Enrichment analysis into module protein showed that TN and HER2 tumors was in fact significantly enriched for glycolysis, vesicle-mediated transport, oligosaccharyl-transferase advanced, steroid biosynthesis, pentose phosphate pathway, and you can ATP joining (Fig. 1A; Second Dining table S3B–S3J). Pyruvate and you will oily acid metabolic process was basically enriched only on the TN subtype. Luminal and you may TP tumors was basically rather graced having electron transport strings, oxidative phosphorylation, TCA duration, and you may ATP synthesis, for the agreement which have earlier studies (36–38). Entirely, WGCNA shown towards the a major international size brand new known cancer of the breast subtype–particular metabolic signatures and you will showcased many paths of competitive subtypes.
To determine the primary drivers you to definitely play a role in brand new aggressiveness of TN subtype, we performed a great position research of the about three modules (blue, black, and you will purple; Fig. 1B). 1C; Secondary Dining table S4). We had been fascinated to find TCA cycle–associated protein in the glycolytic module and that centered our very own research towards engagement of them protein on glycolytic phenotype out of TN cancers. mRNA degrees of IDH2, according to the Cancers Genome Atlas (TCGA) analysis, showed that its phrase synchronised that have tumefaction aggressiveness off luminal so you’re able to HER2, when you are IDH1 mRNA peak try improved merely into the HER2 tumors and ACLY try large during the luminal B and you may HER2 (Fig. 1D). Likewise, brand new TCGA Bowl Cancer tumors Atlas research showed that breast-intrusive carcinoma harbored mutations when you look at the IDH1 and you will ACLY, when you find yourself IDH2 try nonmutated and you may are a great deal more highly shown into the breast cancer compared to almost every other cancer types (cBioportal; Second Fig. S1B-S3D). Examination of almost every other IDH family relations nutrients IDH3A, IDH3B, and you may IDH3G shown inconsistent mRNA term activities between your subtypes (Secondary Fig. S1E). Such results prompted me to create inside-depth studies of your own metabolic reliance away from IDH2, also to select its metabolic vulnerabilities.
In line with increased oxidative metabolic process about TCA course, highest mitochondrial respiration is seen in higher IDH2 tissues (Fig
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 American dating advice. 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.
Top 20 very central protein one to designed the fresh new center of your network provided healthy protein employed in glycolysis (LDHA, LDHB, ENO1, PGK1, GPI, PFKL, PKM, PGM1), TCA duration-related (IDH1, IDH2, ACLY), and you will pentose phosphate path (G6PD, H6PD, PGD, TKT; Fig
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.