TP53 Induced Glycolysis and Apoptosis Regulator and Monocarboxylate Transporter 4 drive metabolic reprogramming with c-MYC and NFkB activation in breast cancer

Abstract

Breast cancer is composed of metabolically coupled cellular compartments with upregulation of TP53 Induced Glycolysis and Apoptosis Regulator (TIGAR) in carcinoma cells and loss of caveolin 1 (CAV1) with upregulation of monocarboxylate transporter 4 (MCT4) in fibroblasts. The mechanisms that drive metabolic coupling are poorly characterized. The effects of TIGAR on fibroblast CAV1 and MCT4 expression and breast cancer aggressiveness was studied using coculture and conditioned media systems and in-vivo. Also, the role of cytokines in promoting tumor metabolic coupling via MCT4 on cancer aggressiveness was studied. TIGAR downregulation in breast carcinoma cells reduces tumor growth. TIGAR overexpression in carcinoma cells drives MCT4 expression and NFkB activation in fibroblasts. IL6 and TGFB drive TIGAR upregulation in carcinoma cells, reduce CAV1 and increase MCT4 expression in fibroblasts. Tumor growth is abrogated in the presence of MCT4 knockout fibroblasts and environment. We discovered coregulation of c-MYC and TIGAR in carcinoma cells driven by lactate. Metabolic coupling primes the tumor microenvironment allowing for production, uptake and utilization of lactate. In sum, aggressive breast cancer is dependent on metabolic coupling.

Keywords: TP53 induced glycolysis and apoptosis regulator; c-MYC; glycolysis; metabolic heterogeneity; mitochondrial metabolism; monocarboxylate transporter 4.

FIGURE 1

TIGAR expression in human breast cancer and effect of TIGAR Knockdown on tumor growth. (A) TIGAR mRNA expression in normal breast tissue and breast cancer. (B) TIGAR immunoblot in T47D, AT-3 and MCF7 carcinoma cells with TIGAR knockdown (sgTIGAR) via CRISPR/Cas9. (C) Tumor volume and weight of TIGAR knockdown T47D cells orthotopically coinjected with BJ1 fibroblasts in nude mice. (D) Tumor volume and weight of TIGAR knockdown AT-3 cells coinjected with MEFs fibroblasts in syngeneic mice. (E-G) Tumor volume and weight of TIGAR knockdown RFP tagged MCF7 cells orthotopically co-injected with BJ1 fibroblasts (E). In vivo imaging for RFP of TIGAR knockdown MCF7 tumors on day 21 after co-injection (F). Tumor volume and weight at resection (G) (*P < .05). Error bars represent the SE of the mean.

FIGURE 2

Modulation of monocarboxylate transporter 4 (MCT4) in fibroblasts by carcinoma TIGAR. (A, B) MCT4 expression by immunofluorescence in BJ1 fibroblasts cocultured with TIGAR knockdown MCF7 cells (A) and TIGAR overexpression T47D cells (B). (C) MCT4 immunoblot in BJ1 fibroblasts cocultured with TIGAR overexpressing T47D cells via a 0.4 μm filter insert. Protein from the fibroblast component was immunoblotted for MCT4. (D) TIGAR immunoblot of MCF7 cells overexpressing TIGAR. (E) MCT4 immunofluorescence in BJ1 fibroblasts cocultured with TIGAR overexpressing MCF7 cells. Quantification of fibroblast MCT4 staining is normalized to cell number (*P < .05). Error bars represent the SE of the mean. N = 4, Scale bar: 20 μm.

FIGURE 3

NFkB activation in fibroblasts by carcinoma TIGAR. (A, B) NFkB activity in NIH3T3 fibroblasts cocultured with TIGAR overexpressing T47D or MCF7 carcinoma cells. NIH3T3 cells were stably transfected with an NFkB luciferase reporter and luminescence is normalized to total protein. (C) TIGAR immunoblot in NIH3T3 wild-type control or sg TIGAR knockdown fibroblasts. (D) NFkB activity in NIH3T3 wild-type control or sg TIGAR knockdown fibroblasts under control conditions and after the addition of 10 mM Lactate. NIH3T3 cells were stably transfected with an NFkB luciferase reporter and luminescence is normalized to total protein. (E) BJ1 fibroblasts were immunoblotted for pP65 and MCT4 after treatment with LPS (1 μg/ml), NFkB inhibitor PS1145 (10 μM) and the combination of LPS and PS1145. (F) MCT4 expression in BJ1 fibroblasts expressing GFP cocultured with TIGAR overexpressing T47D cells with 10 μM PS1145, which is an NFkB inhibitor. (G) Quantification of fibroblast MCT4 staining is normalized to cell number (*P < .05). Error bars represent the SE of the mean. N = 4, Scale bar: 20 μm.

FIGURE 4

Effects of IL6 and TGFB on the expression profile of cancer cells and fibroblasts. (A) Heatmap representation of cytokine expression values determined from conditioned media of T47D carcinoma and BJ1 fibroblast cocultures via an insert. (B) Quantification of IL6 levels in conditioned media from T47D carcinoma and BJ1 fibroblast cocultures via an insert. (C) Quantification of TGFB levels in conditioned media from T47D carcinoma and BJ1 fibroblast cocultures via an insert. (D) TIGAR immunoblot of T47D carcinoma cells exposed to TGFB2 (10 ng/ml) and IL6 (3 ng/ml). (E) TGFB immunoblot in T47D EV empty vector control and TIGAR overexpressing cells. (F) CAV1 and MCT4 immunoblot of BJ1 fibroblast cells exposed to IL6 (3 ng/ml) and TGFB2 (10 ng/ml). (G) TIGAR and TGFB immunoblot in BJ1 Sg CTRL or Sg TIGAR cells. (H) TGFB immunoblot in NIH3T3 Sg CTRL or Sg TIGAR cells. (I) MCT4 and TGFB immunoblot in BJ1 Sg CTRL or Sg MCT4 knockdown cells. (J, K) CAV1 and MCT4 expression by immunofluorescence of BJ1 fibroblasts exposed to the soluble cytokines TGFB2 (10 ng/ml) and IL6 (3 ng/ml) or cocultured with T47D cells. Quantification is normalized to cell number. (L) CAV1 expression by immunofluorescence in BJ1 fibroblasts cocultured with TIGAR overexpressing T47D cells and treated with 10 μM SB431542, which is a TGFB inhibitor. Quantification of fibroblast CAV1 and MCT4 staining is normalized to cell number (*P < .05). Error bars represent the SE of the mean.

FIGURE 5

Effect of Lactate on carcinoma cell compartment. (A) MCT1 immunoblot in MCF7 cells under control conditions and after treatment with 5 mM lactate. (B) c-MYC immunoblot in MCF7 carcinoma cells exposed to 5 mM Lactate compared with control. (C) c-MYC immunoblot in MCF7 and MDA-MB-231 Sg Control or MCF7 sg TIGAR knockdown cells. (D) c-MYC and TIGAR immunoblot in MCF7 and AT-3 TIGAR overexpressing cells compared with EV empty vector control cells. (E) c-MYC and TIGAR immunoblot in MCF7 Sg MYC knockdown cells compared with Sg Control cells. (F) c-MYC immunoblot in AT-3 and HA-cMYC immunoblot PY8119 in c-MYC overexpressing cells compared with LV control cells. (G) AT-3 and PY8119 LV control and c-MYC overexpressing cells were stably transfected with a secrete-pair dual luminescence GLuc/SEAP TIGAR promoter reporter. The GLuc luminescence is produced based on TIGAR promoter luciferase activity. The GLuc luminescence is normalized to SEAP luminescence which allows for transfection and expression normalization (*P < .05). (H) AT-3 LV control and c-MYC overexpressing cells were stably transfected with a secrete-pair dual luminescence GLuc/SEAP mutant TIGAR promoter reporter. The GLuc luminescence is produced based on TIGAR promoter luciferase activity. The GLuc luminescence is normalized to SEAP luminescence which allows for transfection and expression normalization (*P < .05). (I) TIGAR, c-MYC and H3K9 acetylation immunoblot in MCF7 Sg Control and Sg TIGAR knockdown cells under control conditions or after exposure to 2.5 mM acetate. (J) Lactyllysine immunoblot in MCF7 Sg control and Sg TIGAR knockdown cells exposed to 0, 5 and 10 mM Lactate. Quantification of Lactyllysine protein expression normalized to vinculin in MCF7 control and TIGAR knockdown cells exposed to lactate. (K) Lactyllysine immunoblot in MCF7 EV empty vector control or TIGAR overexpressing cells exposed to 0, 5 and 10 mM Lactate. Quantification of Lactyllysine protein expression normalized to vinculin in MCF7 control and TIGAR overexpressing cells exposed to lactate. Error bars represent the SE of the mean.

FIGURE 6

Tumor growth and mitochondrial metabolism induced by TIGAR is mediated by MCT4 in fibroblasts. (A, B) Tumor volume and weight of EV empty vector control or TIGAR overexpressing breast carcinoma cells co-injected with wild-type (WT) or MCT4−/− MEFs into the flank of WT or MCT4−/− C57Bl6 mice. (C) Model of effects of TIGAR expression in carcinoma cells. TIGAR in carcinoma cells positively modulates the expression c-MYC in a cell autonomous fashion and modulates the expression of fibroblast MCT4 and CAV1. Conversely, fibroblast MCT4 and CAV1 expression modulates the expression of TIGAR in carcinoma cells. This cross-talk is mediated by the production of the soluble factors TGFB, IL6 and lactate. (D) Oxygen consumption rate (OCR) of tumors generated from PY8119 EV empty vector control or TIGAR overexpressing breast carcinoma cells co-injected with wild-type (WT) MEFs in WT mice was measured under baseline conditions with 5 mM glucose, after injection of 1.5 μM Oligomycin and after injection of 2.0 μM FCCP (N = 6). (E) Oxygen consumption rate (OCR) of tumors generated from PY8119 EV empty vector control or TIGAR overexpressing breast carcinoma cells co-injected with wild-type (WT) MEFs in WT mice was measured under baseline conditions with 5 mM glucose and 10 mM lactate, after injection of 1.5 μM Oligomycin and after injection of 2.0 μM FCCP (N = 6). (F) Oxygen consumption of tumors generated from PY8119 EV empty vector control or TIGAR overexpressing breast carcinoma cells co-injected with MCT4−/− MEFs in MCT4−/− mice was measured under baseline conditions with 5 mM glucose, after injection of 1.5 μM Oligomycin, and after injection of 2.0 μM FCCP (N = 6). G, Oxygen consumption of tumors generated from PY8119 EV empty vector control or TIGAR overexpressing breast carcinoma cells co-injected with MCT4−/− MEFs in MCT4−/− mice was measured under baseline conditions with 5 mM glucose and 10 mM lactate, after injection of 1.5 μM Oligomycin, and after injection of 2.0 μM FCCP (N = 6). OCR values were normalized to protein concentration. Error bars represent the SE of the mean. (*P < .05). Created with BioRender.com