Section on Cellular Neurobiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Biostatistics & Bioinformatics Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2022 Mar 14;23(6):3113. doi: 10.3390/ijms23063113.
Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model.
Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays.
Exosomes released from cancer cells contain mRNA and protein. mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and , a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells.
We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.
外泌体通过细胞间通讯促进肿瘤生长和转移，尽管其机制尚不清楚。羧肽酶 E（CPE）支持包括肝细胞癌（HCC）在内的不同癌症的进展。在这里，我们使用 HCC 细胞系作为癌症模型，研究了 CPE 是否是外泌体中的生物活性有效成分，以及它是否有助于肿瘤发生。
从癌细胞或人血清的上清液中分离出外泌体。使用 PCR 和 Western blot 分析 mRNA 和蛋白质表达。用来自高转移性 HCC97H 细胞的外泌体孵育低转移性 HCC97L 细胞。在其他实验中，用负载 CPE-shRNA 的外泌体孵育 HCC97H 细胞。使用 MTT、集落形成和基质胶侵袭实验评估细胞增殖和侵袭。
从癌细胞释放的外泌体包含 mRNA 和蛋白质。来自高转移性与低转移性细胞的外泌体中富集了 mRNA 水平，跨越各种癌症类型。在一项初步研究中，与健康受试者相比，癌症患者血清中外泌体的 拷贝数显著更高。用来自 HCC97H 细胞的外泌体处理的 HCC97L 细胞显示出增强的增殖和侵袭；然而，用 CPE-shRNA 预处理的 HCC97H 细胞的外泌体未能促进增殖。当用负载 CPE-shRNA 的 HEK293T 外泌体孵育 HCC97H 细胞时，CPE、细胞周期调节蛋白 Cyclin D1 和原癌基因 的表达受到抑制，导致 HCC97H 细胞增殖减少。
我们确定 CPE 是驱动低转移性 HCC 细胞生长和侵袭的外泌体生物活性分子。负载 CPE-shRNA 的外泌体可通过抑制 Cyclin D1 和 c-MYC 抑制恶性肿瘤细胞增殖。因此，CPE 是外泌体传递肿瘤发生的关键因素，基于外泌体的 CPE-shRNA 递送为肿瘤进展提供了一种潜在的治疗方法。值得注意的是，测量癌症患者血清中外泌体中的 CPE 转录本水平可能具有潜在的液体活检应用。
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