Creatine-mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer

Abstract

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

Keywords: Acsbg1; Gatm; breast cancer; creatine; hypoxia; obesity.

Fig. 1. Diet-induced obesity promotes tumor progression in an orthotopic breast cancer model

A. Body weight of female mice on chow or HFD (n=11 per group). B. Lean and fat mass of chow and HFD mice at necropsy (n=7-8 per group) from a separate, matched cohort. C. Longitudinal volume of orthotopic E0771 tumors in chow and HFD fed mice (n=11 per group). D. Representative image of EdU (red) and DAPI (blue) labeled tumor slides. Quantification of EdU⁺ as a percentage of DAPI nuclei per 20x field (n=3 tumors per group). E. Quantification of cleaved caspase 3 positive cells per 20x field by immunohistochemistry (n= 4-5 tumors per group). F. Immunoblot of lysates from lean or obese tumors. HIF1α protein levels were normalized to β-actin and quantified using ImageJ. Data represent mean +/− standard error of the mean (SEM). * p<0.05, *** p<0.001, **** p<0.0001 For panel C, group, time, and group by time p-values are denoted and *** p<0.001 by post-hoc analysis at the final time point. See also Figure S1.

Fig. 2. Transcriptomic analysis of adipose tissue from obesity-accelerated breast cancer model

A. Diagram of orthotopic tumor location in mammary fat pad (peritumoral) and contralateral fat pad. B. KEGG metabolic pathway analysis of genes differentially expressed between HFD contralateral and peritumoral adipose tissue (n=4 per group). C. Segment of the Arginine and Proline Metabolism KEGG pathway demonstrating the creatine synthesis pathway. D. Relative mRNA expression of Gatm in bulk adipose tissue (n=4 per group). E. Normalized expression of Gatm in lean bulk tissue or adipocyte-specific TRAP purified mRNA (n=5-6 per group). F. Normalized expression of Gatm in obese bulk tissue (input) or adipocyte-specific TRAP purified mRNA (n=5-6 per group). G. Total abundance of creatine in bulk tumor from lean and obese mice by LC/MS. H. Total abundance of phosphocreatine in bulk tumor from lean and obese mice by LC/MS. I. Relative expression of Adiponectin and Gatm in whole adipose tissue or the mature adipocyte fraction (n=3 per group). J. Relative expression of Adiponectin and Gatm in cultured adipocytes differentiated from stromal vascular fraction (n=6 wells of differentiated adipocytes). K. Body weight of control or Adipo-Gatm KO mice on chow or HFD. L. Fat and lean mass of control or Adipo-Gatm KO mice at endpoint. M. Longitudinal volume of orthotopic E0771 tumors in lean or obese control or Adipo-Gatm KO mice. L-M; pooled from two independent experiments (n=20-27 per group). Data represent mean +/− SEM. *p<0.05, **p<0.01, *** p<0.001, ****p<0.0001. For panel M, group, time, and group by time p-values are denoted and *** p<0.001 by post-hoc analysis at the final time point. See also Figure S2 and S3.

Fig 3.. Genetic knockdown of creatine transporter (Slc6a8) in breast cancer cells attenuates tumor growth in obesity

A. Normalized mRNA expression of Slc6a8 in control or Slc6a8 KD cells. B. Immunoblot of Slc6a8 in membrane fractions of control (lane 1) or Slc6a8 KD cells (lane 2). C. Body weight of B6 mice on chow or HFD (n=10-19 per group). D. Longitudinal volume of control or Slc6a8 KD tumors in lean or obese mice (n=10-19 per group). E. Normalized expression of Slc6a8 in bulk control or Slc6a8 KD tumors. F. Relative abundance of creatine as measured by LC/MS (n=5 per group). Data represent mean +/− SEM. *p<0.05, **p<0.01, *** p<0.001, ***p<0.0001. For panel D, group, time, and group by time p-values are denoted in the figure and *** p<0.001 by pairwise post-hoc analysis at the final time point.

Fig 4.. Transcriptomic analysis of cancer cells isolated from lean and obese tumors

A. Schematic depicting FACS to isolate mE0771 cells from bulk tumor. B. Relative expression of a panel of genes differentially regulated in obese mE0771 cells. C. Diagram depicting the role of Acsbg1. D. Immunoblot of control or Acsbg1 OE cells. E. Longitudinal volume of control or Acsbg1 OE tumors in lean or obese animals (n=9-12 per group). F. Longitudinal volume of control or Acsbg1 KD tumors in lean or obese animals (n=8-10 per group). G. Diagram depicting the function of triacsin C. H. Tumor progression in obese animals with control or Acsbg1 KD tumors, treated with vehicle or triacsin C (n=3-4 per group). I. Tumor progression in lean animals with control or Acsbg1 KD tumors, treated with vehicle or triacsin C (n=4-7 per group). Data represent mean +/− SEM. *p<0.05, **p<0.01, *** p<0.001. For panels, E, F, H, and I, group, time, and group by time p-values are denoted in the figure. *p<0.05, **p<0.01 by post-hoc analysis at the final time point. See also Figure S4.

Fig 5.. Acsbg1-overexpressing tumors in obese mice have remodeled metabolism

A. Relative mRNA expression of Slc6a8 in bulk control or Acsbg1 OE tumors in lean or obese animals (n=5 per group). B. Relative oxygen consumption rate of control or Acsbg1 OE cells at baseline and following treatment with oligomycin, FCCP, and rotenone/antimycin. To the right is quantification of ATP generated through oxidative phosphorylation. C. Heatmap of dysregulated polar metabolites extracted from control or Acsbg1 OE tumors from lean or obese mice. D. PCA plot of C. E. Significantly dysregulated (p-value < 0.05, student’s t-test) metabolites from pairwise comparisons as labeled were queried against the KEGG database using MBROLE(López-Ibáñnez, Pazos and Chagoyen, 2016). Data represent mean +/− SEM. *p<0.05, **p<0.01, and ****p<0.0001. See also Figure S5.

Fig 6.. Acsbg1-dependent tumor progression is supported by exogenous creatine

A. Body weight of lean and obese control, Acsbg1 OE, or Acsbg1 OE + Slc6a8 KD animals B. Longitudinal volume of control, Acsbg1 OE, or Acsbg1 OE + Slc6a8 KD tumors in lean animals (n=8-9 per group) C. Longitudinal volume of control, Acsbg1 OE, or Acsbg1 OE + Slc6a8 KD tumors in obese animals (n=5-7 per group). D. SLC6A8 or ACSBG1 expression in human breast tumors by grade of tumor differentiation (well, n=88; moderate, n=156; poor, n=178). E. SLC6A8 or ACSBG1 expression in human breast tumors by tumor subtype in overweight or obese women (Luminal A, n=141; Luminal B, n=26; Her2, n=33; Basal, n=68). F. Schematic depicting proposed mechanism. Data in A-C represent mean +/− SEM. Group, time, and group by time p values are denoted. Data in D and E represents normalized intensity (log2). The bar represents the 95% confidence interval of gene expression. The dot represents average expression level. * represent p value < 0.05; ** represent p value < 0.01. See also Figure S6.