Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo

Abstract

Background: Despite substantial progress made in the last decades in colorectal cancer (CRC) research, new treatment approaches are still needed to improve patients' long-term survival. To date, the promising strategy to target tumor angiogenesis metabolically together with a sensitization of CRC to chemo- and/or radiotherapy by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) inhibition has never been tested. Therefore, initial evaluation and validation of newly developed compounds such as KAN0438757 and their effects on CRC cells are crucial steps preceding to in vivo preclinical studies, which in turn may consolidate new therapeutic targets.

Materials and methods: The efficiency of KAN0438757 to block PFKFB3 expression and translation in human CRC cells was evaluated by immunoblotting and real-time PCR. Functional in vitro assays assessed the effects of KAN0438757 on cell viability, proliferation, survival, adhesion, migration and invasion. Additionally, we evaluated the effects of KAN0438757 on matched patient-derived normal and tumor organoids and its systemic toxicity in vivo in C57BL6/N mice.

Results: High PFKFB3 expression is correlated with a worse survival in CRC patients. KAN0438757 reduces PFKFB3 protein expression without affecting its transcriptional regulation. Additionally, a concentration-dependent anti-proliferative effect was observed. The migration and invasion capacity of cancer cells were significantly reduced, independent of the anti-proliferative effect. When treating colonic patient-derived organoids with KAN0438757 an impressive effect on tumor organoids growth was apparent, surprisingly sparing normal colonic organoids. No high-grade toxicity was observed in vivo.

Conclusion: The PFKFB3 inhibitor KAN0438757 significantly reduced CRC cell migration, invasion and survival. Moreover, on patient-derived cancer organoids KAN0438757 showed significant effects on growth, without being overly toxic in normal colon organoids and healthy mice. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy.

Keywords: KAN0438757; PFKFB3; colon cancer; glycolysis; intestinal organoids; rectal cancer.

Figure 1

PFKFB3 expression in human colon and rectal cancer. (A,B) PFKFB3 mRNA expression on colonic and rectal normal mucosa versus colonic and rectal tumors. (C,D) Kaplan–Meier plots for colon cancer overall survival (patients n = 475), and rectal cancer disease-free survival (patients n = 207), respectively, **** p < 0.0001.

Figure 2

KAN0438757 effects on PFKFB3 expression in cancer cells and HUVECs. (A) KAN0438757 synthetization protocol steps. (B) Immunoblot. PFKFB3 inhibition in colorectal cancer cells and HUVECs upon 12 h treatment with KAN0438757. (C) Immunoblot relative quantification by densitometry (relative to B-ACTIN loading control). (D) RT-PCR analysis. Relative mRNA expression of PFKFB3 in HCT-116, HT-29 and HUVEC cells upon 4, 12, 24 and 48 h treatment with KAN0438757. Data is $\pm$ SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 3

KAN0438757 effectively reduces glycolytic metabolites’ levels. (A) Glycolysis schematic representation. Red arrows mark the metabolites directly affected by KAN0438757 treatment in vitro. (B) LC-MS metabolomic analysis for HCT-116 cancer cells and HUVECs after 6h treatment with KAN0438757. (C) Seahorse analysis for oxygen consumption rate (OCR) evaluation performed with colorectal cancer cells and HUVECS upon 6 h treatment with KAN0438757. (C) Data is representative of three independent experiments. Data is ± SEM. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.0001.

Figure 4

PFKFB3 inhibition by KAN0438757 reduces cancer cell proliferation and induces cell death in a concentration-dependent manner. (A–D) xCELLigence assay performed with HCT-116 and HT-29 colorectal cancer cells, HUVECs and normal colon epithelial cells during 96 h under different concentrations of KAN0438757 (continuous line); the dotted lines represent the fitted saturation curve in logarithm. (E) CellTiterBlue assay performed with HCT-116, HT-29, HUVECs and normal colon epithelial cells during 48 h. (F) LDH assay performed with HCT-116, HT-29, HUVECs and normal colon epithelial during 48 h. Data is $\pm$ SEM. (A–F) Combined data from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

KAN0438757 effects on intestinal patient-derived organoids. (A,B) Representative pictures of normal colon (mucosa) and tumor patient-derived organoids, treated with 30 μM KAN0438757 for 24 h and their size quantification (right-side panel). Scale bars, 200 μm. Data is ± SEM. * p < 0.05; ** p < 0.01; ns, p > 0.05.

Figure 6

PFKFB3 inhibition by KAN0438757 alters cell motility and cancer cell invasion capabilities. (A) Representative pictures of migration assay performed with 25 μM KAN0438757. (B) Migration assay quantification performed with HCT-116, HT-29 and HUVEC cells under different concentrations of KAN0438757 for 42 h (cancer cells) and 12 h HUVEC cells. (C) Boyden chamber invasion assay quantification performed with HCT-116 and HT-29 cancer cells for 96 h. (D) RT-PCR analysis for cell migration-and cytoskeleton-associated genes in HUVECs. Data is $\pm$ SEM. (B,C) combined data from three independent experiments. (D) data is representative of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, p > 0.05.

Figure 7

In vivo toxicity testing of KAN0438757 in immune-competent mice. (A) Relative weight curves of C57BL6/N mice treatment with 10 mg/kg (n = 3), 25mg/kg (n = 3) and 50 mg/kg (n = 3) KAN0438757 (arrows, i.p. injections). Control, DMSO (n = 3). (B,C) Whole blood counts (RBC, WBC, thrombocytes) and serum biochemical evaluation of aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), creatinine and lactate after treatment periods of 24 h to 21 days with KAN0438757. (D) Representative pictures of histological analysis (H&E) of lungs, heart, liver, spleen, kidneys, adrenal glands, duodenum and colon from mice treated 21 days (21 d) with KAN0438757 (25 mg/kg dosage). * p < 0.05; Data is $\pm$ SEM.