Hishida Seiji, Kawakami Kyojiro, Fujita Yasunori, Kato Taku, Takai Manabu, Iinuma Koji, Nakane Keita, Tsuchiya Tomohiro, Koie Takuya, Miura Yuri, Ito Masafumi, Mizutani Kosuke
Department of Urology, Gifu University Graduate School of Medicine, Gifu, Japan.
Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
Prostate. 2021 Jun;81(9):592-602. doi: 10.1002/pros.24138. Epub 2021 Apr 27.
Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines.
Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis.
Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated.
Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.
卡巴他赛（CBZ）目前广泛用于对多西他赛（DOC）耐药的前列腺癌（PC）患者，然而，大多数患者最终会产生耐药性。因此，发现新的耐药治疗靶点将大有裨益。我们旨在通过蛋白质组学分析从后来产生CBZ耐药性的DOC耐药PC患者血清中分离的细胞外囊泡（EVs）以及从DOC和CBZ耐药PC细胞系培养基中收获的EVs，来鉴定CBZ耐药的候选治疗靶点。
使用与磁珠偶联的含T细胞免疫球蛋白结构域和粘蛋白结构域蛋白4（Tim4），从获得CBZ耐药性前后收集的DOC耐药PC患者血清以及DOC耐药（22Rv1DR）和CBZ耐药（22Rv1CR）PC细胞系的条件培养基中纯化EVs。通过纳升液相色谱-串联质谱（nanoLC-MS/MS）对EVs进行蛋白质分析，随后进行蛋白质表达的比较分析和网络分析。通过WST-１法评估磷脂酰肌醇-3-激酶（PI3K）抑制剂ZSTK474的细胞毒性作用。通过蛋白质印迹分析检测PI3K和PTEN的表达及磷酸化。
在差异调节的蛋白质中，CBZ耐药PC细胞系和患者的EVs中分别有77种和61种蛋白质显著增加。两个数据集之间的比较显示，果糖二磷酸醛缩酶、胞质非特异性二肽酶、CD63、CD151、肌球蛋白轻链9和过氧化物酶体增殖物激活受体6这六种蛋白质在细胞系和患者的EVs中均升高。对增加的EV蛋白质进行网络分析，确定了与CBZ耐药相关的途径，包括PI3K信号通路。ZSTK４74显著抑制22Rv1CR细胞的生长并提高其对CBZ的敏感性。在22Rv1CR细胞中，PI3K被激活，而抑制PI3K的PTEN失活。
以Tim-4作为工具成功完成了血清EVs的蛋白质组学分析，以分离高度纯化的EVs。我们的结果表明，CBZ与PI3K抑制剂联合使用可能是CBZ耐药PC患者的一种有前景的治疗选择。
