A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

溶酶体运输（lysosome trafficking）在肿瘤侵袭中发挥关键作用，而肿瘤侵袭是转移发生的核心事件。本实验室既往研究证实，在肝细胞生长因子（hepatocyte growth factor, HGF）或细胞外酸性pH（extracellular pH, pHe）等肿瘤微环境刺激下，溶酶体向细胞表面的顺向（向外）运动可增强组织蛋白酶B（cathepsin B）分泌，进而促进肿瘤细胞侵袭。顺向溶酶体运输依赖于钠氢交换体（sodium-proton exchanger）的活性，且可通过曲格列酮（Troglitazone）或EIPA阻断这类离子泵而实现逆转。鉴于上述药物因毒性问题无法推进临床转化，我们设计了一套经过验证且可重复的高内涵筛选实验，旨在发现能够阻断外周溶酶体运输的药物，以鉴定出可抑制肿瘤细胞侵袭的新型候选药物。我们采用自动化高内涵成像系统（Cellomics）检测溶酶体相对于细胞核的定位情况。在共计筛选的2210种已上市再利用药物与天然产物药物中，共鉴定出18个阳性命中物。其中一种被认定为顺向溶酶体运输抑制剂的化合物为氯硝柳胺（niclosamide），一款已上市的人用抗蠕虫药物。进一步研究显示，在临床相关浓度下，氯硝柳胺可阻断酸性pHe、HGF及表皮生长因子（epidermal growth factor, EGF）诱导的顺向溶酶体重新分布、蛋白酶分泌、细胞运动以及去势抵抗性前列腺癌细胞DU145的侵袭能力。为阐明氯硝柳胺阻断顺向溶酶体运动的具体机制，我们发现该药物对三磷酸腺苷（ATP）水平、微管或肌动蛋白丝均无显著影响，对磷脂酰肌醇3-激酶（PI3K）与丝裂原活化蛋白激酶（MAPK）通路的作用也极为微弱。氯硝柳胺可破坏溶酶体腔内的pH梯度，但不会破坏溶酶体膜；而巴弗洛霉素（bafilomycin）——一种损害溶酶体酸化的试剂——在我们的模型中同样可诱导核周溶酶体聚集（juxtanuclear lysosome aggregation, JLA）。综上，上述数据表明，氯硝柳胺通过调控溶酶体酸化相关通路，促进核周溶酶体聚集（JLA）。综上，我们已设计出一套经过验证且可重复的高内涵筛选实验方法，用于筛选可抑制溶酶体运输并降低肿瘤侵袭能力的药物，并总结了其中一款候选药物的作用机制。