"Moonlighting in the Nucleus": Role of the nuclear SGLT1 glucose transporter in the adaptive radiation response of tumor cells

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The repair of DNA damage requires energy-intensive chromatin restructuring, and as a consequence, tumor cells that carry out aerobic glycolysis are sensitive to genotoxic treatment. A radiation-induced ATP depletion could be detected, to which tumor cells reacted with increased radio sensitivity. The compensatory glucose import through the cell membrane was carried out with the help of the SGLT glucose transporter. After exposure to radiation, SGLT1 was also found in the cell nucleus (nSGLT1) in colocalization with DNA, mRNA, and RNA-processing proteins, suggesting a previously unknown role of SGLT1 in the regulation of tumor cell survival and tumor resistance.To clarify the role of nSGLT1, the molecular mechanism of the nuclear translocation of SGLT1 after exposure to radiation will be investigated. For this purpose, a fluorescence-labeled SGLT1 is expressed in colorectal tumor cells and the subcellular localization is determined after irradiation. The importance of the putative NLS of SGLT1 for nuclear translocation is checked using a deletion mutant. By adding a strong NLS, the SGLT1 is accumulated in the cell nucleus. A possible co-transport with Caveolin1 is evaluated by a Caveolin1 knockdown. To check the functional relevance of nSGLT1, the functional endpoints are the residual DNA damage and the clonogenic cell survival after nSGLT1 knockdown or over-expression. In addition, the nuclear concentrations of the glucose metabolites, acetyl-CoA, ATP and NAD+ are determined. Chromatin restructuring as part of the DNA repair process is visualized by measuring the histone H3 modifications. The effect of SGLT1 inhibition by the clinically approved inhibitor dapagliflozin is tested on the above-mentioned endpoints. To clarify the functional role of the nuclear SGLT1 in the “RNA-induced silencing complex” (RISC), the SGLT1 is immunoprecipitated and the bound mRNA and miRNA are identified and compared with the total transcriptome. A function of SGLT1 as a transcription factor is clarified by characterizing the DNA binding sequences of SGLT1 with the help of TAM chip technology. Unknown binding partners of SGLT1 in the cell nucleus are identified after SGLT1 immunoprecipitation using Maldi-TOF. To check the translational relevance of nSGLT1, the localization of SGLT1 is examined immunohistochemically on 100 paraffin sections from patients with rectal cancer and on a “multi organ tissue microarray”. The influence of nSGLT1 on neoadjuvant radio / chemotherapy of rectal cancer (RT and 5-fluorouracil) is examined in vitro in the presence of dapagliflozin with the above-mentioned endpoints.The results obtained should provide the molecular basis for a combination therapy consisting of inhibition of nSGLT1 and radiation therapy.