TRIM24 links glucose metabolism with transformation of human mammary epithelial cells

Abstract

Tripartite motif 24 protein (TRIM24) is a plant homeodomain/bromodomain histone reader, recently associated with poor overall survival of breast-cancer patients. At a molecular level, TRIM24 is a negative regulator of p53 levels and a co-activator of estrogen receptor. However, the role of TRIM24 in breast tumorigenesis remains largely unknown. We used an isogenic human mammary epithelial cell (HMEC) culture model, derived from reduction mammoplasty tissue, and found that ectopic expression of TRIM24 in immortalized HMECs (TRIM24 iHMECs) greatly increased cellular proliferation and induced malignant transformation. Subcutaneous injection of TRIM24 iHMECs in nude mice led to growth of intermediate to high-grade tumors in 60-70% of mice. Molecular analysis of TRIM24 iHMECs revealed a glycolytic and tricarboxylic acid cycle gene signature, alongside increased glucose uptake and activated aerobic glycolysis. Collectively, these results identify a role for TRIM24 in breast tumorigenesis through reprogramming of glucose metabolism in HMECs, further supporting TRIM24 as a viable therapeutic target in breast cancer.

Figure 1. Aberrant expression of TRIM24 during breast cancer progression

A, TRIM24-Immunohistochemical (IHC) staining was performed on a breast tissue array (BR2082, US Biomax) with tissues from different stages of breast cancer progression such as normal mammary gland, atypical ductal hyperplasia, Intraductal carcinoma and invasive ductal carcinoma, B, Distribution of TRIM24 expression (p-value:1.32e-07) in TCGA breast invasive carcinoma patients (1008) and normal tissues (92) and C, in PAM50 breast cancer subtypes (950, also see Supp. Fig. S1B for additional information). D, TRIM24 expression in breast tissue array (BR1503, US Biomax) by Allred score (left), Correlation of TRIM24 expression with ER, PR, HER2, p53, and Ki67 (right and bottom left, as assessed by IHC). ***p < 0.001.

Figure 2. Aberrant expression of TRIM24 in an isogenic HMEC model

A, Schematic of the isogenic HMEC model showing transition of the cells from finite-lifespan cells to immortal and malignant cells. B, TRIM24 mRNA (left) and protein (right) expression in HMEC lines transitioning from “normal” finite-lifespan cells before stasis to immortal and malignant cells. Data are averages from three biological replicates ± standard deviation (SD). **p < 0.01.

Figure 3. TRIM24 drives iHMEC transformation and survival

A, qRT-PCR analysis of TRIM24 mRNA and Immunoblot of TRIM24 and p53 in Control, TRIM24-1, and TRIM24-2 iHMECs. B, Cell proliferation analysis of Control, TRIM24-1, and TRIM24-2 iHMECs. C, Cell-cycle analysis of Control, TRIM24-1, and TRIM24-2 iHMECs. D, Anchorage-independent proliferation of Control iHMECs, TRIM24-1 iHMECs, and MCF-7 cells as determined using a soft agar colony formation assay (left) and quantification of the colonies per field ± SD (right). The data are averages from two replicates ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4. Regulation of multiple cancer-associated pathways in iHMECs by TRIM24

A, Heat map showing unsupervised hierarchical clustering of 233 differentially expressed genes in TRIM24-1 compared to the Control. B, Table showing the list of Upregulated pathways associated with Growth and Metabolism in TRIM24-iHMECs (for complete list of pathways please see Supp. T3 & T4). C, qRT-PCR analysis of Glycolytic genes in Control, TRIM24-1, and TRIM24-2 iHMECs showing upregulation of their expression. D, Immunoblot for various Glycolytic proteins in Control, TRIM24-1, and TRIM24-2 iHMECs. E, qRT-PCR analysis of TCA-cycle genes in Control, TRIM24-1, and TRIM24-2 iHMECs. The data are averages from three biological replicates ± SD. F, GSEA profiles of different pathways positively correlated to TRIM24 expression in TCGA-BRCA dataset. NES: Normalized Enrichment Score (See Supp. Table T5 for complete list of pathways.) *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. TRIM24 leads to diverse metabolic states and increased glucose uptake

A and B, Extracellular Acidification rate (ECAR) and Oxygen Consumption Rate (OCR) in the presence of Oligomycin (1 μM), FCCP (Carbonylcyanide p-trifluoromethoxyphenylhydrazone, 500 nM) and Rot + Ant [Rotenone (1 μM) + Antimycin A (1 μM) measured using an XF^e96 Extracellular Flux Analyzer (Sea-horse) for Control, TRIM24-1 and TRIM24 iHMECs. Both ECAR and OCR, were normalized using cell number. C, 2-NBDG (Florescent Glucose) uptake assay for Control, TRIM24-1, and TRIM24-2 iHMECs for 0 hours (untreated) and 2 hours. Florescent intensity curves of the 2-NBDG uptake are shown. D, qRT-PCR analysis of GLUT4 and GLUT1 mRNA expression in Control, TRIM24-1, and TRIM24-2 iHMECs. The data are averages from three biological replicates ± SD. ***p < 0.001.

Figure 6. TRIM24 regulates expression of several genes associated with glucose metabolism in multiple breast cancer subtypes

A, MCF-7, SKBR3 and MDA-MB-231 cells were transfected with siControl or siRNA against TRIM24 (siTRIM24) and harvested 48 hours later. Effect of TRIM24 knockdown on expression of a glucose metabolism gene panel (by qRT-PCR analysis) is shown. The data are averages from three replicates ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. B, mRNA expression for ACO1, IDH2, OGDH and HK2 in HMEC lines transitioning from normal finite-lifespan pre-stasis cells to abnormal finite post-stasis, non-malignant immortal and malignant cells. The data are averages from three biological replicates ± SD. *p < 0.05; **p < 0.01.

Figure 7. TRIM24-HMECs form intermediate to high-grade tumors in nude mice

A, Tumor volume after 65 days post-injection of Control and TRIM24-iHMECs in nude mice. **p < 0.01 (all compared to Control). B, H&E Staining and TRIM24-IHC for various grades of tumors obtained from nude mice injected with TRIM24-iHMECs. C, Distribution of various grades of tumors from mice injected with either Control or TRIM24-iHMECs. D, Distribution of epithelial cells in tumors from mice injected with either Control or TRIM24-iHMECs.