Fasting inhibits aerobic glycolysis and proliferation in colorectal cancer via the Fdft1-mediated AKT/mTOR/HIF1α pathway suppression

Abstract

Evidence suggests that fasting exerts extensive antitumor effects in various cancers, including colorectal cancer (CRC). However, the mechanism behind this response is unclear. We investigate the effect of fasting on glucose metabolism and malignancy in CRC. We find that fasting upregulates the expression of a cholesterogenic gene, Farnesyl-Diphosphate Farnesyltransferase 1 (FDFT1), during the inhibition of CRC cell aerobic glycolysis and proliferation. In addition, the downregulation of FDFT1 is correlated with malignant progression and poor prognosis in CRC. Moreover, FDFT1 acts as a critical tumor suppressor in CRC. Mechanistically, FDFT1 performs its tumor-inhibitory function by negatively regulating AKT/mTOR/HIF1α signaling. Furthermore, mTOR inhibitor can synergize with fasting in inhibiting the proliferation of CRC. These results indicate that FDFT1 is a key downstream target of the fasting response and may be involved in CRC cell glucose metabolism. Our results suggest therapeutic implications in CRC and potential crosstalk between a cholesterogenic gene and glycolysis.

Fig. 1. Fasting impairs glycolysis and proliferation of CT26 cells in vitro and in vivo.

Fasting inhibited CT26 cell proliferation as measured by a CCK8 assay (from left to right: P = 0.0009; P = 0.0006). b Cell proliferation was also evaluated using EdU immunofluorescence staining. Proliferating cells were labeled with EdU. n = 3; scale bar: 100 µm. c Bioinformatics analysis of differentially expressed genes identified via iTRAQ proteomics. We magnified the genes related to the glycolysis pathway particularly. d The graph shows the relative fold fraction of EdU-positive cells. e Fasting reduced glucose uptake in CT26 cells. f Fasting decreased lactate production via glycolysis in CT26 cells. g Fasting downregulated the expression of rate-limiting glycolytic enzymes in glucose metabolism (GLUT1, HK2, LDHA, PGK1, and GPI) by western blot. h ECAR, an indicator of glycolysis, was reduced in CT26 cell cultured in the fasting mimic medium. i OCR, which reflects mitochondrial respiration, was increased in CT26 cell cultured in the fasting mimic medium. j CT26 cells were injected into BALB/c mice. When the tumors were palpable, the mice were randomly assigned to the control group or the fasting mimic diet (FMD) group. Photograph of dissected tumors (upper: FMD group; lower: control group; n = 5). k Fasting downregulated the transcription of rate-limiting glycolytic enzymes in glucose metabolism (GLUT1, HK2, LDHA, PGK1, and GPI) by qRT-PCR. l The tumor volumes were measured every day after the 9th day. The FMD attenuated tumor growth in mice (n = 5). m, o Tumor weights and tumor volumes on the 25th day (n = 5; P = 0.001; P = 0.0292). n Representative ¹⁸F-FDG microPET/CT imaging of tumor-bearing mice (n = 3) (upper: control group; lower: FMD group). The tumors are indicated with arrows. p The ratio of the tumor SUVmax in the control group and the FMD group (n = 3; P = 0.0459). Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.

Fig. 2. Fasting upregulates the level of FDFT1, which is correlated with prognosis in CRC.

a The expression of FDFT1 was increased significantly in the fasting group compared with that in the control group in the GSE60653 data set (n = 3). b The relative expression of FDFT1 was also increased greatly in the fasting group compared with that in the control group by iTRAQ (n = 3; P = 0.0319). c The mRNA expression of FDFT1 in dissected tumor tissue from the fasting mimic group and the control group was measured by qRT-PCR (n = 15; P < 0.0001). d, e Fasting mimic medium also increased the protein level of FDFT1 in CT26 and SW620 cells. f Representative graph of the IHC analysis carried out in human CRC and noncancerous tissues (n = 23; upper: scale bar is 200 µm; lower: scale bar is 100 µm). g The expression of FDFT1 was downregulated in most of the tumor tissues (19/23), but was upregulated in most of the adjacent noncancerous tissues (18/23) (n = 23; P = 0.0004). h The relative expression levels of FDFT1 mRNA in CRC tissues and matched adjacent noncancerous tissues were determined by qRT-PCR (n = 81; both P < 0.0001). i Kaplan–Meier analysis of the overall survival of patients with CRC in the FUSCC cohort according to FDFT1 expression. The median expression level was used as the cutoff. High FDFT1 expression predicted better prognoses for CRC patients in the FUSCC cohort. (high FDFT1 patients = 39, low FDFT1 patients = 42; P = 0.0238, log-rank test) j, k The expression of the FDFT1 gene was significantly lower in CRC tissues than in normal tissues in the GDS2609 and GDS4382 data sets (P < 0.0001; P = 0.0049). l Analysis of the correlation of FDFT1 expression with TNM stage in CRC patients. Lower FDFT1 expression was correlated with higher TNM stage (P = 1.91 × 10⁻⁵). m Survival analysis of FDFT1 data from the TCGA database stratified by FDFT1 expression. High FDFT1 expression indicated a better prognosis. (P = 0.018, log-rank test). Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. Box denotes 25th to 75th percentile, horizontal bar is median in h, j, and k. Kaplan–Meier analysis and log-rank tests were used in panels i, m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group (or non-tumor/normal tissue).

Fig. 3. FDFT1 negatively regulates the proliferation of CRC cells.

a, b The efficiency of FDFT1 overexpression in CT26 cells was measured by qRT-PCR and western blotting (P < 0.0001). c, d The efficiency of FDFT1 knockdown in CT26 cells was measured by qRT-PCR and western blotting (both P < 0.0001). e FDFT1 overexpression inhibited CT26 cell proliferation as measured by a CCK8 assay (P = 0.0006). f FDFT1 knockdown increased CT26 cell proliferation as measured by a CCK8 assay (sh1 vs con: P = 0.0008; sh3 vs con: P = 0.0009). g Cell proliferation in control and FDFT1-overexpressing CT26 cells was also evaluated using EdU immunofluorescence staining. Scale bar: 100 µm. h The graph shows the relative fold fraction of EdU-positive cells. i Cell proliferation was also evaluated in control and FDFT1 knockdown CT26 cells using EdU immunofluorescence staining. Scale bar: 100 µm. j The graph shows the relative fold fraction of EdU-positive cells. k FDFT1 overexpression decreased the colony-forming capacity of CT26 cells as measured by a colony-formation assay. l The graph shows the statistical results of the colonies. m FDFT1 knockdown increased the colony-forming capacity of CT26 cells as measured by a colony-formation assay. n The graph shows the statistical results of the colonies (from left to right: P = 0.0003; P = 0.0006). o FDFT1 overexpression inhibited CT26 cell invasion as measured by a Transwell assay. Scale bar: 100 µm. p The graph shows the number of invaded cells. q FDFT1 knockdown increased CT26 cell invasion as measured by a Transwell assay. Scale bar: 100 µm. r The graph shows the number of invaded cells. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ****P < 0.0001, compared with the control group.

Fig. 4. FDFT1 is a downstream target of fasting in suppressing CRC proliferation.

a FDFT1 overexpression and fasting for 48 h inhibited CT26 cell proliferation as measured by a CCK8 assay. Compared with either treatment alone, FDFT1 overexpression combined with fasting for 48 h had the most obvious inhibitory effect on CT26 cell proliferation (from left to right: P = 0.0021; P = 0.0005; P = 0.0003). b Western blotting indicated that fasting 48 h and FDFT1 overexpression increased the protein level of FDFT1 in CRC cells. Fasting exerted an additive effect on FDFT1 expression level in cells overexpressing FDFT1 in the suppression of CRC cell proliferation. c, d Photograph of dissected tumors (first line: CT26 cells + normal diet; second line: CT26 cells + FMD; third line: FDFT1-overexpressing CT26 cells + normal diet; fourth line: FDFT1-overexpressing CT26 cells + FMD; n = 4; both P < 0.0001). Both the FMD and FDFT1 overexpression inhibited tumor growth in the mice. The FMD combined with the implantation of FDFT1-overexpressing CT26 cells had the most obvious inhibitory effect on tumor growth in the mice. e Representative ¹⁸F-FDG microPET/CT imaging of tumor-bearing mice. f The ratio of the tumor SUVmax in the four groups. The SUVmax was decreased most significantly in the FDFT1-overexpressing CT26 cells + FMD group (n = 3; from left to right: P = 0.0018; P = 0.0018; P = 0.0003). g The protein expression of FDFT1 in dissected tumor samples was evaluated by IHC. Scale bar: 100 µm. h Graph shows the quantitative analysis of FDFT1 staining (n = 3). i The effect of FDFT1 knockdown, fasting 48 h and FDFT1 knockdown combined with fasting 48 h on CT26 cell proliferation was evaluated by CCK8 (upper: P = 0.005; lower: P = 0.0045). j, k Photograph of dissected tumors (first line: CT26 cells + normal diet; second line: CT26 cells + FMD; third line: shFDFT1 CT26 cells + normal diet; fourth line: shFDFT1 CT26 cells + FMD; n = 4; P = 0.0006; P = 0.0002; P = 0.0003). The FMD inhibited tumor growth in mice. FDFT1 knockdown promoted tumor growth in mice. The FMD combined with shFDFT1 CT26 cells did not inhibit tumor growth in the mice. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group (or normal diet group). ^#P < 0.05, ^##P < 0.01.

Fig. 5. mTOR expression is inversely correlated with FDFT1 expression.

a FDFT1 overexpression inhibited the protein level of mTOR, whereas FDFT1 knockdown increased the protein level of mTOR in CT26 and SW620 cells. b FDFT1 expression and mTOR expression were negatively correlated in CRC patient samples. Scale bar: 200 µm. c The mTOR silencing efficiency of the siRNA in CT26 and SW620 cells was validated by western blotting. d The effect of mTOR silencing on FDFT1 expression level in CT26 was evaluated by western blotting. e The protein level of mTOR when FDFT1 knockdown combined with or without mTOR inhibitor in CT26. f The protein level of pS6k, S6k, pS6, and S6 under the effect of fasting and FDFT1 overexpression in CRC cells. g, h CCK8 proliferation assays showed that the silencing of mTOR decreased the proliferation of CT26 and SW620 cells. k, n The silencing of mTOR reduced glucose uptake and lactate production in CT26 and SW620 cells. i, j, l, o The silencing of mTOR decreased the ECAR and increased the OCR in CT26 and SW620 cells. m The silencing of mTOR decreased the expression of AKT, HIF1α, and proteins encoded by relevant glycolytic genes, such as GLUT1, HK2, LDHA, GPI, PGK1, in CT26 and SW620 cells. p–t Based on TCGA data set analysis, mTOR expression was positively correlated with AKT1, HIF1α, GLUT1, HK2, and LDHA expression. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, compared with the control group.

Fig. 6. FDFT1 inhibits glycolysis through suppressing the AKT-mTOR-HIF1α pathway in CRC.

a FDFT1 overexpression reduced glucose uptake in CT26 cells. b FDFT1 overexpression decreased lactate production via glycolysis in CT26 cells. c ECAR was reduced when FDFT1 was overexpressed in CT26 cells. d OCR was increased when FDFT1 was overexpressed in CT26 cells. e FDFT1 knockdown increased glucose uptake in CT26 cells. f FDFT1 knockdown increased lactate production via glycolysis in CT26 cells. g ECAR was increased when FDFT1 was knocked down in CT26 cells. h OCR was decreased when FDFT1 was knocked down in CT26 cells. i, m FDFT1 overexpression inhibited the protein and mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, and LDHA, in CT26 cells. j, m FDFT1 knockdown increased the protein and mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, and LDHA, in CT26 cells. k, n FDFT1 overexpression decreased the protein and mRNA expression of AKT, mTOR, and HIF1α. l, n FDFT1 knockdown increased the protein and mRNA expression of AKT, mTOR, and HIF1α. o Photograph of dissected tumors (the first line: normal diet, the second line: FMD + glucose, the third line: FMD, n = 5). p The tumor volumes were measured every 2 days after the 13th day. The FMD + glucose group can reverse the tumor growth inhibition induced by the FMD (n = 5; ns: P = 0.1838; P = 0.0001). q The protein level of FDFT1 and mTOR in dissected tumor samples from normal diet group, FMD group and FMD + glucose group was measured by western blotting. r The glucose level in these three groups. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group (or normal diet group). ^#P < 0.05, ^##P < 0.01.

Fig. 7. Fasting and mTOR inhibitor synergize in suppressing CRC proliferation and clinical significance of the FDFT1/AKT-mTOR-HIF1α pathway in CRC patients.

a CT26 cells were injected into BALB/c mice. When the tumors were palpable, the mice were randomly assigned to the normal diet group, FMD group, the rapamycin 1 mg/kg group and FMD + rapamycin 1 mg/kg group. Photograph of dissected tumors (the first line: normal diet, the second line: FMD, the third line: rapamycin 1 mg/kg, the fourth line: FMD + rapamycin 1 mg/kg, n = 5). b The tumor volumes were measured every 3 days after the 9th day (n = 30; ***P = 0.0008, P = 0.0003; ^#P = 0.0133). On day 9 after inoculation, all the tumor were palpable. c Kaplan–Meier analysis of the overall survival of mice after the inoculation in normal diet group, FMD group, normal diet mice treated with rapamycin 1 mg/kg group and FMD + rapamycin 1 mg/kg group (n = 30; log-rank score: P = 0.0049 for FMD group, P = 0.0058 for rapamycin 1 mg/kg group; P = 0.00069 for FMD + rapamycin 1 mg/kg group.) d, e The expression level of FDFT1 in four groups was evaluated by western blotting and qRT-PCR (**P = 0.0025, P = 0.0097; ***P = 0.0008; ^#P = 0.0133, P = 0.0351). f–j Survival analysis stratified by combining FDFT1 levels with AKT1, mTOR, HIF1α, GLUT1, and HK2 levels from CRC patients in the TCGA cohort. k Proposed model of the mechanism underlying the fasting-mediated regulation of glucose metabolism via the FDFT1/AKT-mTOR-HIF1α axis in colorectal cancer. Fasting upregulates the expression of FDFT1 during the inhibition of colorectal cancer cell aerobic glycolysis and proliferation. FDFT1, whose downregulation is correlated with malignant progression and poor prognosis in CRC, acts as a critical tumor suppressor in CRC. We then observed that FDFT1 is an important downstream target of fasting that mediates the inhibition of CRC cell proliferation. Mechanistically, FDFT1 inhibits the AKT-mTOR-HIF1α pathway, impairing aerobic glycolysis, and thereby suppressing the proliferation of CRC cells. There is also a reverse regulation of FDFT1 by mTOR. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. Kaplan–Meier analysis and log-rank tests were used in panel c. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, compared with normal diet group. ^#P < 0.05, ^##P < 0.01.