Dapagliflozin slows E0771 tumor growth in obese mice in an insulin-dependent manner

To examine the potential utility of dapagliflozin as an anti-tumor agent in vivo, we treated obese mice with dapagliflozin in drinking water beginning on the day of E0771 tumor implantation. Not surprisingly, dapagliflozin caused glycosuria, but did not affect energy expenditure or caloric intake, measured during the first week of treatment before the groups of mice diverged in body weight (Fig. 1a, Additional file 1: Figure S1A-J). As expected, water intake increased in the dapagliflozin-treated group as a compensatory mechanism to avoid dehydration, and a small (1%), physiologically insignificant increase in respiratory exchange ratio was also observed. However, 3 weeks later, sustained glucose wasting in urine was associated with reductions in body weight and fat mass in high-fat fed mice (Additional file 1: Figure S1K-L). SGLT2 inhibition lowered plasma glucose concentrations in 5-h fasted mice by 80 mg/dL and reduced plasma insulin concentrations in fed, 5-h fasted, and 16-h fasted mice (Fig. 1b, c), in contrast to metformin, which lowered plasma insulin only after a prolonged fast (Additional file 1: Figure S1M). To examine the impact of the reduction in plasma insulin on tumor growth and metabolism, we infused insulin subcutaneously to match plasma insulin concentrations in 5-h fasted dapagliflozin-treated mice to those measured in untreated HFD controls. E0771 tumor glucose metabolism was insulin-responsive: glucose uptake and oxidation were increased in tumors of HFD fed, hyperinsulinemic mice but normalized with dapagliflozin treatment; however, restoring hyperinsulinemia via subcutaneous insulin infusion increased tumor glucose uptake and oxidation to rates observed in HFD control mice. Hyperinsulinemia had a profound effect on tumor growth rates: 4 weeks after tumor implantation, E0771 tumors were 1000 mm3 larger in HFD mice than lean controls. However, dapagliflozin treatment reduced rates of tumor growth such that tumor growth in dapagliflozin-treated mice mimicked that of chow fed animals. This effect was insulin-mediated: restoring hyperinsulinemia increased tumor growth rates in dapagliflozin-treated mice to those measured in obese HFD mice.

Fig 1 Dapagliflozin slows E0771 breast tumor growth in an insulin-dependent manner. a, b Urine and plasma glucose concentrations. Unless otherwise designated, all measurements were performed in 5-h fasted mice. c Plasma insulin. d, e Tumor 2-deoxyglucose uptake and V PDH /V CS . f Tumor size. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. chow, ++P < 0.01, ++++P < 0.0001 vs. HFD + dapagliflozin, with the color of the symbols indicating the group compared to the group designated by the symbols. In all panels, data are the mean ± S.E.M. of n = 5 per group. Groups were compared by ANOVA with Bonferroni’s multiple comparisons test Full size image

Next, we aimed to examine whether the ability of dapagliflozin to slow E0771 tumor growth was a cell-autonomous effect. The maximum daily dose of dapagliflozin is 10 mg per day in humans; this dose results in a peak dapagliflozin concentration of less than 0.4 μM [54]. Ten-fold higher dapagliflozin concentrations had no impact on E0771 tumor glucose uptake or oxidation, nor did this dose of dapagliflozin alter cell division in vitro (Fig. 2a–c); however, 10,000-fold higher, suprapharmacologic dapagliflozin concentrations did reduce glucose uptake and oxidation, associated with slower E0771 cell division in vitro. In contrast, canagliflozin showed a dose-dependent effect to reduce tumor glucose uptake and oxidation and to suppress tumor cell division at pharmacologically relevant doses [55] (Fig. 2a–c). These data indicate that SGLT2 inhibitors may have some cell-autonomous effect to slow tumor growth, likely through direct suppression of tumor glucose metabolism. However, the lack of an effect of dapagliflozin at pharmacologically relevant concentrations suggests that most if not all of the impact of this agent occurs through alterations in systemic metabolism. In contrast, insulin—at doses that are supraphysiologic but commonly used in in vitro studies in the literature—promoted both glucose uptake and oxidation in E0771 tumors, accelerating tumor cell division (Fig. 2a–c).

Fig. 2 Impact of SGLT2 inhibitors and insulin on E0771 cell metabolism and division in vitro. a Glucose uptake. b V PDH /V CS . c Cell division. In all cell studies, data are the mean ± S.E.M. of n = 4 replicates. Data were compared by ANOVA with Bonferroni’s multiple comparisons test, in which each group was compared to the mean of the vehicle-treated cells Full size image

CRMP slows E0771 tumor growth in obese mice in an insulin-dependent manner

After observing the ability of dapagliflozin to slow E0771 breast tumor growth by reversing hyperinsulinemia, we asked whether similar effects would be seen as a result of reducing circulating insulin concentrations with an agent that works through a divergent mechanism. To that end, we treated obese, HFD fed, E0771 tumor-bearing mice with CRMP. Consistent with previous data [34, 56] and with an uncoupling effect confined to the liver, CRMP treatment did not alter body weight or fat mass, whole-body energy expenditure, food or water intake, or respiratory exchange ratio in obese mice (Additional file 1: Figure S2A-J). However, mitochondrial uncoupling lowered liver, plasma, and skeletal muscle triglyceride content (Fig. 3a, Additional file 1: Figure S2K-L). This reduction in ectopic lipid content resulted in lower 5-h fasted plasma glucose concentrations, and lower plasma insulin concentrations under both fed and fasted conditions (Fig. 3b, c). Tumor glucose uptake and oxidation were both modulated by circulating insulin concentrations and normalized by insulin sensitization: high fat feeding increased, and CRMP decreased, both parameters. However, restoring hyperinsulinemia by chronic subcutaneous insulin infusion increased tumor glucose uptake and oxidation to levels measured in untreated HFD tumor-bearing mice, confirming that tumor glucose uptake and oxidation are dynamic and insulin-responsive (Fig. 3d, e). These insulin-mediated alterations in tumor glucose metabolism translated to differences in tumor size: high fat feeding accelerated E0771 tumor growth, whereas the reversal of insulin resistance and hyperinsulinemia with CRMP reversed this effect through an insulin-mediated mechanism. However, infusion of insulin via subcutaneous pellet to match plasma insulin concentrations in CRMP-treated mice to those of HFD control animals completely abrogated the effect of CRMP to slow tumor growth (Fig. 3f).

Fig. 3 A controlled-release mitochondrial protonophore slows E0771 breast tumor growth in an insulin-dependent manner. a Liver triglyceride content. In all panels, unless otherwise specified, all mice were fasted for 5 h before they were studied. b, c Plasma glucose and insulin concentrations. d Tumor 2-deoxyglucose uptake and V PDH /V CS . f Tumor size. **P < 0.01 vs. chow, #P < 0.05, ##P < 0.01 vs. HFD + CRMP, with the color of the symbols indicating the group compared to the group designated by the symbols. In all panels, data are the mean ± S.E.M. of n = 5–6 per group. Groups were compared by ANOVA with Bonferroni’s multiple comparisons test Full size image

Next, we aimed to understand whether DNP exerted a direct effect on tumor growth or metabolism independent of insulin. Although tumor DNP concentrations in six CRMP-treated mouse tumors were negligible (0.015 ± 0.003 nmol/g, approximately equivalent to 0.015 μM), we confirmed that DNP did not affect tumor glucose metabolism or growth directly by measuring the rate of glucose uptake, V PDH /V CS , and cell division in vitro in MC38 cells and found each parameter to be unaltered after incubation in 1 μM DNP but increased with high concentrations of insulin (Fig. 4a–c). However, higher, markedly supraphamacologic DNP concentrations were toxic to the cells, reducing tumor glucose uptake, oxidation, and cell number.

Fig. 4 Impact of DNP and insulin on E0771 cell metabolism and division in vitro. a Glucose uptake. b V PDH /V CS . In all samples, this ratio was below the limit of detection (0.5%) in 500 μM DNP-treated cells. c Cell division. In all cell studies, data are the mean ± S.E.M. of n = 4 replicates. Data were compared by ANOVA with Bonferroni’s multiple comparisons test, in which each group was compared to the mean of the vehicle-treated cells Full size image

Dapagliflozin slows MC38 tumor growth in obese mice in an insulin-dependent manner

Having demonstrated that dapagliflozin impedes E0771 tumor growth by reversing systemic hyperinsulinemia, we next asked whether these results would translate to a second obesity-associated mouse tumor model: MC38 colon adenocarcinoma. Dapagliflozin caused profound glucosuria and increased water drinking before divergence in body weight, but did not affect food intake, energy expenditure, or the respiratory exchange ratio (Additional file 1: Figure S3A-J). However, after 4 weeks of treatment, dapagliflozin treatment resulted in lower body weight, an effect partially abrogated by insulin replacement (Additional file 1: Figure S3K-L). Chronic dapagliflozin treatment lowered plasma glucose and insulin concentrations and reduced both tumor glucose uptake and V PDH /V CS in an insulin-dependent manner, whereas obesity increased tumor glucose metabolism and SGLT2 inhibition normalized it, the obesity- and hyperinsulinemia-associated increases in glucose uptake and oxidation were restored by chronic subcutaneous insulin infusion (Fig. 5b–e). Similar to its effect in E0771 breast cancer, dapagliflozin slowed MC38 tumor growth: 4 weeks after tumor implantation, tumor size was reduced by 50% in dapagliflozin-treated obese mice; however, the effect of dapagliflozin to slow tumor growth was reversed by restoring hyperinsulinemia (Fig. 5f).

Fig. 5 Dapagliflozin slows MC38 colon adenocarcinoma tumor growth in an insulin-dependent manner. a, b Urine and plasma glucose concentrations. In all panels, mice were fasted for 5 h before studies were performed. c Plasma insulin concentrations. d, e Tumor glucose uptake and V PDH /V CS . f Tumor size. *P < 0.05, **P < 0.01, ***P < 0.001 vs. chow; +P < 0.05, ++P < 0.01, +++P < 0.001 vs. HFD + dapagliflozin. The color of the symbols indicates the group compared to the group designated by the symbols. In all panels, data are the mean ± S.E.M. of n = 5–6 per group, with groups compared by ANOVA with Bonferroni’s multiple comparisons test Full size image

Next, we explored whether SGLT2 inhibitors exert a direct effect to alter glucose metabolism or cell division in MC38 cells at pharmacologically relevant concentrations. While dapagliflozin did not alter glucose uptake, V PDH /V CS , or cell division at concentrations in the range of those measured in patients treated with the maximum daily dose of the drug (0.5 μM), but reduced all three parameters at a suprapharmacologic concentration (5 mM), canagliflozin reduced both glucose uptake and oxidation at pharmacologically relevant concentrations (Fig. 6a–c). Because SGLT2 inhibitors can cause ketoacidosis [57], albeit typically not in the well-hydrated state [58], and a ketogenic diet may slow tumor growth, at least in animals [59], we then asked whether ketones themselves may alter tumor growth. In contrast to insulin, which promoted MC38 cell division in vitro at supraphysiologic concentrations, incubation in physiologic (1 mM) concentrations of β-hydroxybutyrate (β-OHB) had no impact on cell division (Fig. 6d). Finally, we examined the potential impact of ketones themselves to alter MC38 tumor growth in vivo and found that chronic (4 weeks) ketone supplementation in drinking water had no impact on plasma glucose or insulin concentrations or on tumor size, despite doubling plasma β-OHB concentrations (Fig. 6e, f, Additional file 1: Figure S3M-N).

Fig. 6 Neither SGLT2 inhibitors nor β-OHB directly alter MC38 tumor cell division. a Impact of SGLT2 inhibitors and insulin on MC38 cell glucose uptake in vitro. In panels a–d, n = 4 in vitro replicates, with groups compared by ANOVA with Bonferroni’s multiple comparisons test, in which each group was compared to the vehicle-treated cells (**P < 0.01, ***P < 0.001, ****P < 0.0001). b V PDH /V CS . c Cell division. d Impact of β-OHB and insulin on cell division. e Plasma β-OHB concentrations after 4 weeks of β-OHB supplementation in drinking water, which was removed 2 h before plasma was obtained. f Tumor size. In panels e and f, n = 8 per group, with groups compared by the two-tailed unpaired Student’s t test. All data are presented as the mean ± S.E.M Full size image

Tumor insulin signaling changes dynamically after a meal

The fact that insulin promotes tumor growth in vivo, associated with increased tumor glucose uptake and oxidation, and that two agents that lower both fasting and postprandial insulin concentrations strikingly slow both E0771 and MC38 tumor growth, suggest that tumor insulin signaling may be dynamically regulated. To test this possibility, we performed an oral glucose tolerance test in MC38 tumor-bearing mice and observed a transient postprandial increase in phosphorylation of Akt in tumor (pSer473), which was slightly delayed as compared to plasma insulin concentrations, and an increase in tumor pThr389 p70 S6K (Fig. 7a, b, Additional file 1: Figure S5A-B), demonstrating that tumor insulin signaling indeed is acutely altered in response to normal physiological changes in insulin concentrations.