Figures and data
Experimental design and canonical HER2 signaling.
A) Schematic of mammary tumors from the mammary fat pad of female MMTV-neundl-YD5 mice and adjacent 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 transmission electron microscopy (TEM). 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. G,H) 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 (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 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; p70S6k1 – 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.
Assessment of HER2-driven mammary tumor and benign mammary tissue transcriptomes.
A) PCA of benign mammary tissue and mammary tumors, 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.
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. B) 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) 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.
Overlapping downregulated genes and proteins between HER2-driven mammary tumor transcriptome and proteome.
A) Venn diagram showing 144 overlapping downregulated targets between the assessed HER2-driven tumor and benign mammary tissue transcriptomes and proteomes. B) KEGG analysis for common downregulated targets. Top 10 downregulated C) pathways (Reactome 2022) and D) 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 C,D which were sorted by p-value.
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/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. E-J) Heatmaps of E) Complex I, F) Complex II, G) Complex III, H) Complex IV, I) Complex V (ATP synthase) and J) TCA cycle transcriptomes. All proteomic analyses (panels A-C) were conducted on n=5/group. All transcriptomic analyses (panels D-J) were conducted on n=6/group. Panels B,C were analyzed using an unpaired two tailed Student’s t-test (*p<0.05). In Panel D, * and coloured (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.
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 complex II-supported respiration normalized to tissue wet weight, C) lipid-supported respiration normalized to tissue wet weight, D) Complex I and complex II-supported respiration normalized to total protein content, E) lipid-supported respiration normalized to total protein content, F) Complex I and complex 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/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).
Lipid and carbohydrate linked respiration and lipid metabolism transcriptome and proteome.
A) Maximal 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).
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, coloured 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/group) and transcripts (n=6/group).
Absolute phosphorylated protein targets downstream of HER2 activation.
A) Quantified western blots for phosphorylated canonical HER2 signaling proteins (n=5-6/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).
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) 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), 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.
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/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.
Proteomic analysis of detected OXPHOS subunits.
Heatmaps showing subunits detected for A) Complex I, B) Complex II, C) Complex III, D) Complex IV, 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/group). G) OXPHOS complex I-V absolute abundance as a percentage of the total detected proteome (n=5/group). Data in panels F,G are presented as means ± SD and analyzed with an unpaired, two tailed Student’s t-test (*p<0.05).
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.
Reactive oxygen species emission in benign mammary tissue and HER2-driven mammary tumors.
A) Schematic depicting major sites of superoxide emission from respiratory complexes I and III. Superoxide dismutase 2 (SOD2) subsequently catalyzes the dismutation of superoxide 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).
List of 9 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.
