Transcriptomic insight into salinomycin mechanisms in breast cancer cell lines: synergistic effects with dasatinib and induction of estrogen receptor beta. Transcriptomic insight into salinomycin mechanisms in breast cancer cell lines: synergistic effects with dasatinib and induction of estrogen receptor beta

Purpose:The use of a single-drug to encounter cancer progression is generally ineffective due to the tumors heterogeneity. To improve the clinical outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. In this study, we proposed to use a combination of salinomycin (which targets anticancer stem cells) and dasatinib (an Src kinase inhibitor) for the treatment of breast cancer. The use of a RNA-seq is a combinatorial technique that allows to quantify global gene expression in biological samples. We employed this next generation sequencing technique to provide an initial insight into how Sal and Das alone, as well as in combination, modulated gene expression in the MDA-MB-468 human triple negative breast cancer cell line. Method: mRNA profiles of MDA-MB-468 cells treated with PBS (control), salinomycin, dasatinib, or the drug combination Sal + Das were generated by deep sequencing, in quadruplicate, using HiSeq4000 sequencer with single-end 50 bps reads. The results were validated by RT-qPCR analysis using StepOnePlus Real-timePCR system and SYBR Green assays. Results: RNA-seq data revealed that both salinomycin and dasatinib inhibit the expression of many downstream targeted genes of the STAT3, Wnt/beta-catenin, and hedgehog cell signaling pathways. The drug combination exhibited synergism through suppression of multiple pathways, leading to a promotion of cell cycle arrest at the G1/S phase mainly via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway. We also identified new salinomycin mechanisms involved in upregulating transcription factor 2. The study further led to a discovery of a new drug-induced targeting of estrogen receptor beta approach for triple-negative breast cancer treatment. Conclusion: Using RNA-seq, we identified for the first time the responsible pathways, including the estrogen-mediated S-phase entry pathway, that contributed to the synergistic effects of the drug combination Sal + Das. The discoveries of potential therapeutic targets, such as E2F2 as well as a novel drug-induced targeting of estrogen receptor beta (ESR2) approach were also described herein.We believe that using next generation approach to study drug mechanisms will allow us to identify more specific disease-relevant biomarkers for precision treatment of breast cancer, as well as other cancers in the future. Overall design: mRNA profiles of MDA-MB-468 cells treated with PBS (control), salinomycin, dasatinib, or the drug combination Sal + Das were generated by deep sequencing, in quadruplicate, using HiSeq4000 sequencer with single-end 50 bps reads.

研究目的：由于肿瘤异质性，单一药物用于对抗肿瘤进展通常疗效不佳。为改善临床结局，临床常采用作用机制各异但抗癌活性互补的多种药物进行联合治疗，以增强抗肿瘤疗效并降低获得性耐药风险。本研究拟采用沙利霉素（salinomycin，靶向抗癌干细胞）与达沙替尼（dasatinib，一种Src激酶抑制剂）联合方案治疗乳腺癌。RNA测序（RNA-seq）是一种可定量分析生物样品中全局基因表达水平的综合技术，本研究采用该下一代测序技术，旨在初步阐明沙利霉素（Sal）、达沙替尼（Das）单药及联合给药时，在人三阴性乳腺癌细胞系MDA-MB-468中对基因表达的调控机制。
实验方法：采用HiSeq4000测序仪进行单端50bp读长的深度测序，对经PBS（对照组）、沙利霉素、达沙替尼及沙利霉素+达沙替尼联合给药处理的MDA-MB-468细胞进行四次生物学重复的mRNA谱分析。采用StepOnePlus实时荧光定量PCR系统与SYBR Green染料法进行RT-qPCR验证实验，以确认测序结果的可靠性。
实验结果：RNA测序数据显示，沙利霉素与达沙替尼均可抑制信号转导与转录激活因子3（STAT3）、Wnt/β-连环蛋白（Wnt/β-catenin）及Hedgehog等多条细胞信号通路的众多下游靶基因的表达。联合给药则通过抑制多条通路展现出协同抗肿瘤效应，主要经雌激素介导的S期进入通路、部分经乳腺癌易感基因1（BRCA1）与DNA损伤应答通路，促进细胞周期在G1/S期发生阻滞。本研究还发现了沙利霉素上调转录因子2（transcription factor 2）表达的全新作用机制。此外，本研究进一步发现了一种全新的药物诱导靶向雌激素受体β（estrogen receptor beta，ESR2）的治疗策略，可用于三阴性乳腺癌的治疗。
研究结论：本研究通过RNA测序首次明确了沙利霉素与达沙替尼联合给药产生协同效应的关键通路，其中包括雌激素介导的S期进入通路。本研究同时还发现了包括E2F转录因子2（E2F2）在内的潜在治疗靶点，并阐明了上述全新的药物诱导靶向雌激素受体β的治疗策略。我们认为，采用下一代测序技术研究药物作用机制，将有助于未来为乳腺癌及其他癌症的精准治疗筛选出更多特异性疾病相关生物标志物。
整体实验设计：采用HiSeq4000测序仪进行单端50bp读长的深度测序，对经PBS（对照组）、沙利霉素、达沙替尼及沙利霉素+达沙替尼联合给药处理的MDA-MB-468细胞进行四次生物学重复的mRNA谱分析。