Signal Transduction of Cellular Motility, Internal Medicine IV, Science Unit for Basic and Clinical Medicine, Justus Liebig University Giessen, Aulweg 128, D-35392, Giessen, Germany.
Molecular Oncology of Solid Tumors, Internal Medicine IV, Justus Liebig University Giessen, Aulweg 128, D-35392, Giessen, Germany.
Cell Commun Signal. 2024 Jan 30;22(1):85. doi: 10.1186/s12964-024-01484-2.
K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.
K-Ras 是胰腺、结肠和非小细胞肺癌腺癌中最常发生突变的 Ras 变体。K-Ras 的激活突变导致活性 Ras-GTP 的增加，从而导致效应蛋白和下游信号通路的过度激活。在这里，我们证明致癌 K-Ras(V12) 通过激活磷脂酰肌醇 3-激酶 (PI3-K)/Akt 途径来调节肿瘤细胞迁移，并通过上调 Akt3 诱导 E-钙粘蛋白和神经细胞粘附分子 (NCAM) 的表达。体外相互作用和共沉淀测定鉴定出 PI3-Kα 是活性 K-Ras4B 的真正效应物，但不是 H-Ras 或 N-Ras 的效应物，导致 Akt 磷酸化增强。此外，K-Ras(V12)诱导的 PI3-K/Akt 激活增强了所有分析的细胞系中的迁移。有趣的是，使用 Akt 同工型特异性抗体的 Western blot 分析和 qPCR 研究表明，在 EGFP-K-Ras(V12)表达细胞克隆中，Akt3 的量和活性明显增加，而 Akt1 和 Akt2 的量下调。为了更详细地研究每个 Akt 同工型的功能作用和可能的同工型间的串扰，我们在 PANC-1 胰腺和 H23 肺癌细胞中稳定耗尽每个同工型。在大多数细胞系中表达最少的 Akt 同工型 Akt3 在 Akt2 耗尽的细胞中特别上调和激活。由于 EGFP-K-Ras(V12)的表达通过诱导多涎酸化的 NCAM 降低了 E-钙粘蛋白介导的细胞间粘附，因此分析了 Akt3 作为 E-钙粘蛋白和 NCAM 的调节剂。Western blot 分析显示 Akt3-kd 细胞中 E-钙粘蛋白和 NCAM 的表达明显减少，而 Akt1 和 Akt2 的缺失上调了 E-钙粘蛋白，特别是在 H23 肺癌细胞中。总之，我们确定致癌 K-Ras4B 是 PI3-Kα-Akt 信号的关键调节剂，Akt3 是 K-Ras4B 诱导的 E-钙粘蛋白和 NCAM 表达和定位调节的关键调节剂。
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