Department of Pathology and Laboratory Medicine (JFL, LB, RCW, SJW, SLC), Medical University of South Carolina, 171 Ashley Avenue, MSC 908, Charleston, SC, 29425-9080, USA.
Department of Pathology (SNB, KAR) and the Medical Scientist Training Program (SNB), University of Alabama at Birmingham, Birmingham, AL, 35294-0017, USA.
Cell Commun Signal. 2019 Jul 10;17(1):74. doi: 10.1186/s12964-019-0388-5.
We have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1-null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades.
ErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P-GGFβ3;Trp53;Erbb4 mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4-null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation.
Aberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P-GGFβ3 and P-GGFβ3;Trp53 mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4-null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4-null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1β-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation.
ErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.
我们发现 erbB 受体酪氨酸激酶驱动 NF1 缺失的恶性外周神经鞘瘤（MPNST）中的 Ras 过度激活和生长。然而，MPNST 可变地表达具有不同功能特征的多种 erbB 受体，尚不清楚哪些受体驱动 MPNST 发病机制。在这里，我们通过测试改变 erbB4 表达通过独特地激活关键细胞质信号级联来促进 MPNST 发病机制的假设。
使用免疫组织化学、免疫细胞化学、免疫印迹和实时 PCR 评估 erbB4 表达。为了定义 erbB4 功能，我们生成了具有 floxed Erbb4 等位基因（P-GGFβ3；Trp53；Erbb4 小鼠）并在这些肿瘤中消除 Erbb4 的 MPNST 发生的小鼠。使用 H-胸腺嘧啶掺入、MTT 测定、实时 Glo 和细胞计数测定评估 MPNST 细胞的增殖和存活。将对照和 Erbb4 缺失的 MPNST 细胞原位异种移植到免疫缺陷小鼠中，并分析这些移植物的生长、增殖（Ki67 标记）、凋亡（TUNEL 标记）和血管生成。使用细胞质激酶的抗体阵列来鉴定 erbB4 反应性激酶。使用药理学或遗传学抑制来鉴定驱动增殖的 erbB4 反应性激酶。
在 30 例手术切除的人类 MPNST 中以及在基因工程小鼠模型（P-GGFβ3 和 P-GGFβ3；Trp53 小鼠）中均可见异常 erbB4 表达；在源自 MPNST 的细胞系中存在多种 erbB4 剪接变体，其在激活 PI3 激酶和核信号方面的能力不同。与未消融对照相比，Erbb4 缺失的 MPNST 细胞表现出增殖和存活减少以及形态改变。Erbb4 缺失细胞的原位同种异体移植物明显小于对照，增殖、存活和血管生成减少。在人类 MPNST 细胞中敲低 ERBB4 同样抑制了 DNA 合成和活力。尽管我们之前已经表明广谱 erbB 抑制剂抑制 Ras 激活，但 Erbb4 消融并不影响 Ras 激活，这表明 erbB4 通过非 Ras 依赖性途径驱动肿瘤发生。对 43 种候选激酶的分析确定了多个 NRG1β 反应性和 erbB4 依赖性信号通路，包括 PI3K、WNK1、STAT3、STAT5 和磷脂酶-Cγ 通路。尽管 WNK1 抑制不会改变增殖，但抑制 STAT3、STAT5 和磷脂酶-Cγ 会显著降低增殖。
ErbB4 通过激活关键的非 Ras 依赖性信号级联，包括 STAT3、STAT5 和磷脂酶-Cγ 途径，促进 MPNST 的生长。因此，ErbB4 及其效应途径是 MPNST 中潜在有用的治疗靶点。
