PhD Scientific Days 2023

Pharmaceutical Sciences I.

Copper Trafficking of KRAS Mutant Cell Lines

Text of the abstract

Introduction: KRAS mutations have an increasing significance in oncology. However it remains challenging to target therapeutically. Despite its importance in human tumors, especially colorectal, pancreatic and lung cancers, no selective treatment has been developed, except for KRAS G12C mutations. Recent studies have shown differences between KRAS mutant and wild type cells regarding copper bioavailability, which suggests altered trafficking, uptake and storage mechanisms in mutant cells.
Aims: Our aims were to examine the differences in short term (2h) and long term (72h) copper uptake in different media (1); sensitivity to intracellular copper chelators (2); macropinocytosis (3); and ATP7A copper transporter expression (4) in various wild type and KRAS mutant tumor cell lines.
Methods: Experiments were carried out using the following KRAS mutant and wild type cell lines: SW48 wild type, SW48 G12C, SW48 G12D, SW48 G12V, HCT-116, HKH-2, HT-29, SW480, DLD-1 and Caco-2. IC50 values of copper chelator treatment were determined by PrestoBlue cell viability assay. After copper treatment, copper levels were measured with total reflection X-ray fluorescence analysis (TXRF). Macropinocytosis was examined with fluorescent activated cell sorting (FACS) in SW48 cell lines, after dextran-FITC treatment. ATP7A expression was measured with Western Blot technique.
Results: No significant difference was found in sensitivity to intracellular copper chelator treatment between wild type and mutant cells. After 72 hours of low concentration copper treatment, wild type SW48 cells had significantly higher copper contents than KRAS mutant SW48 cells. The copper levels of cells treated with 5 µM Cu(II) compared to non-treated cells after 72h were: 617% in SW48 wild type cells, 204%, 207%, and 248% in SW48 G12C, SW48 G12D, and SW48 G12V cells respectively. Various mechanisms can be responsible for this effect, including altered uptake with active transport or via macropinocytosis, or increased efflux transport in mutant cells through ATP7A copper transporter. In our studies we carried out experiments to examine these assumed effects.
Conclusion: KRAS mutant and wild type cell lines show clear differences in copper trafficking.
Funding: This work was supported by the SE 250+ fellowship.