Fasting-mimicking diet blocks triple-negative breast cancer and cancer stem cell escape

Abstract

Metastatic tumors remain lethal due to primary/acquired resistance to therapy or cancer stem cell (CSC)-mediated repopulation. We show that a fasting-mimicking diet (FMD) activates starvation escape pathways in triple-negative breast cancer (TNBC) cells, which can be identified and targeted by drugs. In CSCs, FMD lowers glucose-dependent protein kinase A signaling and stemness markers to reduce cell number and increase mouse survival. Accordingly, metastatic TNBC patients with lower glycemia survive longer than those with higher baseline glycemia. By contrast, in differentiated cancer cells, FMD activates PI3K-AKT, mTOR, and CDK4/6 as survival/growth pathways, which can be targeted by drugs to promote tumor regression. FMD cycles also prevent hyperglycemia and other toxicities caused by these drugs. These data indicate that FMD has wide and differential effects on normal, cancer, and CSCs, allowing the rapid identification and targeting of starvation escape pathways and providing a method potentially applicable to many malignancies.

Keywords: CDK4/6; PI3K/AKT; PKA; cancer stem cells; fasting; fasting-mimicking diet; glucose; mTOR; starvation escape pathways; triple-negative breast cancer.

Conflict of interest statement

Declaration of interests V.D.L. is a scientific advisor and has equity interest in L-Nutra, a company that develops medical food. V.D.L. and G.S. have submitted a patent related to this work. C.V. is an inventor of an FMD regimen (patent pending).

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. FMD reduces mammosphere growth, the expression of CSC markers, and stem cell frequency in SUM159 human triple-negative breast cancer (TNBC), and its effect is potentiated by 2-deoxy-D-glucose

(A) SUM159 tumor masses were processed for ex vivo primary mammosphere (n = 7–9) and ex vivo serial sphere-forming assay (n = 4). (B) Aldefluor analyses were performed by flow cytometry using the ALDEFLUOR kit to measure ALDH1 expression in SUM159 tumor masses (n = 5). (C) Growth of SUM159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL diet or 5 cycles of FMD alone or plus 3% glucose supplementation in drinking water (n = 9). (D) Tumor masses were excised and processed for ex vivo primary mammosphere assay (n = 9). (E) Growth of SUM159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL diet or 5 cycles of FMD alone or combined with WZB117 (10 mg/kg) once a day intraperitoneally (i.p.) (n = 6). (F) Tumor masses were excised and processed for ex vivo primary mammosphere assay (n = 6). (G) Growth of SUM159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL diet or 5 cycles of FMD alone or combined with 2DG (200 mg/kg) once a day i.p. (n = 15). (H) Tumor masses were excised and processed for ex vivo primary mammosphere assay (n = 10–15) and serial mammosphere assay (n = 5). (I) SUM159 tumor cells derived from in vivo xenografts were injected in recipient mice at different dilutions to perform the limiting dilution assay (n = 6–14). p values were determined by log-rank (Mantel-Cox)test (100,000 cells: ad libitum versus FMD, p =0.0024; ad libitum versus 2DG, p = 0.0660; ad libitum versus FMD + 2DG, p = 0.0008; FMD versus 2DG, p = 0.1007; FMD versus FMD + 2DG, p = 0.1657; 2DG versus FMD + 2DG, p = 0.0177; 10,000 cells: ad libitum versus FMD, p = 0.0011; ad libitum versus 2DG, p = 0.0003; ad libitum versus FMD + 2DG, p < 0.0001; FMD versus 2DG, p = 0.0428; FMD versus FMD + 2DG, p = 0.3120; 2DG versus FMD + 2DG, p = 0.0192; 1,000 cells: ad libitum versus FMD, p = 0.0891; ad libitum versus 2DG, p = 0.3981; ad libitum versus FMD + 2DG, p = 0.0001; FMD versus 2DG, p = 0.4714; FMD versus FMD + 2DG, p = 0.0123; 2DG versus FMD + 2DG, p = 0.0067). The stem cell frequency was calculated using ELDA software. Data are represented as mean ± SEM. p values were determined by two-tailed unpaired t test (A and B) and one-way ANOVA (C–H). See also Figures S1 and S2.

Figure 2.. FMD acts independently of the immune system and reduces metastasis in syngeneic TNBC models

(A) Growth of 4T1-luc cells in the mammary fat pad of 6-week-old female Balb-c mice treated with AL diet or 4 cycles of FMD. Tumor masses were excised and processed for ex vivo primary mammosphere-forming assay (n = 7). (B) 4T1 cells were grown under CTR (1 g/L glucose, 10% FBS), STS (0.5 g/L, 1% FBS), and STS + 1 g/L glucose conditions for a total of 48 h. Cells were then plated to perform the in vitro sphere-forming assay (n = 4 biological replicates). (C) Growth of 4T1-luc cells in the mammary fat pad of 6-week-old female Balb-c mice treated with AL diet or 4 cycles of FMD alone or combined with 2DG (200 mg/kg) once a day i.p. (n = 15 per group). (D) Fluorescence-activated cell sorting (FACS) analysis was performed to measure CD44 and CD24 expression in 4T1 TNBC in vivo (n = 8). (E) Aldefluor analysis was performed by flow cytometry using the ALDEFLUOR kit to measure ALDH1 expression in 4T1 tumor masses (n = 4–5). (F) Growth of 4T1-luc cells intravenously in 6-week-old female Balb-c mice treated with AL diet or 4 cycles of FMD alone or combined with 2DG (200 mg/kg) oncea day i.p. Tumor progression was monitored with bioluminescent imaging 17 days post-cell injection (n = 5 per group). Data are represented as mean ± SEM. p values were determined by two-tailed unpaired t test (A) and one-way ANOVA (B–E). See also Figure S3.

Figure 3.. Hyperglycemia is associated with worse overall survival in advanced TNBC patients

(A) Kaplan-Meier curves for overall survival (OS) according to baseline blood glucose levels in advanced TNBC patients treated with first-line platinum-based doublet chemotherapy. Normoglycemia and hyperglycemia were defined according to 100 mg/dL threshold. The + symbol in Kaplan Meier curves indicates patients who were censored at the time of data cut-off analysis. (B) Multivariable analysis adjusting the impact of hyperglycemia on OS according to clinical characteristics previously selected on the basis of univariate analysis. For each covariate the hazard ratio (HR), 95% confidence interval (CI), and p value are indicated. CIs not crossing the value of 1 indicate a statistically significant impact of that variable on patient OS. The p value is indicated in bold when statistically significant. See also Figure S4.

Figure 4.. PKA is downregulated by FMD selectively in CSCs

(A) Detection of phosphorylated CREB levels and total CREB, KLF5, and VINCULIN, as loading control, in SUM159 and 4T1 tumor masses (n = 5). (B) Volcano plot showing the significance versus the log2 fold change between CD44+CD24– and CD44+CD24+. Up- and downregulated genes (|log2FC| > 0.58 and adj. p < 0.05) are displayed in red and green, respectively. Deregulated genes involved in the PKA pathway are highlighted. (C) Detection of phosphorylated CREB levels and total CREB, KLF5, and VINCULIN, as loading control, in SUM159 CD44+CD24+ and CD44+CD24– subpopulations. (D) Detection of phosphorylated CREB levels and total CREB, KLF5, and VINCULIN, as loading control, in 4T1 CD44+CD24+ and CD44+CD24– subpopulations. Data are represented as mean ± SEM. p values were determined by two-tailed unpaired t test (A) and multiple unpaired t test (C and D). See also Figure S5.

Figure 5.. PKA activation through 8Br-cAMP reverses STS-dependent sphere reduction and CD44+CD24– cell lowering

(A and B) SUM159 and 4T1 cells were grown under CTR (1 g/L glucose, 10% FBS) and STS (0.5 g/L, 1% FBS) conditions for a total of 48 h. At 24 h cells were treated with 8-Br-cAMP. Cells (A) were then plated to perform the in vitro sphere-forming assay (n = 4–6 biological replicates) or (B) were used to perform flow cytometry analysis to measure the percentage of CD44+CD24+ and CD44+CD24– cells in SUM159 or 4T1 TNBC models (n = 6–9 biological replicates). (C) Detection of G9A, H3meK9 levels, and VINCULIN, as loading control, in SUM159 and 4T1 tumors (n = 7–8). Data are represented as mean ± SEM. p values were determined by two-tailed unpaired t test (C) and one-way ANOVA (A and B).

Figure 6.. FMD activates starvation escape mechanisms that can be targeted by drugs

(A) Volcano plot showing the significance versus the log2 fold change in SUM159 tumor masses, by comparing FMD versus AL. Up- and downregulated genes (|log2FC| > 0.58 and adj. p

Figure 7.. FMD reverts late-stage TNBC progression by potentiating the effect of kinase inhibitors and reversing the lethality caused by hyperglycemia

(A) Growth of SUM 159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL dietor FMD, alone or combined with pictilisib (100 mg/kg, 5 consecutive days a week by oral gavage), ipatasertib (75 mg/kg, 5 consecutive days a week by oral gavage), and rapamycin (2 mg/kg, every other day i.p.) (n = 10–13). Progression-free survival was monitored over time and causes of death are reported. (B) Blood glucose level was determined through Accu chek guide instrument. (C) Growth of SUM159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL diet or FMD, alone or combined with palbociclib (62.5 mg/kg, byoral gavage, every other day) and pictilisib (100 mg/kg, by oral gavage, 5 consecutive days a week) (n = 10). Tumor masses were used to perform the ex vivo sphere-forming assay (n = 5). (D) Growth of SUM159 xenografts in 8-week-old female NOD scid (NSG) mice treated with AL diet or FMD. Thirty-five days post-cell injection, mice started to be treated with pictilisib (100 mg/kg, 5 consecutive days a week by oral gavage), ipatasertib (75 mg/kg, 5 consecutive days a week by oral gavage), and rapamycin (2 mg/kg, every other day i.p.) plus FMD (n = 15). (E) Putative model for the effect of FMD on SUM159 TNBC cells and CSCs. Data are represented as mean ± SEM. p values were determined by two-tailed unpaired t test (C). See also Figure S7.