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

“细胞核兼职”：核SGLT1葡萄糖转运蛋白在肿瘤细胞适应性辐射反应中的作用

• 批准号: 460552618
• 负责人: Professor Dr. Klaus Dittmann
• 金额: --
• 依托单位: Labor Experimentelle Radioonkologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/460552618?language=en
• 关键词: Moonlighting; Nucleus; Role; nuclear; SGLT1

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.

DNA损伤的修复需要能量密集的染色质重组，因此，进行有氧糖酵解的肿瘤细胞对遗传毒性治疗敏感。可以检测到辐射诱导的ATP耗竭，肿瘤细胞对辐射敏感性增加。在SGLT葡萄糖转运蛋白的帮助下，通过细胞膜进行代偿性葡萄糖输入。放射后，SGLT 1也出现在细胞核（nSGLT 1）中，与DNA、mRNA和RNA加工蛋白共定位，提示SGLT 1在调节肿瘤细胞存活和肿瘤抵抗中的作用是未知的。为了阐明nSGLT 1的作用，将研究放射后SGLT 1核转位的分子机制。为此，在结肠直肠肿瘤细胞中表达荧光标记的SGLT 1，并在照射后确定亚细胞定位。使用缺失突变体检查SGLT 1的推定NLS对核转位的重要性。通过加入强NLS，SGLT 1在细胞核中积累。通过Caveolin 1敲低来评估与Caveolin 1的可能的共运输。为了检查nSGLT 1的功能相关性，功能终点是nSGLT 1敲低或过表达后的残留DNA损伤和克隆形成细胞存活。此外，还测定了葡萄糖代谢产物乙酰辅酶A、ATP和NAD+的核浓度。染色质重组作为DNA修复过程的一部分，通过测量组蛋白H3修饰来可视化。对上述终点检测了临床批准的抑制剂达格列净对SGLT 1的抑制作用。为了阐明核SGLT 1在“RNA诱导沉默复合物”（RISC）中的功能作用，对SGLT 1进行免疫沉淀，鉴定结合的mRNA和miRNA，并与总转录组进行比较。利用TAM芯片技术对SGLT 1的DNA结合序列进行了分析，阐明了SGLT 1作为转录因子的功能。使用Maldi-TOF进行SGLT 1免疫沉淀后，鉴定细胞核中SGLT 1的未知结合伴侣。为了检查nSGLT 1的翻译相关性，在来自直肠癌患者的100个石蜡切片和“多器官组织微阵列”上用化学方法检查SGLT 1的定位。在存在达格列净的情况下，通过上述终点，在体外研究了nSGLT 1对直肠癌新辅助放疗/化疗（RT和5-氟尿嘧啶）的影响。获得的结果应为nSGLT 1抑制和放疗组成的联合治疗提供分子基础。