The analysis was performed on two BC cell lines, MDA-MB-231 and MCF-7. DEGs were identified between the control of the two cell lines, and between treated cells, i.e., 2-DG, MET, or glucose-starved cells. DEGs were determined using TAC or SAM. DEGs were visualized either on a volcano or SAM plot (Fig. 2 a, b) (only the representation of MDA-MB-231-GS versus the MDA-MB-231 control is shown). The lists of DEGs and the others volcano and SAM plot representations are shown in Additional file 1: Table S1. Biological significance was extracted from the gene lists by systematically enriching their DEGs with GO and pathway terms (KEGG and Reactome for DAVID, only Reactome for binary clustering). The top enriched clusters are only described in this section. Modulators for the most significant terms are also shown in parentheses.
One should mind the language we used to describe the regulation of functions. This is recommended because the regulation of other regulations is highlighted. Regulation of regulation should be interpreted as if we were multiplying positive and negative numbers, e.g. if a cluster exhibits down-regulation, and if a function in that group negatively regulates a property A, the resulting regulation of A should tend towards a positive regulation. It should be noted that biological functions fall into two distinct categories: the modulation of a process and the process itself. Thus, for phrases such as "[negative regulation of] A", process A is under activity, coexisting with another process negatively modulating A. Furthermore, during the analysis of each cluster, terms describing positive and negative regulation coexisted. The regulation with a BH-adjusted p-value that was at least twenty times (i.e., 1:0.05) greater than the other was assumed to be the dominant modulation. If this FC was less than twenty or if the p-value was >0.005, the modulations were interpreted as canceling each other out.
The comparison between the cell line’s controls can be reviewed with Additional file 2 and Additional file 3: Figure S1.
1. MDA-MB-231 cell line
1.1. Regulation with 2-DG
For the MDA-MB-231 cell line, the comparison of 2-DG-treated cells with the control (Fig. 3a) revealed that most clusters are up-regulated and very few are down-regulated. For the upregulated biological processes: [negative regulation of] response to stress and stimulus (lipids, hormones, chemicals), radiation, ER stress, and UPR (chaperone activation); [positive regulation of] cellular component traffic (protein and nitrogen compound localization, vesicle-mediated), cell migration; [negative regulation of] immune system processes (antigen presentation, toll-like receptors (TLRs), and interleukin (IL) signaling pathway); [positive regulation of] cell-cell and cell-substrate adhesion (laminin and proteoglycans); autophagy; [negative regulation of] metabolic processes of proteins and nitrogen compounds (phosphorylation, proteolysis); [negative regulation of] protein catabolism by ubiquitination; apoptotic processes; [positive regulation of] cell population proliferation (epithelial cells); [negative regulation of] homeostatic processes (temperature); and [positive regulation of] organization of extracellular components (collagen and hemidesmosome). These processes were found to recruit molecular functions describing protein binding, enzymes (kinase, ubiquitin ligase), adhesion molecules (cadherin), and glutamate receptors. In addition, oxidoreductase, protein disulfide isomerase, mitogen-activated protein kinase (MAPK), and transmembrane transporter activities were observed. Cellular compartments included cytoplasmic vesicles and lysosomes, nucleus and ER network, ER lumen and chaperone complex, cell adhesion foci, and the extracellular region.
Down-regulated clusters show DNA replication, DNA completion, cell cycle (G1/S phase transition), and purine/pyrimidine metabolism.
1.2. Regulation with metformin
Also comparing MDA-MB-231 cells, DEGs in MET-treated cells versus control (Fig. 3b) yielded mostly up-regulated clusters with a few down-regulated clusters. For the up-regulated clusters, the biological processes were [negative regulation of] RNA biosynthesis, phosphate metabolic process, and protein phosphorylation; [down-regulation of] stress and stimulus response (hypoxia, lipids, ROS, UPR, hormones, radiation, chemicals) and associated signal transduction (cyclic adenosine monophosphate (cAMP), calcium ion); [negative regulation of] catalytic activity (ubiquitin-protein transferase/ligase); [negative regulation] of cell death and apoptosis; [positive regulation] of cell motility and movement of cellular components; antigen processing and presentation via the major histocompatibility complex class I (MHC class I) (interferon (IFN) signaling); and transcription mediated by the forkhead box O (FOXO) transcription factor. Molecular functions were primarily binding mechanisms of "sequence-specific” transcriptional DNA cis-regulatory regions, proteins such as transcription (co-)factors or repressors, identical proteins (homo-dimerization), and kinases. Cellular compartments of activity included the extracellular exosome, focal adhesion, and cell-substrate junctions.
For the down-regulated clusters, the biological processes were DNA replication, DNA completion (removal of the flap intermediate); DNA damage response; DNA repair (resolution of D-loop structures, homologous DNA repair (HDR) by homologous recombination (HRR), Double-strand breaks (DSBs)) and [negative regulation of] cell cycle and division (cyclin-dependent G1/S phase transition, initiation of telomere c-strand synthesis and polymerase switching); chromatin organization; and senescence-associated secretory phenotype (SASP).
1.3. Regulation during glucose starvation
Finally, for glucose-starved cells (Fig. 3c), clustering also yielded mostly up-regulated with slightly more down-regulated clusters. For the up-regulated clusters, the biological functions were [positive regulation of] protein metabolism and modification process (post-translational modification (PTM), phosphorylation, proteolysis) and macromolecule biosynthesis; and [positive regulation of] nitrogen compound and nucleic acid metabolism and biosynthesis process (RNA transcription by RNA polymerase II); [negative regulation of] response to stress and stimulus (ER-related, UPR and chaperones, hypoxia, chemicals), organic substances (hormone, cytokine); [positive regulation of] cell localization and transport of vesicles and proteins; [negative regulation of] cell death and apoptotic process and pathway; [negative regulation of] catalytic, transferase, and [positive regulation of] binding activities; antigen processing and presentation; temperature and protein stability homeostasis; [positive regulation of] cell adhesion; and [negative regulation of] transforming growth factor-beta (TGFβ) receptor signaling. Molecular functions were mainly binding and homodimerization of "domain-specific" proteins (c-terminal) and binding of enzymes (protein kinase, GTPase and ubiquitin(-like) ligase), anions, misfolded proteins, ribonucleoprotein complexes, and cadherins. Cellular compartments include the cytoplasm (organelle membranes, mitochondria, vesicles and lysosomes, ER and its chaperone complex, Golgi apparatus), protein complexes (transferase, ubiquitin ligase), focal adhesions, and anchoring junctions.
For the down-regulated clusters, biological processes showed DNA methylation and replication; cell cycle and division (regulation of polo-like kinase 1 (PLK1) activity during the G2/M transition, checkpoints, initiation of telomere C-strand synthesis, telomere end packaging); [positive regulation of] chromatin and kinetochore, nuclear fission, microtubule and centriole organization; DNA damage response (Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) upregulation in response to replication stress, DNA damage and telomere stress-induced senescence, oxidative stress-induced senescence); DNA repair (DSBs processing); DNA completion; [positive regulation of] cellular protein, nuclear, RNA localization and chromosome segregation; NOTCH signaling; cholesterol biosynthesis via sterol regulatory element protein (SREBP) gene expression; glycolysis; FOXO-mediated transcription; and SASP.
2. MCF-7 cell line
2.1. Regulation with 2-DG
As with the MCF-7 cell line, comparison of the 2-DG-treated cells with the control (Fig. 4a) yielded only down-regulated clusters, in contrast to what the same comparison yielded for the MDA-MB-231 cell line. The biological processes were metal ion sequestration and zinc ion homeostasis, cell differentiation, and Rho GTPase-activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidases.
2.2. Regulation with metformin
Comparison of MET versus control (Fig. 4b) yielded mostly down-regulated and a few up-regulated clusters. For the up-regulated clusters, the biological properties were as follows: cellular responses to stress (oxygen-containing compound, drug, and steroid hormone); UPR (protein kinase R (PKR)-like ER kinase (PERK)-mediated); ER stress (transcription factor 4 (ATF4)-mediated gene activation, intrinsic apoptotic signaling) and signal transduction by the class mediator p53; cellular autophagy; apoptotic process; cellular differentiation and development; and temperature homeostasis. No molecular function terms were shown due to the paucity of DEGs. Vesicles, lytic vacuoles, and cytoplasm were compartments of activity.
For the down-regulated clusters, the biological functions were: [negative regulation of] mitotic cell cycle and division (checkpoints, G1/S phase transition and associated P53 regulation, ATR in response to replication stress, related to cyclin E); DNA damage response; chromosome segregation; DNA replication (mediated by E2F, up-regulation of the pre-replication complex, DNA unwinding); RNA biosynthesis and transcription via the RNA polymerase II promoter; [negative regulation of] nuclear division, chromosome and cytoskeleton organization; cell population proliferation; and Rho GTPase effectors (formin activation).
2.3. Regulation during glucose starvation
The last comparison, namely glucose starvation versus control (Fig. 4c), resulted in predominantly up-regulated clusters and some down-regulated clusters. For the up-regulated clusters, the biological processes were [positive regulation of] cell response to ER stress (ATF4 activation, intrinsic apoptotic signaling), UPR (Activating transcription factor 6-alpha (ATF6α) and X-box 1(S) binding protein (XBP1[S]) activate chaperone genes, PERK-mediated response, inositol-requesting enzyme 1 α (IRE1 α)-mediated response), and other stresses and stimuli (organic and cyclic chemicals, lipids, hypoxia, hormones); cell growth; transcription via RNA polymerase II, metabolic process and protein phosphorylation, serine family amino acid biosynthetic process; transmembrane transport of amino acids and hexoses (mediated by ABC family proteins) and cellular localization; regulation of chemical and ion (metals, inorganics, cations) homeostatic process; and FOXO mediated transcription. Molecular functions include transmembrane transport of amino acids and anions, binding of misfolded proteins, kinases and ubiquitin(-like) ligases, chaperones, homodimerization of proteins, and DNA binding of cofactors and transcription repressors. Cellular compartments include the cytoplasm, the ER chaperone complex, and kinase complexes (cyclin-dependent holoenzyme, serine/threonine).
As for the down-regulated clusters, their biological functions were as follows: [negative regulation of] apoptotic process; mitotic cell cycle (checkpoints, G1/S phase transition and associated transcriptional regulation by P53, and sister chromatid separation); and [positive regulation of] cell population proliferation.
3. Overlapping cell line
To show the functional similarities between the two BC cell types, a gene overlap sham cell line was established. Overlapping up-and down-regulated genes between the two cell lines were identified for each treatment. They were analyzed and cross-referenced in the same manner as the true cell lines.
Overlapping DEGs in the 2-DG treatment revealed too few genes, allowing no enrichment.
3.1. Regulation with metformin
Enrichment with MET resulted primarily in up-regulated clusters and some down-regulated clusters (Fig. 5a). For the up-regulated clusters, the biological processes were a cellular response to UPR, ER stress (intrinsic apoptotic signaling), chemicals, and other stimuli (glucocorticoids, hormones, lipids, chemicals), and RNA biosynthesis and metabolic process. Molecular functions describe the binding of "sequence-specific" DSBs to DNA (transcription regulatory region), and transcription factors. Cellular compartments include the nucleus, chromosome, RNA poly II transcription regulator, and ubiquitin ligase protein complexes.
For the down-regulated clusters, the biological processes were DNA replication, and [negative regulation of the] cell cycle (chromosome segregation, response to DNA damage, cyclin E-associated events during the G1/S transition, Skp1-Cullin-1-F-box (SCF)-mediated degradation of p27/p21, S-phase kinase-associated protein (Skp2)).
3.2. Regulation during glucose starvation
DEGs in the GS primarily resulted in up-regulated clusters (Fig. 5b). For these, the biological processes were as follows: [positive regulation of] ER stress response (ATF4-mediated gene activation, intrinsic apoptotic signaling) and UPR; protein folding in the ER (chaperone activation via ATF6α); [negative regulation of] stimulus response (starvation, steroids, chemicals) ; [negative regulation of] apoptotic signaling pathway; [negative regulation of] protein phosphorylation (cyclin-dependent kinase and MAPK activity) and RNA polymerase II biosynthesis in response to stress; [negative regulation of] transferase activity and catalysis; cell development and differentiation; [positive regulation of] cell localization (ER to cytosol transport, chromosome), epithelial cell motility and migration; cellular redox reaction, chemicals, glucose, and other carbohydrate homeostasis; and FOXO-mediated transcription. Molecular functions describe the binding of misfolded proteins, carbohydrate derivatives, and anions; intramolecular disulfide isomerase activity (S-S bond transposition); chaperone-mediated protein folding; and transmembrane transport of neutral amino acids. Cellular compartments include the cytoplasm, granule (melanosome), ER smooth membrane, chaperone complex, ER quality control compartment, and cell ruffle and leading edge.
For the down-regulated clusters, the biological processes were mitotic cell cycle arrest (p53 regulation of transcription of genes involved in G2 cell cycle arrest, PLK-mediated events, cyclin-associated events), and associated checkpoints and phase transitions; [positive regulation of] chromosome segregation and cell localization; response to DNA damage (G1 mitotic damage, unique transduction by the class mediator p53); cell development and differentiation; and nuclear fission and organelle organization.
4. Preserved functions
The relatively intact functions in the MDA-MB-231 cell line differed between treatments. With all treatments, the G protein-coupled receptor signaling pathway and the detection of chemical stimuli involved in the sensory perception of odor were preserved, the latter being signal transducers and integral components of the cell membrane. For 2-DG, the processes of melanin biosynthesis and [positive regulation of] transmembrane glucose transport were detected. As for GS, nervous system processes (sensory perception of smell) and cell adhesion were described.
For the MCF-7 cell line, actin organization was called, with activities occurring primarily at the plasma membrane, anchor junctions (cell-cell), and the leading edge of the cell. These activities occur specifically at the cell periphery, plasma membrane, extracellular matrix, and collagen trimer. Finally, GS demonstrated a multicellular organization process (sensory odor perception, olfactory signaling pathway), activity in the G protein-coupled receptor signaling pathway, developmental process (nervous system), and chemical stimuli detection. These processes act through the activity of transmembrane signaling receptors and occur at the membrane as integral components.
5. Intersection of DEGs
For this part, genes from the three treatments were intersected and Venn diagrams constructed. These give the number of intersected DEGs in the MDA-MB-231 (Fig. 6a) and MCF-7 (Fig. 6b), and overlap cell lines (Fig. 6c). The gene lists are present in Additional file 4: Table S2, Additional file 5: Table S3, and Additional file 6: Table S4).
5.1. Intersection in the MDA-MB-231 cell line
5.1.1. Common genes between 2-DG and metformin
Starting with the MDA-MB-231 cell line (Fig. 6a), genes common between MET and 2-DG treatments were grouped only into down-regulated clusters. Enrichment only showed activity in pathways related to vitamin metabolism and transport, and bile acid and bile salt synthesis via 24-hydroxycholesterol.
5.1.2. Common genes between 2-DG and glucose starvation
Enrichment of the intersected genes of 2-DG and GS treatments yielded only up-regulated clusters. We cite cellular response to [negative regulation of] organo-nitrogen compound, ER stress (XBP1[S]- and IRE1α-mediated UPR), stimuli (chemicals, cytokines, cell growth, regulation of insulin-like growth factor (IGF) transport, and uptake by insulin-like growth factor-binding proteins (IGFBP)); protein localization, vesicle-mediated transport, and cell migration; HER2 and TGFβ tyrosine kinase signaling; cell development and differentiation; cell adhesion and related regulation; autophagy; phosphorus metabolism and protein PTM; cell death and apoptosis.
5.1.3. Common genes between metformin and glucose starvation
Genes resulting from the intersection of MET and GS yielded predominantly up-regulated clusters with functions such as [negative regulation of] response to stress and stimuli (Lipopolysaccharides (LPS) and lipids); [positive regulation of] innate immune response (tumor necrosis factor (TNF) signaling pathway, NOD-like receptor (NLR) signaling pathway); cell death, apoptosis, and related pathways; cell proliferation; cell development and differentiation, cell aging; and [positive regulation of] cell-cell adhesion. For down-regulated clusters, we have a response to DNA damage stimuli (checkpoint, DSBs processing, p53 class mediator signaling), response to X-ray and UV light; [negative regulation of] DNA replication; [positive regulation of] nitrogen compounds and nucleic acid metabolic process, DNA repair (resolution of D-loop structures by Holliday junction intermediates and synthesis-dependent strand annealing (SDSA), DSBs, HDR), RNA biosynthesis, protein phosphorylation, and ubiquitination; mitotic cell cycle (phase transition, checkpoints, telomere end packaging); organelle nuclear division and chromosome organization; cell division; and SASP.
5.1.4. Common genes between 2-DG, metformin, and glucose starvation
Finally, the genes shared by all treatments mainly exhibited up-regulated clusters with biological functions such as [negative regulation of] stress and stimulus response (chemical, lipid and LPS, glucocorticoid, cytokine, hypoxia, ER and UPR stress, and IFN-alpha and beta type I and associated pathways); [negative regulation of] catalytic and transferase activity; [negative regulation] of kinase activity (zymogen activation, extracellular signal-regulated kinase ½ (ERK1/2) cascade, RAF-independent MAPK1/3 activation), phosphorus and protein metabolism; biosynthesis of nitrogenous compounds, transcription by RNA polymerase II and ROS production; endogenous peptide antigen presentation by MHC class I; cell population proliferation; [negative regulation] of cell component movement and cell motility; cell migration and differentiation; cell death and apoptosis.
As for the down-regulated clusters, we have the cell cycle (G1/S transition) and DNA repair (HRR).
5.2. Intersection in the MCF-7 cell line
5.2.1. Common genes between 2-DG and metformin
Moving to the MCF-7 cell line (Fig. 6b), few or no genes were found in common between the MET and 2-DG comparison, and thus no significant enrichment resulted.
5.2.2. Common genes between 2-DG and glucose starvation
Comparison of 2-DG and GS treatments yielded only upregulated clusters, namely neurotrophic tropomyosin receptor kinase (NTRKs) signaling.
5.2.3. Common genes between metformin and glucose starvation
Common DEGs between MET and GS treatments yield a predominantly up-regulated enrichment cluster and a down-regulated cluster. For the upregulated, biological functions showed cellular response to chemical stress (oxygen-containing compounds, arsenic), and [positive regulation of] UPR (PERK-mediated), ER stress (ATF response, chaperone activation), corticosteroids, starvation (saccharides, lipids, peptides); cell death and apoptosis, signaling via the class mediator p53; cell proliferation and associated regulation; temperature homeostasis; metabolic processes of amino acids, cyclic and carboxylic, and regulation of transcription from the RNA polymerase II promoter in response to stress; cell differentiation and development; bile acid and bile salt metabolism; transmembrane transport of amino acids; and autophagy.
The down-regulated cluster exhibited cell cycle processes (microtubule-binding).
5.2.4. Common genes between 2-DG, metformin, and glucose starvation
Genes common to all treatments showed only clusters of down-regulation. The cellular processes involved were protein nitrosylation, lipid response, cellular zinc ion homeostasis, cell development and differentiation, [positive regulation of] intrinsic apoptotic signaling pathway, and activity of the cysteine-type endopeptidase involved in the apoptotic process (NFκB activity), Rho GTPase effectors.
5.3. Intersection in the Overlap cell line
The intersection of treatment genes was only possible between MET and GS (Fig. 6c). One up-regulated cluster resulted from enrichment, having biological functions expressing [positive regulation of] cellular response to ER stress (ATF4 activates genes in response to ER stress) and chemical UPR (PERK regulates gene expression), corticoids, and signaling through apoptotic signaling pathways.
An additional histogram (Fig. 6d) also shows the difference in the number of DEGs between the overlap and the two cell lines.