Enrichment analysis into the component proteins indicated that TN and you will HER2 cancers was notably graced to have glycolysis, vesicle-mediated transport, oligosaccharyl-transferase advanced, steroid biosynthesis, pentose phosphate pathway, and you will ATP binding (Fig. 1A; Second Dining table S3B–S3J). Pyruvate and you can oily acid metabolism was indeed graced merely regarding TN subtype. Luminal and you will TP tumors have been rather graced having electron transportation chain, oxidative phosphorylation, TCA cycle, and ATP synthesis, in arrangement with previous studies (36–38). Altogether, WGCNA demonstrated into a worldwide scale the new understood cancer of the breast subtype–particular metabolic signatures and showcased one particular paths from competitive subtypes.
To spot the key vehicle operators one contribute to the latest aggression away from TN subtype, we did a great position studies of the around three modules (blue, black colored, and you will red-colored; Fig. 1B). 1C; Second Table S4). We had been fascinated to obtain TCA duration–relevant healthy protein associated with glycolytic module and that focused the research into the engagement ones healthy protein in the glycolytic phenotype away from TN tumors. mRNA levels of IDH2, in accordance with the Cancer Genome Atlas (TCGA) research, indicated that its phrase coordinated with cyst aggression regarding luminal in order to HER2, whenever you are IDH1 mRNA height was enhanced just into the HER2 cancers and ACLY was highest during the luminal B and HER2 (Fig. 1D). While doing so, this new TCGA Bowl Cancers Atlas analysis showed that breast-intrusive carcinoma harbored mutations in the IDH1 and you will ACLY, when you find yourself IDH2 are nonmutated and you can is actually a lot more very shown into the nipple malignant tumors compared to other cancers types (cBioportal; Supplementary Fig. S1B-S3D). Study of almost every other IDH relatives minerals IDH3A, IDH3B, and you may IDH3G exhibited contradictory mRNA expression patterns within subtypes (Supplementary Fig. S1E). This type of abilities prompted us to would into the-depth study of the metabolic dependence off IDH2, in order to choose the metabolic vulnerabilities.
We perturbed IDH2 levels by overexpression, shRNA-based silencing, and CRISPR-Cas9 knockout in TNBC cell lines. IDH2 was stably overexpressed in stage II HCC38 cells with low endogenous expression, silenced in stage III HCC1599 cells with high endogenous expression and knocked-out using CRISPR-cas9 in stage II HCC1143 cells with high endogenous levels (Fig. 2A). Overexpression of IDH2 increased the anchorage-independent growth in soft agar and IDH2 knockout reduced the colony-forming ability (Fig. 2B and C). In addition, high IDH2 expression increased cell survival under oxidative stress and reduced cell survival upon IDH2 knockout (Fig. 2D). Given that each cell degrades H2O2 differently, H2O2 levels were calibrated per cell lines and furthermore, the antioxidant response was evaluated by cellROX staining after induced oxidative stress. IDH2-high cells had reduced cellROX staining with increased antioxidant capacity compared with increased cellROX staining in IDH2-low cells (Fig. 2E; Supplementary Fig. S2A and S2B). Interestingly, proliferation rate in two-dimensional cultures showed reduced proliferation of IDH2-knockout cells compared with control, but no significant proliferation change was observed in IDH2-stable overexpression, or upon transient overexpression of IDH2 in three additional stage II cell lines, HCC1500 (TN), HCC1937 (TN), and HCC1954 (HER2; Fig. 2F; Supplementary Fig. S2C–S2F). Rescue of IDH2 expression in the knockout cells showed increased resistance to oxidative stress compared with the knockout counterparts (Supplementary Fig. S2G and S2H). Functional assays were not performed in HCC1599 due to their aggregated growth with large clumps in suspension culture. Altogether, these functional assays showed that IDH2 promotes the protumorigenic phenotypes of breast cancer cells.
Examination of the metabolic effects of IDH2 perturbation showed increased glycolysis upon IDH2 high expression, as measured by the ECAR, glucose uptake, and lactate secretion (Fig. 2G–I; Supplementary Fig. S2I–S2K). To study the changes in a global manner, we analyzed the proteomes of cells with perturbed IDH2 levels. We identified 9,695 proteins from triplicate analyses of all the six cell lines HCC38 (Control-ox and IDH2-ox), HCC1599 (Control-kd and IDH2-kd), and HCC1143 (Control-ko and IDH2-ko; Supplementary Table S5A). A comparison of significantly changing proteins between IDH2-high and IDH2-low cells identified 948 differentially expressed proteins (FDR 13 C5-glutamine and monitored the isotopologue distribution of TCA cycle metabolites. In concordance with the elevated TCA cycle and oxidative phosphorylation proteins in IDH2-high cells, isotope tracing from 13 C5-glutamine depicted increased alpha-ketoglutarate (m5), citrate (m4), and aspartate (m4) (Fig. 3A–C). Citrate (m4) and aspartate (m4) are derived from the forward, oxidative glutamine metabolism of the TCA cycle (Fig. 3D). Reductive metabolism of glutamine mediated by IDH1/2 has been observed during hypoxia, mitochondrial dysfunction, and during redox homeostasis in anchorage-independent growth (14, 39–41). In parallel to the increased oxidative metabolism, cells with high IDH2 had increased levels of citrate (m5) and aspartate (m3), which indicated reductive carboxylation even under normoxic conditions with active mitochondrial function (Fig. 3B and C). In accordance, the fractional contribution of Glutamine (m5) to citrate (m5), aKG (m5) and aspartate (m3) and the ratios of citrate 5/4 and aspartate 3/4 increased with IDH2 overexpression and reduced with IDH2 knockout (Supplementary Fig. S4A-S4E). 3E; Supplementary Fig. S4F-S4H). In agreement with the genetically perturbed cells, a comparison between the basal IDH2 levels in the different cell lines correlated with isotopologue labeling patterns. Glutamine (m5) tracing in HCC38 with low basal IDH2 showed that >80% of total citrate is citrate (m4) and >60% of aspartate is aspartate (m4) (Supplementary Fig. S4A). In contrast, HCC1599 and HCC1143 cells with high basal IDH2, showed similar proportion of oxidative and reductive metabolism (Supplementary Fig. S4B and S4C). In addition, citrate (m4) and (m5) labeling correlated with basal IDH2 levels (Supplementary Fig. S4I). Overall, these results show higher induction of reductive TCA cycle metabolism in IDH2-high cells.