Institute of Biochemistry, Friedrich-Alexander-University Erlangen-Nürnberg, Fahrstrasse 17, Erlangen, 91054, Germany.
Department of Dermatology, Faculty of Medicine and University Hospital Carl Gustav Carus at TU Dresden, Dresden, Germany.
Cell Commun Signal. 2024 May 6;22(1):256. doi: 10.1186/s12964-024-01632-8.
Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma.
Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis.
We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1.
In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.
黑色素瘤是一种高度异质性的癌症，其中信号通路的频繁激活导致其对不断变化的肿瘤微环境具有很高的适应性。阐明癌症特异性信号通路非常重要，如 PLX4032 抑制剂对黑色素瘤治疗中常见的 BrafV600E 突变的抑制作用所证明的那样。因此，我们研究了受神经营养因子 NRN1 影响的信号通路，NRN1 在黑色素瘤中被上调。
我们使用 NRN1 过表达的细胞培养模型系统，研究了 NRN1 对黑色素瘤细胞功能和信号的影响。我们采用实时细胞分析和球体形成测定法，同时使用激酶磷酸化试剂盒以及启动子活性分析，随后进行 mRNA 和蛋白质分析，来研究分子机制。
我们揭示了 NRN1 与 Notch1 和 Notch3 的裂解细胞内结构域（NICD）直接相互作用，导致 NICD 在细胞质中潜在保留，从而减少其直接下游靶标 Hes1 的表达。这导致 Hes1 驱动的磷酸化复合物中 JAK 和 STAT3 的隔离减少。因此，我们的数据显示，NRN1 过表达时，STAT3 的磷酸化减少，同时与 NRN1 过表达相关的 STAT3 总蛋白水平积累。STAT3 信号通路具有肿瘤抑制和致癌作用的潜力，促使我们研究特定的下游靶标 - 即 Vegf A、Mdr1、cMet - 这些靶标在 NRN1 的致癌水平下被上调。
总之，我们能够表明 NRN1 在黑色素瘤中连接了 Notch 和 STAT3 之间的致癌信号事件。我们还建议，在未来的研究中，应该更加关注信号分子的细胞调节，而不仅仅是经典的磷酸化事件。
