Adipose Tissue-Breast Cancer Crosstalk Leads to Increased Tumor Lipogenesis Associated with Enhanced Tumor Growth

Abstract

We sought to identify therapeutic targets for breast cancer by investigating the metabolic symbiosis between breast cancer and adipose tissue. To this end, we compared orthotopic E0771 breast cancer tumors that were in direct contact with adipose tissue with ectopic E0771 tumors in mice. Orthotopic tumors grew faster and displayed increased de novo lipogenesis compared to ectopic tumors. Adipocytes release large amounts of lactate, and we found that both lactate pretreatment and adipose tissue co-culture augmented de novo lipogenesis in E0771 cells. Continuous treatment with the selective FASN inhibitor Fasnall dose-dependently decreased the E0771 viability in vitro. However, daily Fasnall injections were effective only in 50% of the tumors, while the other 50% displayed accelerated growth. These opposing effects of Fasnall in vivo was recapitulated in vitro; intermittent Fasnall treatment increased the E0771 viability at lower concentrations and suppressed the viability at higher concentrations. In conclusion, our data suggest that adipose tissue enhances tumor growth by stimulating lipogenesis. However, targeting lipogenesis alone can be deleterious. To circumvent the tumor's ability to adapt to treatment, we therefore believe that it is necessary to apply an aggressive treatment, preferably targeting several metabolic pathways simultaneously, together with conventional therapy.

Keywords: E0771; adipocytes; adipose tissue; breast cancer; lipogenesis; lipolysis; luminal B; mouse.

Figure 1

(A) E0771 tumor and (B) body weights two weeks post-implantation in lean and high-fat-diet-induced obese female mice (N = 8–10/group). Panel (C) shows an oral glucose tolerance test performed prior to E0771 implantation (N = 4–6/group). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2

(A) Glycerol released from 3T3-L1 adipocytes treated with conditioned media (representing media from 1 × 10⁶ million cells/mL media) from breast cancer cell lines of different subtypes (N = 9–35/group). (B) Control and tumor-associated mammary adipose tissue weight in lean and high-fat-diet-induced obese female mice two weeks after orthotopic E0771 implantation (N = 10/group). (C) Serum free fatty acids levels in lean female mice before and two weeks after orthotopic E0771 implantation with and without Acipimox treatment (N = 10/group). (D) Tumor weights in control and Acipimox-treated lean and high-fat-diet-induced obese female mice two weeks after orthotopic E0771 implantation (N = 10/group). (E) Tumor weights in control and Atglistatin-treated lean female mice two weeks after orthotopic E0771 implantation (N = 8–10/group). Data are presented as mean ± SEM; ** p < 0.01 and *** p < 0.001.

Figure 3

(A) Total lipid and glucose uptake in orthotopic and ectopic E0771 tumors from lean female mice two hours after an oral load of Intralipid emulsion containing [³H]–triolein and [¹⁴C]–glucose. The lipid ([³H]–triolein) and glucose ([¹⁴C]–glucose) counts in the (B) organic and the (C) aqueous phase of E0771 tumors (N = 5–7/group). (D) Fatty acid synthase (Fasn) mRNA expression in orthotopic and ectopic E0771 tumors from lean female mice two weeks after E0771 implantation (N = 6–7/group). Data are presented as mean ± SEM; * p < 0.05 and *** p < 0.001.

Figure 4

(A) Mitochondrial function as judged by changes in non-mitochondrial respiration, basal respiration, maximal respiration, proton leak-related respiration, ATP production-linked respiration, spare respiratory capacity and coupling efficiency (CE) determined from the oxygen consumption rate (OCR) in response to subsequent addition of oligomycin (O), FCCP and rotenone (R)/antimycin A (A) as indicated in the left panel of control and 24 h mammary adipose tissue co-cultured E0771 cells. Data are presented as OCR normalized to total protein levels (N = 30/group). (B) Glycolytic function, as determined by basal glycolysis rate, glycolytic capacity and glycolytic reserve, was estimated from the extracellular acidification rate (ECAR) of control and 24 h mammary adipose tissue co-cultured E0771 cells in response to subsequent addition of glucose (G), oligomycin (O) and 2-Deoxy-D-glucose (2-DG) as indicated in the left panel. Data are presented as ECAR normalized to total protein levels (N = 30/group). (C) Endogenous and (D) exogenous fatty acid oxidation estimated from the OCR of control and 24 h mammary adipose tissue co-cultured E0771 cells under low-glucose conditions (1.5 mM glucose, with or without the carnitine palmitoyltransferase-1 (CPT-1) inhibitor Etomoxir (eto), with or without BSA-conjugated palmitate (PA:BSA) in response to subsequent addition of oligomycin (O), FCCP and rotenone (R)/antimycin A (A) as indicated in the left panel A (N = 12/group). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01 and ***p < 0.001.

Figure 5

(A) [¹⁴C] counts in aqueous and organic phase (representing de novo lipogenesis) in control and 24 h mammary adipose tissue co-cultured E0771 cells in response to 10 min [¹⁴C]–glucose treatment at a glucose concentration of 11.1 mM (N = 6/group, * p < 0.05 for cultured alone vs. adipose tissue co-culture). Lactate levels in (B) 24 h culture media from differentiating 3T3-L1 adipocytes at the indicated time points (0 days = preadipocytes; 8 days = mature adipocytes) (N = 10/group) and in (C) culture media from isolated mammary (MA) and gonadal adipocytes (GA) (N = 6–10/group) cultured with the so-called MAAC method [41] (# p < 0.05, ## p < 0.01 for lean vs. obese GA; * p < 0.05 for obese MA vs. obese GA), and in (D) serum from lean (N = 7) and high-fat-diet-induced obese (N = 6) female mice (* p < 0.05 for lean vs. obese). Notably, the serum lactate levels are higher than expected; this may reflect that the sera came from terminal blood obtained under deep isoflurane anesthesia. (E) [¹⁴C] counts in aqueous and organic phase in control and 24 h 25 mM lactate-pretreated E0771 cells in response to 15 min [¹⁴C]–glucose treatment at a glucose concentration of 11.1 mM (N = 4–6/group, ** p < 0.01 for control vs. 25 mM lactate pretreatment). (F) [¹⁴C] counts in aqueous and organic phase in orthotopic and ectopic E0771 tumors from lean female mice two hours after an oral load with a [¹⁴C] lactate tracer (N = 5/group). Data are presented as mean ± SEM.

Figure 5

(A) [¹⁴C] counts in aqueous and organic phase (representing de novo lipogenesis) in control and 24 h mammary adipose tissue co-cultured E0771 cells in response to 10 min [¹⁴C]–glucose treatment at a glucose concentration of 11.1 mM (N = 6/group, * p < 0.05 for cultured alone vs. adipose tissue co-culture). Lactate levels in (B) 24 h culture media from differentiating 3T3-L1 adipocytes at the indicated time points (0 days = preadipocytes; 8 days = mature adipocytes) (N = 10/group) and in (C) culture media from isolated mammary (MA) and gonadal adipocytes (GA) (N = 6–10/group) cultured with the so-called MAAC method [41] (# p < 0.05, ## p < 0.01 for lean vs. obese GA; * p < 0.05 for obese MA vs. obese GA), and in (D) serum from lean (N = 7) and high-fat-diet-induced obese (N = 6) female mice (* p < 0.05 for lean vs. obese). Notably, the serum lactate levels are higher than expected; this may reflect that the sera came from terminal blood obtained under deep isoflurane anesthesia. (E) [¹⁴C] counts in aqueous and organic phase in control and 24 h 25 mM lactate-pretreated E0771 cells in response to 15 min [¹⁴C]–glucose treatment at a glucose concentration of 11.1 mM (N = 4–6/group, ** p < 0.01 for control vs. 25 mM lactate pretreatment). (F) [¹⁴C] counts in aqueous and organic phase in orthotopic and ectopic E0771 tumors from lean female mice two hours after an oral load with a [¹⁴C] lactate tracer (N = 5/group). Data are presented as mean ± SEM.

Figure 6

(A) Viability of cultured E0771 cells after 24 h continuous treatment with Fasnall at the indicated concentrations (N = 12/group). (B) Orthotopic and ectopic E0771 tumor weights after 23 days of vehicle or Fasnall treatment (15 mg/kg, daily i.p. injections, N = 7–10/group, * p < 0.05 for the orthotopic vs. ectopic tumors in the vehicle group). The dashed lines indicate the division between Fasnall “non-responders” and “responders”. (C,D) Orthotopic and (E,F) ectopic E0771 tumor growth curves and tumor weights after 23 days of Fasnall treatment comparing vehicle with Fasnall “non-responders” and “responders” separately (* p < 0.05, ** p < 0.01 and *** p < 0.001 for vehicle vs. Fasnall). (G) Viability of cultured E0771 cells after three days of intermittent treatment (4 h/day) with Fasnall at the indicated concentrations (N = 10–12/group, ** p < 0.01 and *** p < 0.001 for control vs. Fasnall). Data are presented as mean ± SEM.

Figure 6

(A) Viability of cultured E0771 cells after 24 h continuous treatment with Fasnall at the indicated concentrations (N = 12/group). (B) Orthotopic and ectopic E0771 tumor weights after 23 days of vehicle or Fasnall treatment (15 mg/kg, daily i.p. injections, N = 7–10/group, * p < 0.05 for the orthotopic vs. ectopic tumors in the vehicle group). The dashed lines indicate the division between Fasnall “non-responders” and “responders”. (C,D) Orthotopic and (E,F) ectopic E0771 tumor growth curves and tumor weights after 23 days of Fasnall treatment comparing vehicle with Fasnall “non-responders” and “responders” separately (* p < 0.05, ** p < 0.01 and *** p < 0.001 for vehicle vs. Fasnall). (G) Viability of cultured E0771 cells after three days of intermittent treatment (4 h/day) with Fasnall at the indicated concentrations (N = 10–12/group, ** p < 0.01 and *** p < 0.001 for control vs. Fasnall). Data are presented as mean ± SEM.