Nucleus-Translocated GCLM Promotes Chemoresistance in Colorectal Cancer through a Moonlighting Function. Nucleus-Translocated GCLM Promotes Chemoresistance in Colorectal Cancer through a Moonlighting Function

Emerging evidence indicates that metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. In this study, utilizing a genome-scale metabolic CRISPR-Cas9 knockout library screen, we observed that loss of Glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably heightens the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B-repressing factor (NKRF), a known inhibitor of NF-κB signaling, to promote NF-κB activity and subsequently facilitate chemoresistance. In response to platinum drug treatment, P38 phosphorylates GCLM at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM is evident in CRC tissues and correlates with poor prognosis and heightened P38 activity. Overall, our findings shed light on the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance. Overall design: The human metabolic enzyme sgRNA library, targeting 1773 metabolic enzyme-encoding genes with 4 sgRNAs per gene, was designed and synthesized by GENEWIZ (Suzhou, China) and was then packaged into lentiviruses as previously described. For the in vitro CRISPR-Cas9 knockout screenin the presence of oxaliplatin, 4×107 HCT116 cells were plated, transduced with the lentiviral library and selected with puromycin (0.8 µg/mL) as previously reported3. The selected cells were divided into 4 groups and treated with PBS or 10 µM oxaliplatin for 7 days. Thereafter, the remaining surviving cells were harvested, and their genomic DNA was isolated and amplified by 2-step PCR using NEBNext Ultra II Q5 Master Mix (M0544L, New England Biolabs, Ipswich, MA, USA) and the primers listed in Supplementary Table 4. The PCR products containing the sgRNA sequences were extracted from the gel, quantified, sequenced, and analyzed as previously described

越来越多的证据表明，代谢酶在肿瘤进展过程中发挥兼职功能（moonlighting function），其中包括调控化疗耐药性。然而，这类功能的潜在分子机制仍未明确。本研究利用全基因组规模代谢酶CRISPR-Cas9敲除文库开展筛选实验，结果发现，谷胱甘肽生物合成的限速酶——谷氨酸-半胱氨酸连接酶修饰亚基（Glutamate-cysteine ligase modifier subunit, GCLM）的缺失，可显著增强结直肠癌（colorectal cancer, CRC）细胞对铂类化疗药物的敏感性。
机制层面，本研究揭示了一条非经典通路：细胞核内的GCLM可竞争性结合核因子κB抑制因子（NF-kappa-B-repressing factor, NKRF），该因子是已知的NF-κB信号通路抑制剂，进而促进NF-κB的活化，最终介导化疗耐药性的产生。在铂类药物处理后，P38可使GCLM在T17位点发生磷酸化，使其被importin α5识别并随后转位进入细胞核。此外，结直肠癌组织中核GCLM的表达水平显著升高，且与不良预后及增强的P38活性呈正相关。
综上，本研究的发现阐明了GCLM在增强NF-κB活性及后续化疗耐药过程中所发挥的关键非代谢功能，以及其翻译后调控机制。
实验整体设计：由中国苏州的GENEWIZ（苏州金唯智生物科技有限公司）设计并合成了靶向1773个代谢酶编码基因的人类代谢酶sgRNA文库，每个基因对应4条sgRNA。随后按照此前报道的方法将其包装为慢病毒。在奥沙利铂存在的条件下开展体外CRISPR-Cas9敲除筛选实验：接种4×10^7个HCT116细胞，用上述慢病毒文库进行转导，并以0.8 μg/mL嘌呤霉素进行筛选，操作流程同此前发表的文献3。将筛选得到的细胞分为4组，分别用PBS或10 μM奥沙利铂处理7天。随后收集存活的剩余细胞，提取其基因组DNA，采用NEBNext Ultra II Q5 高保真DNA聚合酶预混液（货号M0544L，新英格兰生物实验室，美国马萨诸塞州伊普斯维奇）以及补充表4中所列的引物，通过两步PCR进行扩增。将包含sgRNA序列的PCR产物从凝胶中回收、定量并测序，随后按照此前报道的方法进行数据分析。