PO.ET01.02 · 实验与分子治疗
靶向RUNX转录活性可降低三阴性乳腺癌的肿瘤异质性和侵袭性
Targeting RUNX transcriptional activity reduces tumor heterogeneity and aggressiveness in triple-negative breast cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
三阴性乳腺癌（TNBC）是一种高度侵袭性疾病，其特征为早期复发并富含癌症干细胞（CSC），从而导致化疗耐药和转移。我们的研究聚焦于RUNX在TNBC侵袭性、促进细胞迁移和调控基因表达中的作用。我们曾报道RUNX1影响雄激素反应型TNBC细胞系的化疗耐药性，Ferrari（2016）则显示其与TNBC患者的不良预后相关。Lv（2024）表明RUNX2也参与化疗耐药，Halperin（2022）确立了RUNX1在乳腺癌患者的癌症相关成纤维细胞中上调并预示不良疾病结局。我们的研究探讨了抑制RUNX转录活性如何影响TNBC细胞系的侵袭性和药物反应。使用RUNX-CBFbeta商品化抑制剂（AI-10-104和AI-10-49），我们观察到MDA-MB-231和-468细胞系的活力和迁移显著降低，凋亡增加。在强制悬浮细胞模型中，抑制RUNX转录活性降低了对CSC维持至关重要的基因的mRNA水平。在MDA-MB-231细胞中，RUNX抑制完全阻碍了乳腺球（mammosphere）的形成。RUNX1的mRNA和蛋白表达在多柔比星（Doxo）和紫杉醇（Px）处理的细胞中升高。RUNX抑制增强了药物毒性，降低活力并促进凋亡。AI-10-104与Doxo联用比单用Doxo更显著地减少了乳腺球数量，而AI-10-104则恢复了Px耐药细胞对Px的敏感性。通过流式细胞术，我们观察到RUNX抑制显著下调PD-L1表达，提示其在肿瘤免疫逃逸中起重要作用。此外，RUNX1抑制改变了线粒体自噬相关基因的表达，并在SUM-159PT细胞中诱导线粒体碎裂。最后，通过使用长读长牛津纳米孔技术的转录组分析（cDNA-seq），我们比较了经AI-10-104处理的贴壁生长与强制悬浮生长的MDA-MB-468细胞的表达谱，并深入了解了其潜在机制。分析已鉴定出10个基因，包括代谢、应激、耐药基因等，其表达受RUNX转录抑制的差异性影响取决于培养模型。这些发现指向在不同肿瘤亚群中可能存在不同的RUNX依赖性调控机制，我们目前正在研究这一点。总之，我们的结果表明，RUNX通过影响细胞内异质性和免疫监视等癌症标志，在产生化疗耐药性TNBC细胞中起关键作用。
查看英文原文 English abstract
Triple-negative breast cancer (TNBC) is a highly aggressive disease, characterized by early relapse and enriched with cancer stem cells (CSCs), contributing to chemoresistance and metastasis. Our research focuses on RUNXs role in TNBC aggressiveness, cell migration promotion and regulation of gene expression. We reported that RUNX1 influences chemoresistance in androgen-responsive TNBC cell lines and Ferrari (2016) showed its correlation with poor prognosis in TNBC patients. Lv (2024) showed that RUNX2 is also implicated in chemoresistance, and Halperin (2022) established that RUNX1 is upregulated in cancer-associated fibroblasts of breast cancer patients and predicts poor disease outcomes. Our study explores how inhibiting RUNX transcriptional activity impacts TNBC cell lines aggressiveness and drug response. Using RUNX-CBFbeta commercial inhibitors (AI-10-104 and AI-10-49) we observed significant reduction in viability and migration, and increased apoptosis in MDA-MB-231 and -468 cell lines. In a forced-suspended cell model, inhibiting RUNX transcriptional activity decreased mRNA levels of crucial genes for CSC maintenance. In MDA-MB-231 cells, RUNX inhibition completely hindered mammosphere formation. RUNX1 mRNA and protein expression increased in doxorubicin (Doxo) and paclitaxel (Px) treated cells. RUNX inhibition enhances drug toxicity, reducing viability and boosting apoptosis. Combined AI-10-104 and Doxo significantly reduced the number of mammospheres even more than Doxo alone, while AI-10-104 restored Px sensitivity in Px-resistant cells. By flow cytometry we observed that RUNX inhibition significantly downregulates PD-L1 expression, suggesting an important role in tumor immune escape. Furthermore, RUNX1 inhibition alters mitophagy-related gene expression and induces mitochondrial fragmentation in SUM-159PT cells. Finally, by transcriptomic analysis (cDNA-seq) using long-read oxford nanopore technology, we compare the expression profiles of adherent with forced-suspension growing MDA-MB-468 cells, treated with AI-10-104, and gain insights into the underlying mechanisms. Analyses have identified 10 genes including metabolic, stress, drug resistance' genes, among others, whose expression is differentially affected by RUNX transcriptional inhibition depending on the culture model. These findings point to potentially distinct RUNX-dependent regulatory mechanisms across tumor subpopulations, which are currently under our investigation. In summary, our results suggest that RUNXs play a critical role in generating chemoresistant TNBC cells by influencing intracellular heterogeneity and immune surveillance cancer hallmarks.
利益披露 Disclosure
N. B. Fernandez, None..
F. L. Couto, None..
M. S. Sosa, None..
N. Rubinstein, None.