Modulation of metabolite transport in human cancer cells by carbonic anhydrase

• Applicant: Privatdozent Dr. Holger M. Becker
• Subject Area: Cell Biology ; Biochemistry ; Biophysics
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 327616187

The most aggressive and invasive tumor types, which often reside in a hypoxic environment, rely on extensive glycolysis to meet their increased demand for energy and biosynthetic precursors. The excessive augmentation in glycolytic activity is often triggered by hypoxia, which derives from enhanced cell proliferation, accompanied by insufficient or chaotic vascularization. The tremendous increase in glycolysis leads to vast production of lactate and protons, both of which have to be removed from the cell to prevent intracellular acidification and inhibition of glycolysis. Efflux of lactate from cancer cells is primarily mediated by the monocarboxylate transporters MCT1 and MCT4, both of which carry lactate in co-transport with a proton. MCT-mediated proton efflux exacerbates extracellular acidification and supports the formation of a hostile environment which further fosters tumor growth.The proposed project aims on a better understanding of the processes that facilitate transport capacity of MCTs in tumour tissues. Thereby special emphasis will be put on the function the carbonic anhydrases CAIX and CAXII which could form a transport metabolon with MCTs to drive lactate efflux. Hypoxia-regulated CAIX is almost exclusively found in tumor cells and commonly linked to poor prognosis. CAXII is also overexpressed in many cancer cells and has recently found to be involved in the acquisition of chemoresistance. We will first elucidate which changes within the tumour environment influence the expression levels of these proteins and lead to formation of the transport metabolon. Second objective is to understand the molecular mechanisms behind this non-catalytic transport metabolon with special emphasis on the formation of the protein complex and its function as proton antenna. The third and final objective is to elucidate which role this transport metabolon plays in tumour metabolism and cell survival. To reach these objectives we apply various physiological and biochemical techniques, including intracellular pH imaging with fluorescent dyes and real-time metabolite imaging with FRET sensors in single cancer cells and multi-cellular spheroids, mRNA knockdown, in situ proximity ligation assays on cell lines and human tissue samples, and pull-down assays, heterologous protein expression in Xenopus oocytes, as well as cell proliferation, migration, and invasion assays.The findings in this project should help us to a better understanding of the complex interplay between metabolite transporters and carbonic anhydrases in tumor cells and provide the basis for an exploitation of these interactions as potential drug targets in modern cancer therapy.

• DFG Programme: Research Grants