微卫星不稳定性高（MSI-H）结直肠癌（CRC）患者具有较高的肿瘤突变负荷和肿瘤免疫原性，对免疫治疗表现出更高的反应率和更好的生存率。然而，部分 MSI-H CRC 患者仍然会出现不良的疾病结果。我们的目的是确定决定这些异质临床结果的肿瘤自主调节因子。癌症基因组图谱 (TCGA) 数据集用于识别 MSI-H CRC 患者预后不良的调节因子。建立了表达靶向调节因子的稳定 CRC 肿瘤克隆，以评估迁移和干性特性、免疫细胞脆弱性和细胞内 (CIC) 结构形成。 RNA 测序 (RNA-seq) 用于识别稳定的 CRC 肿瘤克隆中丰富的生物学途径。对福尔马林固定石蜡包埋 (FFPE) MSI-H CRC 标本进行临床病理学表征，以探索所涉及的潜在机制。我们发现，在生存结果较差的 MSI-H CRC 患者中，癌症/睾丸抗原家族 45 成员 A1 (CT45A1) 表达上调。表达 CT45A1 的微卫星稳定 (MSS) CRC 细胞显示出增强的迁移能力。然而，表达 CT45A1 的 MSI-H CRC 细胞（而非 MSS CRC 细胞）对自然杀伤 (NK) 细胞的细胞毒性表现出更高的抵抗力，并充当同型 CIC 结构中的外部细胞，防止外源或治疗性抗体进入内部 CRC 细胞。使用小分子抑制剂或针对肌球蛋白轻链激酶 (MYLK) 的短发夹 RNA (shRNA) 灭活 RHO-ROCK/MLCK-MLC2 信号传导可消除 NK 细胞耐药性并减少表达 CT45A1 MSI-H CRC 细胞的外细胞命运。在 MSI-H CRC 患者中，CT45A1 阳性肿瘤表现出 MLC2 磷酸化增加、外细胞命运增加和生存率降低。我们证明 CT45A1 会增强 MSI-H CRC 的晚期进展，而靶向 MLC2 磷酸化可能会增强 CT45A1 阳性 MSI-H CRC 患者的免疫治疗效果。© 2024 作者。约翰·威利出版的《分子肿瘤学》

Patients with microsatellite instability-high (MSI-H) colorectal cancer (CRC) have high tumor mutation burden and tumor immunogenicity, exhibiting a higher response rate to immunotherapy and better survival. However, a portion of MSI-H CRC patients still experience adverse disease outcomes. We aimed to identify the tumor-autonomous regulators determining these heterogeneous clinical outcomes. The Cancer Genome Atlas (TCGA) dataset was used to identify regulators in MSI-H CRC patients with unfavorable outcomes. Stable CRC tumor clones expressing targeted regulators were established to evaluate migratory and stemness properties, immune cell vulnerability, and cell-in-cell (CIC) structure formation. RNA-sequencing (RNA-seq) was used to identify enriched biological pathways in stable CRC tumor clones. Clinicopathological characterization of formalin-fixed paraffin-embedded (FFPE) MSI-H CRC specimens was performed to explore the underlying mechanisms involved. We showed that cancer/testis antigen family 45 member A1 (CT45A1) expression was upregulated in MSI-H CRC patients with poor survival outcomes. CT45A1-expressing microsatellite stable (MSS) CRC cells showed enhanced migratory ability. However, CT45A1-expressing MSI-H CRC cells, but not MSS CRC cells, showed higher resistance to natural killer (NK) cell cytotoxicity and served as outer cells in homotypic CIC structures, preventing exogenous or therapeutic antibody access to inner CRC cells. Inactivating RHO-ROCK/MLCK-MLC2 signaling with small-molecule inhibitors or short-hairpin RNAs (shRNAs) targeting myosin light chain kinase (MYLK) abolished NK cell resistance and reduced the outer cell fate of CT45A1-expressing MSI-H CRC cells. In MSI-H CRC patients, CT45A1-positive tumors exhibited increased MLC2 phosphorylation, increased outer cell fate, and decreased survival. We demonstrated that CT45A1 potentiates the advanced progression of MSI-H CRC, and targeting MLC2 phosphorylation may enhance immunotherapy efficacy in CT45A1-positive MSI-H CRC patients.© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.