Laboratorio Biología Tumoral, Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5110566, Chile.
Millennium Institute on Immunology and Immunotherapy, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5110566, Chile.
Cells. 2023 Feb 3;12(3):506. doi: 10.3390/cells12030506.
Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1c mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1c behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1c-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1c cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.
胶质母细胞瘤（GBM）是最常见和侵袭性最强的脑肿瘤，因为其在治疗后会复发。这主要是由具有干性特征的细胞亚群介导的，这些细胞被称为胶质母细胞瘤干细胞样细胞（GSCs），它们对抗肿瘤药物具有极强的抵抗力。因此，对 GSC 发生的分子过程的理解的进步应该对降低侵袭性有重大贡献。高水平的内皮素转换酶 1（ECE1）与 GBM 的恶性进展有关，ECE1 是内皮素 1（ET-1）肽激活的关键。ECE1 有四个已知的同工型，它们在细胞质 N 端序列上有所不同，可激活 ET-1。同工型 ECE1c 被蛋白激酶 CK2 磷酸化丝氨酸 18 和丝氨酸 20，从而增加其稳定性，从而促进结肠癌细胞的侵袭性特征。为了研究 ECE1c 是否在 GBM 中发挥恶性作用，我们设计了一个 ECE1c 突变体，通过将靠近磷酸丝氨酸的推定泛素化赖氨酸（即 K6R）转换为精氨酸。该 ECE1c 突变体在 U87MG、T98G 和 U251 GBM 细胞中稳定表达，并将其行为与模拟或野生型 ECE1c 表达克隆细胞进行比较。ECE1c 在所有细胞系中均表现为高度稳定的蛋白质，其表达促进了自我更新，并富集了具有增强的神经球形成能力的干细胞样群体，以及增强的干细胞样表面标志物的表达。这些源自 ECE1c 的 GSC 样细胞还表现出对 GBM 相关化疗药物替莫唑胺和吉西他滨的增强抗性，以及 ABCG2 外排泵的表达增加。此外，ECE1c 细胞还表现出增强的转移相关特征，例如粘附的调节以及细胞迁移和侵袭的增强。总之，获得 GSC 样表型以及增强的化疗耐药性和侵袭性特征，使我们能够提出磷酸化 ECE1c 作为预后不良的新标志物以及 GBM 的潜在治疗靶点。
