Discerning MCT1s role in chromatin remodelling and metabolomics changes in cancer progression

单羧酸转运体1(MCT1)是一种参与细胞代谢的跨膜蛋白，参与单羧酸盐(如丙酮酸代谢底物和乳酸代谢副产物)的细胞内外转运。据报道，在包括子宫内膜癌(EC)在内的几种癌症类型中，MCT1的质膜(PM)表达增加，并与患者存活率降低有关。虽然细胞核不是MCT的常见位置(根据目前对其功能的了解)，但我们和其他人报告了核MCT1(NMCT1)在子宫内膜[2]和软肉瘤[3]活检组织中的表达。这两项研究都表明，患有nMCT1的患者比没有nMCT1的患者存活时间更长。此外，许多研究表明，在细胞核中发现的代谢蛋白可以作为辅助因子或通过为这些过程提供底物(如乳酸、乙酰辅酶A)来调节转录和染色质重塑(见文献[4，5])。有趣的是，我们还发现，当在细胞核中时，MCT1与其他蛋白质相互作用，这些蛋白质改变染色质重塑并调节基因表达。因此，了解nMCT1的S在核中的新角色对于发现和开发癌症治疗的新方法以提高患者的预期寿命至关重要。该项目旨在确定nMCT1在潜在的染色质重塑和/或代谢改变中的作用，涉及癌症进展和患者生存。为此，我们将首先将CRISPR-Cas9技术与分子克隆相结合，将MCT1蛋白定向定位于细胞核(NMCT1)或质膜(PM MCT1)，以改造癌细胞系(Ishikawa和HEC1A)。然后，我们将对这些细胞系进行一些体外方法(例如免疫荧光、免疫印迹、电子显微镜)，以表征工程细胞系中的亚细胞定位和表达水平。带有nMCT1棒和PM MCT1的工程细胞株的代谢谱将使用质谱学进行，数据分析将使用R包进行。染色质重塑的变化将使用ATAC测序进行评估。代谢组学和染色质重塑的结果将通过R或任何其他相关的生物信息学工具进行比较和关联，以评估MCT1亚细胞定位、代谢变化和染色质结构之间的相关性。到这个跨学科项目结束时，被选中的学生将被配备在热门的湿实验室和生物信息学技能的博士后职位。

Monocarboxylate transporter 1 (MCT1) is a transmembrane protein involved in cell metabolism and mediates transport of monocarboxylates (such as pyruvate {a metabolic substrate} and lactate {a metabolic by product}) in and out of the cell. Increased plasma membrane (PM) expression of MCT1 has been reported in several cancer types including endometrial cancer (EC) and is associated with reduced patient survival. Although nucleus is not a usual location for MCTs (based on current knowledge on their function), we and others reported nuclear MCT1 (nMCT1) expression in endometrial [2] and soft sarcoma [3] biopsies. Both studies showed that patients with nMCT1 survive longer than the patients without it. Further, a number of studies showed that metabolic proteins found in the nucleus can regulate transcription and chromatin remodelling by acting as co-factors or by providing the substrates (such as lactate, acetyl-coA) for these processes (reviewed in [4, 5]). Interestingly, we also found that when in nucleus, MCT1 interacts with other proteins that modify chromatin remodelling and regulate gene expression. Therefore, understanding nMCT1's novel role in nucleus is vital to identify and exploit novel approaches in cancer treatment to enhance life-expectancy amongst patients. This project aims to identify nMCT1s role in relation to potential chromatin remodelling and/or metabolomic changes involved in cancer progression and patient survival. For this purpose, we will first combine CRISPR-Cas9 technology with molecular cloning to engineer cancer cell lines (Ishikawa and HEC1A) with MCT1 protein targeted specifically to the nucleus (nMCT1) or plasma membrane (PM MCT1). We will then subject these cell lines to a number of in vitro methods (e.g. immunofluorescence, western blotting, electron microscopy) to characterise subcellular localisation and expression levels in engineered cell lines. Metabolic profile of the engineered cell lines with nMCT1 wand PM MCT1 will be performed using mass spectroscopy and data analysis will be performed using R package. Changes in chromatin remodelling will be assessed using ATAC-sequencing. Results of metabolomics and chromatin remodelling will be compared and correlated to evaluate correlation between MCT1 subcellular localisation, changes in metabolism and chromatin structure by using R or any other relevant bioinformatics tools. By the end of this interdisciplinary project, the selected student will be equipped in sought after wet lab and bioinformatics skills for post-doctoral positions.