Prokarenkaite Rimvile, Kuodyte Karolina, Gudoityte Greta, Budginaite Elzbieta, Naumovas Daniel, Strainiene Egle, Velickevicius Kristijonas, Dulskas Audrius, Sileika Ernestas, Venius Jonas, Tunaitis Virginijus, Pivoriunas Augustas, Starkuviene Vytaute, Stankevicius Vaidotas, Suziedelis Kestutis
Laboratory of Molecular Oncology, National Cancer Institute, Vilnius, Lithuania.
Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
J Exp Clin Cancer Res. 2025 Jul 11;44(1):199. doi: 10.1186/s13046-025-03439-y.
Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Despite substantial advancements in CRC therapy in recent years, ionizing radiation (IR) continues to be the predominant treatment for colon malignances. However, it still lacks the precision required for excellent therapeutic outcomes, ultimately resulting in tumor radioresistance. This study seeks to explore the potential of atypical PARPs including PARP9, PARP12, PARP13 and PARP14 as innovative radiosensitizing targets for CRC.
We utilized CRISPR/Cas9-mediated gene editing to knockout the PARP9, PARP12, PARP13 and PARP14 in HT29 and DLD1 cells. The cells were exposed to either a single dose of 6-10 Gy or to fractionated dose of 5 × 2 Gy X-ray radiation cultivating cells in 2D, laminin-rich ECM 3D and multicellular spheroid models. The transcriptomes of nonirradiated and irradiated cells were analyzed using microarrays. Gene set enrichment analysis was conducted to determine the pathways in which PARP13 is engaged. Cell viability was assessed using a clonogenic assay. Gene expression levels in cells and patient samples were quantified using RT-qPCR.
The expression of PARP9, PARP12, PARP13 and PARP14 was particularly elevated in irradiated colorectal cancer HT29 cells in a microenvironment-dependent manner. PARP13 deficiency significantly enhanced the sensitivity of HT29 cells to both single-dose and multifractionated irradiation regimens, resulting in reduced colony formation and spheroidal integrity. Microarray analysis indicated that PARP13 may modulate the expression genes associated with immune response signaling pathways, including members of PARP family. Furthermore, PARP13 loss in HT29 cells markedly impaired the expression of immune response related genes following multifractionated ionizing irradiation. Finally, chemoradiotherapy significantly elevated the expression of PARP9, PARP12, PARP13 and PARP14 in rectal tumors, while having no effect on adjacent normal colon tissues. Elevated pre-treatment PARP9 expression levels and a blunted post-treatment increase in PARP9 and PARP14 expression predicted poor overall survival in rectal cancer patients, while PARP13 emerged as the most significant discriminator between tumor and healthy tissue.
Collectively, the PARP9/13/14 axis is implicated in the response of CRC to radiation treatment in both preclinical and clinical settings, demonstrating the atypical members of the PARP family as attractive targets for neoadjuvant radiotherapy.
结直肠癌（CRC）是全球第三大常见癌症。尽管近年来CRC治疗取得了重大进展，但电离辐射（IR）仍然是结肠恶性肿瘤的主要治疗方法。然而，它仍然缺乏实现优异治疗效果所需的精准性，最终导致肿瘤放射抗性。本研究旨在探索包括PARP9、PARP12、PARP13和PARP14在内的非典型PARP作为CRC创新放射增敏靶点的潜力。
我们利用CRISPR/Cas9介导的基因编辑技术在HT29和DLD1细胞中敲除PARP9、PARP12、PARP13和PARP14。将细胞暴露于单剂量6 - 10 Gy或分次剂量5×2 Gy的X射线辐射下，在二维、富含层粘连蛋白的细胞外基质三维和多细胞球体模型中培养细胞。使用微阵列分析未照射和照射细胞的转录组。进行基因集富集分析以确定PARP13参与的途径。使用克隆形成试验评估细胞活力。使用RT-qPCR定量细胞和患者样本中的基因表达水平。
PARP9、PARP12、PARP13和PARP14的表达在受照射的结直肠癌HT29细胞中以微环境依赖的方式特别升高。PARP13缺陷显著增强了HT29细胞对单剂量和分次照射方案的敏感性，导致集落形成减少和球体完整性降低。微阵列分析表明，PARP13可能调节与免疫反应信号通路相关的基因表达，包括PARP家族成员。此外，HT29细胞中PARP13的缺失在分次电离辐射后显著损害了免疫反应相关基因的表达。最后，放化疗显著提高了直肠肿瘤中PARP9、PARP12、PARP13和PARP14的表达，而对相邻正常结肠组织没有影响。治疗前PARP9表达水平升高以及治疗后PARP9和PARP14表达增加不明显预示着直肠癌患者的总生存期较差，而PARP13是肿瘤组织与健康组织之间最显著的区分标志物。
总体而言，PARP9/13/14轴在临床前和临床环境中均与CRC对放射治疗的反应有关，表明PARP家族的非典型成员是新辅助放疗的有吸引力的靶点。
