Development of a novel therapeutic agent that exploits specific vulnerabilities in claudin low breast cancer

Claudin-低乳腺癌（CLBC）的特征是间充质和癌干细胞样的性质，是一种恶性肿瘤。 预后差的侵袭性亚型。目前，没有CLBC特异性治疗方案。我们 第一个显示银纳米颗粒（AgNPs）具有选择性细胞毒性和细胞毒性的理想组合， 以及在对非癌性细胞无毒的剂量下治疗CLBC的辐射剂量增强效应 乳腺和其他细胞。已知没有其他纳米材料具有乳腺癌亚型特异性细胞毒性 或辐射致敏特性。经过广泛的筛选和表征过程，我们现在有 在肿瘤中静脉内注射后显示出针对CLBC的体内功效的领先AgNP制剂 这为安全使用这种纳米材料提供了治疗窗口的证据。 AgNPs对CLBC的选择性部分是由于CLBC细胞不能减轻DNA和蛋白质损伤 由AgNPs引起，并进一步增强了间充质癌的普遍脆弱性 与CLBC一样，我们发现内质网（ER）应激在CLBC中被AgNPs选择性诱导。 我们的中心假设是，AgNPs可以作为一种精确的医学形式，用于治疗 claudin-low和其他间叶乳腺癌。值得注意的是，CLBC细胞系和患者样品表达 ESRP 1（内皮剪接调节蛋白）显著低于其他乳腺癌亚型。ESRP 1 调节上皮向间充质转化（EMT）所必需的转录程序。我们发现 基线ESRP 1表达与AgNP敏感性负相关，ESRP 1敲低可增加AgNP敏感性。 灵敏度虽然CLBC仅占乳腺癌的5%，但我们对1954例患者的临床数据显示， 所有乳腺癌中有13%为ESRP 1低（定义为CLBC中≤平均ESRP 1 mRNA）。因此，AgNP 治疗可能对更广泛的患者群体有益。在AIM 1中，我们将测试我们的假设 优化的AgNP可有效用于CLBC的治疗和放射增敏，而不诱导细胞毒性， 正常乳腺上皮细胞的DNA损伤。我们将对摄取，亚细胞定位， AgNPs对CLBC和正常乳腺上皮细胞的细胞毒性、DNA损伤和放射增敏作用 3D细胞培养和鼠原位肿瘤模型。在AIM 2中，我们将检验特定效应 AgNP暴露对氧化还原敏感蛋白、途径和细胞器的影响有助于CLBC特异性 AgNPs的作用机制。我们将使用最先进的试剂和新的蛋白质组学方法， 鉴定CLBC和非CLBC细胞中由AgNP暴露诱导的氧化损伤。在AIM 3中，我们将测试 假设间充质癌细胞对ER应激的潜在敏感性是导致 AgNP对CLBC治疗的特异性。我们将评估ESRP 1表达对AgNP的影响。 诱导指示ER应激的未折叠蛋白质应答的激活。这项工作可以发现新的 CLBC的治疗策略，并为未来的临床试验铺平道路。

Claudin-low breast cancer (CLBC), characterized by mesenchymal and cancer stem cell-like qualities, is an aggressive subtype with a poor prognosis. Currently, there are no CLBC-specific therapeutic regimens. We are the first to show that silver nanoparticles (AgNPs) possess a desirable combination of selective cytotoxicity and radiation dose enhancement effects for treatment of CLBC at doses that are non-toxic to non-cancerous breast and other cells. No other nanomaterial is known to possess a breast-cancer subtype specific cytotoxic or radiation sensitization profile. Following an extensive screening and characterization process, we now have a lead AgNP formulation that shows in vivo efficacy against CLBC following intravenous injection in tumor bearing mice, which provides evidence that a therapeutic window exists for the safe use of this nanomaterial. The selectivity of AgNPs for CLBC is due in part to a failure of CLBC cells to mitigate DNA and protein damage caused by AgNPs, and is further enhanced by what may be a general vulnerability of mesenchymal cancers like CLBC to endoplasmic reticulum (ER) stress, which we found is selectively induced in CLBC by AgNPs. Our central hypothesis is that AgNPs can be used as a form of precision medicine for the treatment of the claudin-low and other mesenchymal breast cancers. Notably, CLBC cell lines and patient samples express significantly less ESRP1 (endothelial splicing regulatory protein) than other breast cancer subtypes. ESRP1 regulates a transcriptional program necessary for epithelial to mesenchymal transition (EMT). We find that baseline ESRP1 expression inversely correlates with AgNP sensitivity and ESRP1 knockdown increases AgNP sensitivity. Although CLBC represents only 5% of breast cancers, our clinical data set of 1954 patients shows that 13% of all breast cancers are ESRP1low (defined as ≤ mean ESRP1 mRNA in CLBC). Therefore, AgNP treatment could be of benefit to a broader patient population. In AIM 1, we will test the hypothesis that our optimized AgNPs are effective for treatment and radiosensitization of CLBC without inducing cytotoxicity or DNA damage in normal breast epithelia. We will image and quantify the uptake, subcellular localization, cytotoxicity, DNA damage and radiosensitizing effects of AgNPs on CLBC and normal breast epithelia grown in 3D cell culture and in murine orthotopic tumor models. In AIM 2, we will test the hypothesis that specific effects of AgNP exposure on redox sensitive proteins, pathways and organelles contribute to the CLBC-specific mechanism of action of AgNPs. We will use the most advanced reagents and novel proteomic approached to identify oxidative damage induced by AgNP exposure in CLBC and non-CLBC cells. In AIM 3, we will test the hypothesis that an underlying sensitivity to ER stress in mesenchymal cancer cells is responsible for the specificity of AgNPs for CLBC treatment. We will evaluate the influence of ESRP1 expression on AgNP- induced activation of the unfolded protein response indicative of ER stress. This work could identify new therapeutic strategies for CLBC and pave the way for future clinical trials.