Exploiting choline kinase activity for detection of anticancer drug pharmacodynamics: investigation of physiologic and pharmacological intervention
Creators: name: Li, Yunqing
Publisher: name: Imperial College London
Year: 2020

description: Overexpression of choline kinase α (CHKA) is associated with cancer malignant transformation. We endeavoured to employ CHKA activity as a pharmacological biomarker for anticancer drug intervention. A choline-based radiotracer, [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH), is employed to monitor CHKA activity, as the cell uptake level of 18F-D4-FCH in vitro is largely dependent on phosphorylation to [18F]-fluoromethyl-[1,2-2H4]-phosphocholine (18F-D4-FCHP) by CHKA, and transport of radio-choline content (18F-D4-FCH and 18F-D4-FCHP). Of note, the physiologic context, including hypoxia, needs to be considered when using choline-based tracers. Hypoxia is a complex microenvironmental condition known to regulate CHKA activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α), and therefore may confound 18F-D4-FCH uptake. In this thesis, three underlying mechanisms by which hypoxia alters 18F-D4-FCH uptake were investigated, which were 18F-D4-FCH import, CHKA phosphorylation activity, and efflux of 18F-D4-FCH and 18F-D4-FCHP. HIF-1α-dependent regulation of CHKA was initially studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer, MDA-MB-231, using CoCl2 that stabilises HIF-1α level. CoCl2 treatment or hypoxic exposure did not change CHKA protein level in both cell lines, but CoCl2 treatment reduced the overall uptake of 18F-D4-FCH in PC-3 cells by 50%, in contrast to the non-significant change of 18F-D4-FCH uptake in MDA-MB-231 cells. The reduction of 18F-D4-FCH uptake in PC-3 cells was linked to diminished accumulation of the dominant radio-choline species, 18F-D4-FCHP, through reduction of CHKA activity and/or increase of efflux of radioactivity. A novel finding was described that a greater proportion of 18F-D4-FCHP than 18F-D4-FCH was exported via HIF-1α-responsive efflux transporters including ABCB4 when HIF-1α level augmented. Efflux blockade with A non-specific ABCB4 inhibitor, cyclosporin A (cys A), was used to block HIF-1α-activated efflux of 18F-D4-FCH and 18F-D4-FCHP (particularly the latter). Cys A was envisaged to largely increase the retention of radio-choline content, thus addressing the confounding effects of HIF-1α on 18F-D4-FCH uptake. The efflux blockade with cys A might aid in 18F-D4-FCH-oriented assessments of anticancer therapy that targets CHKA-associated effectors. Pharmacological responses to EGFR- or c-Src-targeted treatment with gefitinib or dasatinib were monitored using 18F-D4-FCH, based on the functional interaction of CHKA with EGFR and c-Src. Cys A was initially tested under gefitinib treatment; it increased 18F-D4-FCH uptake in ABCB4-overexpressing non-small-cell lung cancer (NSCLC) cells following gefitinib treatment but decreased the uptake in gefitinib-treated NSCLC xenografts. The difference between in vitro and in vivo findings underscores the need for investigating the function of cys A in imaging with 18F-D4-FCH.18F-D4-FCH was also exploited to monitor the interplay between CHKA and androgen receptor in androgen-sensitive prostate cancer cell lines. The 18F-D4-FCH uptake level was not correlated with androgen dependence or sensitivity to co-treatment with CHKA inhibitor and androgen deprivation therapy across prostate cancer cell lines. In summary, further characterisation of mechanisms of choline transport and CHKA activity may be needed for utilising 18F-D4-FCH as a pharmacodynamic marker.