Ungefroren Hendrik, Braun Rüdiger, Lapshyna Olha, Konukiewitz Björn, Wellner Ulrich F, Lehnert Hendrik, Marquardt Jens-Uwe
First Department of Medicine, University Hospital Schleswig-Holstein, Campus Lübeck, D-23538 Lübeck, Germany.
Institute of Pathology, University Hospital Schleswig-Holstein, Campus Kiel, D-24105 Kiel, Germany.
Biomedicines. 2022 Oct 20;10(10):2640. doi: 10.3390/biomedicines10102640.
Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells' migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment.
胰腺导管腺癌（PDAC）细胞以其高侵袭/转移潜能而闻名，这部分受转化生长因子β1（TGFβ1）调控。至少两种I型受体，即ALK5和ALK2参与其中，它们分别通过不同的Smad蛋白SMAD2/3和SMAD1/5传递TGFβ的下游信号，这就引发了它们在调节细胞运动中相对贡献的问题。实时细胞迁移分析表明，通过RNA干扰或用激酶失活突变体（ALK2-K233R）进行显性负性干扰选择性抑制ALK2，在有无TGFβ1刺激的情况下均能强烈增强细胞的迁移活性。异位表达ALK2-K233R与RAC1及其可变剪接异构体RAC1b的蛋白水平升高有关，这两者都与驱动细胞迁移和侵袭有关。相反，RNA干扰介导的RAC1b敲低或CRISPR/Cas9介导的RAC1b敲除导致ALK2表达上调，但相关的骨形态发生蛋白I型受体ALK3或ALK6的表达未上调，并提高了SMAD1/5的磷酸化水平。PDAC是一种异质性疾病，包括具有不同组织形态学亚型的肿瘤，从上皮/经典型到极间充质型。用ALK2抑制剂LDN-193189处理代表这些亚型的各种已建立的和原发性PDAC细胞系时，分化良好的上皮细胞系对基础和TGFβ1依赖性迁移活性的增强反应比分化差的间充质细胞系更强。这些数据表明：（i）ALK2通过抑制RAC1/RAC1b蛋白来抑制迁移；（ii）ALK2和RAC1b在一个自我延续的自动调节负反馈回路中共同作用，以相互控制它们的表达；（iii）ALK2的抗迁移功能在保护上皮亚型细胞不自发地以及在富含TGFβ的肿瘤微环境中不发生侵袭方面似乎尤为关键。
