BACKGROUND Signaling pathways critical for embryonic development re-emerge in adult pancreas during tumorigenesis. Aspartate β-hydroxylase (ASPH) drives embryonic cell motility/invasion in pancreatic development/differentiation. We explored if dysregulated ASPH is critically involved in pancreatic cancer pathogenesis. METHODS To demonstrate if/how ASPH mediates malignant phenotypes, proliferation, migration, 2-D/3-D invasion, pancreatosphere formation, immunofluorescence, Western blot, co-immunoprecipitation, invadopodia formation/maturation/function, qRT-PCR, immunohistochemistry (IHC), and self-developed in vitro metastasis assays were performed. Patient-derived xenograft (PDX) models of human pancreatic ductal adenocarcinoma (PDAC) were established to illustrate in vivo antitumor effects of the third-generation small molecule inhibitor specifically against ASPH's β-hydroxylase activity. Prognostic values of ASPH network components were evaluated with Kaplan-Meier plots, log-rank tests, and Cox proportional hazards regression models. RESULTS ASPH renders pancreatic cancer cells more aggressive phenotypes characterized by epithelial-mesenchymal transition (EMT), 2-D/3-D invasion, invadopodia formation/function as demonstrated by extracellular matrix (ECM) degradation, stemness (cancer stem cell marker upregulation and pancreatosphere formation), transendothelial migration (mimicking intravasation/extravasation), and sphere formation (mimicking metastatic colonization/outgrowth at distant sites). Mechanistically, ASPH activates SRC cascade through direct physical interaction with ADAM12/ADAM15 independent of FAK. The ASPH-SRC axis enables invadopodia construction and initiates MMP-mediated ECM degradation/remodeling as executors for invasiveness. Pharmacologic inhibition of invadopodia attenuates in vitro metastasis. ASPH fosters primary tumor development and pulmonary metastasis in PDX models of PDAC, which is blocked by a leading compound specifically against ASPH enzymatic activity. ASPH is silenced in normal pancreas, progressively upregulated from pre-malignant lesions to invasive/advanced stages of PDAC. Expression profiling of ASPH-SRC network components independently/jointly predicts clinical outcome of PDAC patients. Compared to a negative-low level, a moderate-very high level of ASPH, ADAM12, activated SRC, and MMPs correlated with curtailed overall survival (OS) of pancreatic cancer patients (log-rank test, ps < 0.001). The more unfavorable molecules patients carry, the more deleterious prognosis is destinated. Patients with 0-2 (n = 4), 3-5 (n = 8), 6-8 (n = 24), and 9-12 (n = 73) unfavorable expression scores of the 5 molecules had median survival time of 55.4, 15.9, 9.7, and 5.0 months, respectively (p < 0.001). CONCLUSION Targeting the ASPH-SRC axis, which is essential for propagating multi-step PDAC metastasis, may specifically/substantially retard development/progression and thus improve prognosis of PDAC.

中文翻译：

---

天冬氨酸β-羟化酶通过激活SRC信号通路促进胰腺导管腺癌转移。

背景技术对于胚胎发育至关重要的信号传导途径在成瘤过程中在成年胰腺中重新出现。天冬氨酸β-羟化酶（ASPH）在胰腺发育/分化中驱动胚胎细胞运动/侵袭。我们探讨了ASPH失调是否严重参与了胰腺癌的发病机理。方法证明ASPH是否/如何介导恶性表型，增殖，迁移，2-D / 3-D侵袭，胰球形成，免疫荧光，蛋白质印迹，共免疫沉淀，侵染伪足形成/成熟/功能，qRT-PCR，免疫组织化学（IHC） ），并进行了自行开发的体外转移试验。建立了人类胰腺导管腺癌（PDAC）的患者源异种移植（PDX）模型，以说明第三代小分子抑制剂在体内针对ASPH的β-羟化酶活性的体内抗肿瘤作用。用Kaplan-Meier图，对数秩检验和Cox比例风险回归模型评估ASPH网络组件的预后价值。结果ASPH使胰腺癌细胞更具侵略性，表现为上皮-间质转化（EMT），2-D / 3-D侵袭，侵袭伪足形成/功能，如细胞外基质（ECM）降解，干性（癌症干细胞标志物上调和胰腺球形成），跨内皮迁移（模仿血管内/外渗），和球体形成（模仿远处的转移性定植/生长）。从机理上讲，ASPH通过与ADAM12 / ADAM15的直接物理交互（独立于FAK）激活SRC级联。ASPH-SRC轴可实现伪足构造并启动MMP介导的ECM降解/重塑，作为侵入性的执行者。侵染伪足的药理学抑制作用减弱了体外转移。ASPH在PDAC的PDX模型中促进原发性肿瘤的发展和肺转移，这被专门针对ASPH酶活性的领先化合物所阻断。在正常胰腺中，ASPH处于沉默状态，从恶性前病变到PDAC的侵入/进展阶段逐渐上调。ASPH-SRC网络组件的表达谱独立/共同预测PDAC患者的临床结局。与负低水平相比，ASPH，ADAM12，活化的SRC和MMPs的中等至非常高的水平与胰腺癌患者的总体生存期（OS）降低相关（对数秩检验，ps <0.001）。患者携带的不良分子越多，预后就越有害。5个分子的表达评分为0-2（n = 4），3-5（n = 8），6-8（n = 24）和9-12（n = 73）的患者的中位生存时间为分别为55.4、15.9、9.7和5.0个月（p <0.001）。结论以ASPH-SRC轴为靶点，这对于传播多步PDAC转移是必不可少的，它可以特异性地/实质上延迟发育/进展，从而改善PDAC的预后。MMPs与胰腺癌患者总生存期（OS）降低有关（log-rank test，ps <0.001）。患者携带的不良分子越多，预后就越有害。5个分子的表达评分为0-2（n = 4），3-5（n = 8），6-8（n = 24）和9-12（n = 73）的患者的中位生存时间为分别为55.4、15.9、9.7和5.0个月（p <0.001）。结论以ASPH-SRC轴为靶点，这对于传播多步PDAC转移是必不可少的，它可以特异性地/实质上延迟发育/进展，从而改善PDAC的预后。MMPs与胰腺癌患者总生存期（OS）降低有关（log-rank test，ps <0.001）。患者携带的不良分子越多，预后就越有害。5个分子的表达评分为0-2（n = 4），3-5（n = 8），6-8（n = 24）和9-12（n = 73）的患者的中位生存时间为分别为55.4、15.9、9.7和5.0个月（p <0.001）。结论以ASPH-SRC轴为靶点，这对于传播多步PDAC转移是必不可少的，它可以特异性地/实质上延迟发育/进展，从而改善PDAC的预后。和9-12（n = 73）的5个分子的不良表达得分的中位生存时间分别为55.4、15.9、9.7和5.0个月（p <0.001）。结论以ASPH-SRC轴为靶点，这对于传播多步PDAC转移是必不可少的，它可以特异性地/实质上延迟发育/进展，从而改善PDAC的预后。和9-12（n = 73）的5个分子的不良表达得分的中位生存时间分别为55.4、15.9、9.7和5.0个月（p <0.001）。结论以ASPH-SRC轴为靶点，这对于传播多步PDAC转移是必不可少的，它可以特异性地/实质上延迟发育/进展，从而改善PDAC的预后。