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Abstract：
BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers.
METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing.
RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2.
CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.
METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing.
RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2.
CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.
摘要：
背景：铂耐药是卵巢癌（OC）患者生存不良的主要原因。化疗耐药的靶向治疗和生物标志物对OC患者的治疗至关重要。我们以前的研究确定了细胞表面CD55，补体调节蛋白的成员，驱动癌症干细胞（CSC）的化学抗性和维持。CSC与多种癌症的肿瘤复发和转移有关。
方法：使用包括免疫荧光和亚细胞分级分离的蛋白质定位测定法来鉴定癌细胞表面和细胞核处的CD55。蛋白质半衰期测定用于比较细胞表面和核CD55的稳定性。产生CD55缺失突变体并将其引入癌细胞中以鉴定核运输代码，顺铂敏感性,和使用体外和体内模型测定的干细胞频率。CD55结合蛋白的检测通过免疫沉淀和随后的质谱分析。通过RNA测序鉴定由CD55激活的靶途径。
结果：CD55定位于OC标本子集的细胞核，化疗耐药患者的腹水，并富含化学抗性OC细胞。我们确定核CD55是糖基化的并且来源于CD55的细胞表面池。核定位由含有CD55的丝氨酸/苏氨酸(S/T)结构域的运输密码驱动。核CD55是顺铂耐药所必需的，stemness,和OC细胞中的细胞增殖。CD55S/T结构域对于在体外和体内模型中核进入和诱导对顺铂的化学抗性是必需的。CD55S/T结构域的缺失足以使化学抗性OC细胞对顺铂敏感。在细胞核中，CD55结合并减弱表观遗传调节因子和肿瘤抑制因子ZMYND8，同时增加H3K27三甲基化和Polycomb阻遏复合物2的成员。
结论：第一次，我们显示CD55定位于OC的细胞核，并促进CSC和化疗耐药。我们的研究确定了通过阻断CD55核进入治疗铂耐药卵巢癌的治疗机制。
方法：使用包括免疫荧光和亚细胞分级分离的蛋白质定位测定法来鉴定癌细胞表面和细胞核处的CD55。蛋白质半衰期测定用于比较细胞表面和核CD55的稳定性。产生CD55缺失突变体并将其引入癌细胞中以鉴定核运输代码，顺铂敏感性,和使用体外和体内模型测定的干细胞频率。CD55结合蛋白的检测通过免疫沉淀和随后的质谱分析。通过RNA测序鉴定由CD55激活的靶途径。
结果：CD55定位于OC标本子集的细胞核，化疗耐药患者的腹水，并富含化学抗性OC细胞。我们确定核CD55是糖基化的并且来源于CD55的细胞表面池。核定位由含有CD55的丝氨酸/苏氨酸(S/T)结构域的运输密码驱动。核CD55是顺铂耐药所必需的，stemness,和OC细胞中的细胞增殖。CD55S/T结构域对于在体外和体内模型中核进入和诱导对顺铂的化学抗性是必需的。CD55S/T结构域的缺失足以使化学抗性OC细胞对顺铂敏感。在细胞核中，CD55结合并减弱表观遗传调节因子和肿瘤抑制因子ZMYND8，同时增加H3K27三甲基化和Polycomb阻遏复合物2的成员。
结论：第一次，我们显示CD55定位于OC的细胞核，并促进CSC和化疗耐药。我们的研究确定了通过阻断CD55核进入治疗铂耐药卵巢癌的治疗机制。
