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

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基因毒性治疗敏感。可以检测到辐射诱导的ATP耗竭，肿瘤细胞对其反应增加了放射敏感性。通过细胞膜代偿性葡萄糖输入是在SGLT葡萄糖转运体的帮助下进行的。暴露于辐射后，SGLT1还在细胞核（nSGLT1）中与DNA、mRNA和rna加工蛋白共定位，提示SGLT1在调节肿瘤细胞存活和肿瘤耐药中的作用是以前未知的。为了明确nSGLT1的作用，我们将研究辐照后SGLT1核易位的分子机制。为此，在结直肠肿瘤细胞中表达荧光标记的SGLT1，并在照射后确定其亚细胞定位。假设的SGLT1的NLS对核易位的重要性是用缺失突变体来检验的。通过加入强NLS， SGLT1在细胞核中积累。通过敲低Caveolin1来评估与Caveolin1可能的共转运。为了检验nSGLT1的功能相关性，我们以nSGLT1敲低或过表达后的残留DNA损伤和克隆细胞存活为功能终点。此外，还测定了葡萄糖代谢物、乙酰辅酶a、ATP和NAD+的核浓度。染色质重组作为DNA修复过程的一部分是通过测量组蛋白H3修饰可视化。临床批准的抑制剂达格列净对SGLT1的抑制作用在上述终点上进行了测试。为了阐明核SGLT1在“rna诱导沉默复合体”（RISC）中的功能作用，对SGLT1进行免疫沉淀，鉴定结合的mRNA和miRNA，并与总转录组进行比较。通过TAM芯片技术表征SGLT1的DNA结合序列，阐明SGLT1作为转录因子的功能。使用Maldi-TOF对SGLT1进行免疫沉淀后，鉴定出细胞核中未知的SGLT1结合伙伴。为了检查nSGLT1的翻译相关性，我们在100个直肠癌患者石蜡切片和“多器官组织芯片”上进行了SGLT1定位的免疫组织化学检测。在dapaglilozin存在的情况下，以上述终点在体外研究nSGLT1对直肠癌新辅助放化疗（RT和5-氟尿嘧啶）的影响。研究结果将为抑制nSGLT1和放射治疗的联合治疗提供分子基础。

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.