The personalised road to anticancer therapy: utilizing the LAT1 transporter as a selective means for iron deprivation

Abstract

Cancer cells undergo metabolic reprogramming to meet the demands of uncontrolled proliferation. Aberrant iron metabolism is commonly observed across many types of cancer - including breast, ovarian and prostate – manifesting in an upregulation of iron import and a downregulation of iron export, thereby allowing maximum iron accumulation. To counteract aberrant iron metabolism our group designed and synthesised SK4 – a novel iron chelator. SK4 consists of two key moieties: an iron chelation moiety and an amino acid moiety to allow entry through LAT1: an amino acid transporter which is highly expressed in many cancer tissues. SK4 was therefore hypothesised to represent a personalised therapy for LAT1 overexpressing tumours. The aims of this thesis were to: characterise if LAT1 protein expression levels correlate with SK4 sensitivity and therefore serving as a potential biomarker for sensitivity to SK4, investigate the mode of action of SK4 and determine if tumour suppressor NDRG1 is involved in the mechanism of action.

panel of cell lines representative of different tissues were treated with increasing concentrations of SK4 to determine their sensitivity.
SK4 displayed GI50 values ranging from 51.8 - >500μM and IC50s ranging from 25.1- >500μM generated using Sulphorhodamine B assays and cell counting respectively. Normal cell lines were more sensitive to SK4 than cancer cell lines with the most sensitive cell line being MCF10A cells (normal breast) and the least sensitive being the normal human dermal fibroblasts (NHDF). Overall, the pattern of sensitivity was MCF10A > PNT1A (normal prostate) > IOSE397 (normal ovarian) > HeLa (cervical cancer) > PC3 (prostate cancer) > SKOV3 (ovarian cancer) > IOSE80 (normal ovarian) > DU145 (prostate cancer) > MDA MB 231 (breast cancer) > HDF. LAT1 protein expression poorly correlated with SK4 sensitivity demonstrating LAT1 expression alone is not sufficient as a biomarker. To validate SK4 structure activity I used control compounds SK4C1 and SK4C2 each lacking a key moiety, with SK4C1 lacking the iron chelation moiety and SK4C2 lacking the amino acid moiety which allows entry through LAT1. Viability assays with SK4C1 indicated no change in cell viability whereas a slight decline in cell viability was detected at the higher concentrations of SK4C2, thus suggesting cytotoxicity is driven by the iron chelation moiety.

MDA MB 231 cells, MCF10A cells and SKOV3 cells were taken for further experiments as cell lines with respectively low, high and intermediate sensitivity to SK4. Phenotypic assays (PARP-1 cleavage, caspase 3/7 activity and Annexin V assays) demonstrated that breast cancer MDA MB 231 cells and normal breast MCF10A cells underwent apoptosis but that ovarian SKOV3 cells did not display any signs of apoptosis at any timepoints suggesting a different mechanism of cell death is behind SK4 driven cytotoxicity in the SKOV3 cell line. All cell lines tested displayed S phase arrest, potentially due to ribonucleotide reductase inhibition. Treating MDA MB 231, MCF10A and SKOV3 cells with SK4 resulted in a time-dependent upregulation of NDRG1 protein expression.

Bioinformatic analysis contrasting breast cancer samples with the top 10% and bottom 10% gene expression of NDRG1 highlighted extracellular matrix organisation and mitosis in pathway enrichment, which could be further investigated with experimental work. Proteomic analysis demonstrated SK4 treatment inhibited oxidative phosphorylation and the TCA cycle, which was further supported by an increase in AMP detected by metabolomics. PC, PE, and PS lipid species were found to decrease and SM to increase. We propose SK4 has multiple modes of action rather than just one: including upregulation of oxidative phosphorylation, inhibition of ribonucleotide reductase, oxidative phosphorylation, and the TCA cycle.

“Iron addiction” represents a potential target for cancer therapy which warrants further investigation as to its mechanisms of action and improvement of its selectivity. SK4 represents the first iron chelator designed to gain cell entry through the LAT1 transporter exploiting “iron addiction” as well as dysregulated amino acid metabolism. SK4 serves as a good starting point for the further development of more iron chelation compounds which enter through LAT1 exerting a double pronged approach of targeting “iron addiction” to induce cytotoxicity and dysregulated amino acid metabolism across a range of tumour derived cell lines although the mechanism of cell death may vary.

Date of Award	27 Apr 2023
Original language	English
Awarding Institution	Northumbria University
Supervisor	Stephany Veuger (Supervisor) & David Tetard (Supervisor)