor and benign tissue samples. Heatmaps depict all detected proteins (n = 5 biological replicates/group) and transcripts (n = 6 biological replicates/group).
Figure 10
Mitochondrial respiration is suppressed by tyrosine kinase inhibitor lapatinib.
(A) Schematic showing mechanism of lapatinib mediated inhibition of HER2 tyrosine kinase activity. (B) NF639 cell viability assessed using a crystal violet assay expressed as a percentage of untreated viability (0 nM lapatinib). (C) Complex I- and II-supported respiration in the presence of 20 µg digitonin in NF639 cells untreated (gray) or treated with 250 nM lapatinib for 24 hr. Left inset: Schematic of substrates supporting Complex I–II respiration, schematic of lapatinib or DMSO treatment. Right inset: Percent inhibition of maximal CI + II supported respiration (PMDGSC) with oligomycin (OMY), antimycin A (AMA), and rotenone (ROT). Following maximal rates of Complex I- and II-supported respiration, 1 µM OMY was added to inhibit Complex V (ATP synthase), followed by 2.5 µM AMA and 1 µM ROT to inhibit Complexes III and I, respectively. Each datapoint represents a technical replicate (duplicate), and data are presented as means ± SD. Statistical analyses were conducted using an unpaired, two-tailed Student’s t-test (*p < 0.05). Abbrev: E = Endogenous, PMD = pyruvate (5 mM), malate (2 mM), D = ADP (5 mM), G = glutamate (10 mM), S = succinate (10 mM), CC = cytochrome c (10 µM).
Additional files
-
Supplementary file 1
List of nine significantly upregulated proteins between tumor and benign samples, sorted in order of fold change from benign (smallest to largest fold change).
General pathways or functions are listed per protein based on ID mapping from the Uniprot protein sequence database. Adjusted p-values are listed per target.
- https://cdn.elifesciences.org/articles/104079/elife-104079-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104079/elife-104079-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
HER2-driven mammary tumorigenesis enhances bioenergetics despite reductions in mitochondrial content
eLife 14:RP104079.
https://doi.org/10.7554/eLife.104079.3
