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The role of aldolase in breast cancer (Master thesis)

Τσαμούρ, Ατσέλια/ Tsamour, Atselia/ Κλινική Φαρμακολογία - Θεραπευτική

Aldolase is one of the key enzymes in glycolysis, catalyzing the fourth reaction of glycolysis pathway, the cleavage of Fructose 1,6-biphosphate (F1,6BP/FBP) into two triose sugars, Glyceraldehyde 3-phosphate (G3P/GAP) and Dihydroxyacetone phosphate (DHAP). In addition to this fundamental metabolic role, aldolase also seems to have “non-glycolytic” functions within cells. One of these functions is the ability to bind to the lysosomal surface to monitor in-cell glucose levels. Through this mechanism and under low glucose conditions, AMPK is activated before cellular energy levels even start to decline. Aim The aim of this thesis is to investigate the anticancer effect of the pharmacological inhibition of lysosomal aldolase in breast cancer. Specifically, it aims to examine the underlying mechanism of inhibition and the subsequently activated pathways. Finally, it evaluates whether the pharmacological inhibition of lysosomal aldolase can make cancer cells more sensitive to radiation. Methods. In order to investigate the anticancer activity of lysosomal aldolase inhibition, the breast cancer cell lines SUM-159PT, MDA-MB-231, HCC1806, MCF7 and T47D were used. Cells were treated with Aldometanib, in concentrations of 0.125 μΜ, 0.25 μΜ, 0.5 μΜ and 1 μΜ. Cell viability assays were performed in 2D cultures to determine the IC50 values for each of cell lines used. Radiosensitivity was evaluated in the SUM- 159PT cell line in both monolayer - clonogenic assay and in 3D - spheroid assay. The DNA damage marker γΗ2ΑΧ was also analyzed through western blot analysis. Finally, to evaluate the induction of cellular energy stress, western blot analysis was also used for the proteins p-AMPK, p-ACC and p-S6. Results. Inhibition of lysosomal aldolase caused a significant reduction in breast cancer cell proliferation in a dose-dependent manner, with the most sensitive cell line being T47D (IC50 0.08μM) and the most tolerant being HCC1806 (IC50: 0.35 μM). Aldometanib also caused a significant reduction in cancer cell population after exposure to 6 Gy and 10 Gy of radiation, both in monolayer cell cultures and 3D cultures. Furthermore, no difference was observed in the levels of γΗ2ΑΧ in combination with radiation or monotherapy. Finally, an increase in the levels of p-AMPK and p-ACC and a decrease in the levels of p-S6 wad observed. Conclusions Inhibition of lysosomal aldolase with Aldometanib exhibits an anticancer effect, reducing cell proliferation and increasing their sensitivity to radiation. These results form the basis for the development of new therapeutic approaches.