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

克拉丁-低位乳腺癌(CLBC)是一种以间充质和肿瘤干细胞样性质为特征的肿瘤。 侵袭性亚型预后较差。目前，还没有针对CLBC的治疗方案。我们是 第一个表明银纳米颗粒(AgNPs)具有理想的选择性细胞毒性组合 和治疗慢性淋巴细胞性癌的辐射剂量增强效应，剂量从无毒到非癌症 乳房和其他细胞。目前还没有其他纳米材料具有乳腺癌亚型特异性细胞毒。 或辐射敏化特征。经过广泛的筛选和鉴定过程，我们现在有 静脉注射肿瘤后显示体内抗CLBC疗效的先导型AgNP制剂 这提供了安全使用这种纳米材料的治疗窗口的证据。 AgNPs对CLBC的选择性部分归因于CLBC细胞不能减轻DNA和蛋白质损伤 由AgNPs引起，并可能因间充质癌的普遍易感性而进一步增强 如CLBC对内质网(ER)的应激，我们发现这种应激是由AgNPs选择性地诱导的。 我们的中心假设是，AgNPs可以作为一种精确的药物形式用于治疗 克拉丁-低和其他间叶性乳腺癌。值得注意的是，CLBC细胞系和患者样本表达 与其他乳腺癌亚型相比，ESRP1(内皮剪接调节蛋白)显著减少。ESRP1 调节上皮细胞向间充质细胞转化(EMT)所必需的转录程序。我们发现 基线ESRP1表达与AgNP敏感性呈负相关，ESRP1基因敲除可增加AgNP 敏感度。尽管CLBC只占乳腺癌的5%，但我们对1954名患者的临床数据集显示 13%的乳腺癌ESRP1low(定义为≤平均ESRP1mRNAin CLBC)。因此，AgNP 治疗可能会使更多的患者受益。在目标1中，我们将检验我们的假设 优化的AgNPs对CLBC的治疗和放射增敏有效，而不会引起细胞毒性或 正常乳腺上皮细胞DNA损伤。我们将成像并量化摄取，亚细胞定位， AgNPs对CLBC和正常乳腺上皮细胞的细胞毒性、DNA损伤和辐射增敏作用 3D细胞培养和小鼠原位肿瘤模型。在AIM 2中，我们将检验特定效应的假设 氧化还原敏感蛋白、途径和细胞器上的AgNP暴露有助于CLBC特异性 AgNPs的作用机制。我们将使用最先进的试剂和接近的新蛋白质组学 确定AgNP暴露在CLBC和非CLBC细胞中引起的氧化损伤。在AIM 3中，我们将测试 假设间充质癌细胞对内质网应激的潜在敏感性是导致 AgNPs对慢性淋巴细胞性白血病治疗的特异性。我们将评估ESRP1表达对AgNP-1的影响。 诱导激活指示内质网应激的未折叠蛋白反应。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.