PhD Scientific Days 2022

Pharmaceutical Sciences I.

Copper Metabolism of KRAS Mutant Cell Lines

Text of the abstract

Introduction: Oncogenic KRAS mutations play a major role in human tumors, especially colorectal, lung and pancreatic cancers. Despite its importance, KRAS specific targeting has yet to be developed. However, recent studies have shown differences between the copper bioavailability of KRAS wild type cell lines and those that carry KRAS mutations which have alterations in copper trafficking and storage. It is believed that there is a higher copper uptake rate in these mutant cells for which various mechanisms may be responsible, presumably macropinocytosis and active transport with the ATP7A copper transporter.
Aims: Our aims were to examine the differences in (1) copper uptake, (2) sensitivity to copper and copper chelator treatments, and (3) macropinocytosis process between tumor cells with and without KRAS mutation.
Methods: Experiments were carried out using the following cell lines: SW48 wild type, SW48 G12C, SW48 G12D, SW48 G12V, BXPC-3, PANC-1, HCT-116 and HT-29. IC50 values of copper and copper chelator (ammonium-tetrathiomolibdate) treatment were determined by PrestoBlue cell viability assay. After copper treatment, copper levels were measured with total reflection X-ray fluorescence analysis (TXRF) in tumor cells. Macropinocytosis was examined with fluorescent activated cell sorting (FACS) in SW48 cell lines, after albumin-fluorescein isothiocyanate (BSA-FITC) and dextran-FITC treatment.
Results: Comparing mutant KRAS and wild type KRAS cells, mutant cells proved to be more sensitive to copper depletion and more resistant to copper treatment. TXRF analysis results showed that HCT116 KRAS mutant colorectal tumor cells accumulated more copper compared to control cells. After BSA-FITC and dextran-FITC treatment, fluorescent intensity increased in SW48 KRAS mutant cells. It suggests that BSA-FITC and dextran-FITC were internalized by macropinocytosis in the mutant cells while this mechanism is less characteristic of wild type cells.
Conclusion: KRAS mutant and wild type cell lines show differences in copper metabolism. Further research into copper metabolism would be worthwhile in order to develop KRAS selective tumor therapy.
Funding: This work was supported by the SE 250+ scholarship, EFOP-3.6.3-VEKOP-16-2017-00009.

Email address: uvegeselina@gmail.com
Semmelweis University, Doctoral School of Pharmaceutical Sciences
Supervisor: Dr. Szoboszlai Norbert