DataSheet_1_Patient Derived Organoids Confirm That PI3K/AKT Signalling Is an Escape Pathway for Radioresistance and a Target for Therapy in Rectal Cancer.docx

ObjectivesPartial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. Several novel or repurposed drugs have been trialled to improve cancer cell sensitivity to radiotherapy, with limited success. We aimed to understand the mechanisms of resistance to chemoradiotherapy in rectal cancer using patient derived organoid models.
DesignTo understand the mechanisms underlying this resistance, we compared the pre-treatment transcriptomes of patient-derived organoids (PDO) with measured radiotherapy sensitivity to identify biological pathways involved in radiation resistance coupled with single cell sequencing, genome wide CRISPR-Cas9 and targeted drug screens.
ResultsRNA sequencing enrichment analysis revealed upregulation of PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes in radioresistant PDOs. Single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 and PI3K/AKT upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. We then tested the efficiency of dual PI3K/mTOR inhibitors in improving cancer cell sensitivity to radiotherapy. After irradiation, significant AKT phosphorylation was detected (p=0.027) which was abrogated with dual PI3K/mTOR inhibitors and lead to significant radiosensitisation of the HCT116 cell line and radiation resistant PDO lines.
ConclusionsThe PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its targeted pharmacological inhibition leads to significant radiosensitisation in CRC organoids, making it a potential target for clinical trials.

研究目的：超过半数的局部晚期直肠癌患者会出现术前放化疗的部分或完全耐药。目前已有多款新型或老药新用的药物被试用于提升癌细胞对放疗的敏感性，但收效有限。本研究旨在借助患者来源类器官（Patient-Derived Organoid, PDO）模型，解析直肠癌对放化疗产生耐药性的机制。
研究设计：为解析该耐药性背后的分子机制，我们将患者来源类器官（PDO）的治疗前转录组与实测的放疗敏感性进行比对，结合单细胞测序、全基因组CRISPR-Cas9筛选及靶向药物筛选，以明确与放疗耐药相关的生物学通路。
研究结果：RNA测序富集分析显示，放疗耐药性PDO中PI3K/AKT/mTOR通路及上皮间质转化（epithelial mesenchymal transition）相关基因表达上调。对辐照前后PDO的单细胞测序结果表明，mTORC1及PI3K/AKT通路存在激活现象，这一结论通过使用辐照结直肠癌（colorectal cancer, CRC）细胞系开展的全基因组CRISPR-Cas9敲除筛选得到验证。随后我们验证了双靶点PI3K/mTOR抑制剂提升癌细胞放疗敏感性的效果：辐照后可检测到显著的AKT磷酸化水平升高（p=0.027），而双靶点PI3K/mTOR抑制剂可阻断该磷酸化过程，并显著增强HCT116细胞系及放疗耐药性PDO株的放疗敏感性。
研究结论：PI3K/AKT/mTOR通路的激活可导致结直肠癌放疗耐药，对该通路进行靶向药理学抑制可显著提升结直肠癌类器官的放疗敏感性，因此该通路可作为临床试验的潜在靶点。