Dissecting ALK4 Function in Cancer Progression

 描述（由申请人提供）：激活素受体样激酶4（ALK 4）是I型转化生长因子-β（TGF-β）超家族受体，其介导几种TGF-β超家族配体（包括激活素、nodal和GDF 1）的信号传导。ALK 4的失活突变发生在一小部分乳腺癌和胰腺癌中。此外，ALK 4表达在乳腺癌和胰腺癌患者中经常降低/丢失，丢失与不良预后相关。ALK 4也被鉴定为一种基因，其破坏与致癌Ras合作，以促进小鼠模型中的胰腺癌进展。虽然这些数据表明ALK 4功能丧失在乳腺癌和胰腺癌进展中的重要作用，但ALK 4促进癌症进展的机制尚不清楚。我们已经证明，在乳腺和胰腺模型中，ALK 4表达的丧失与转移潜力一致，ALK 4表达的沉默增加了癌细胞的迁移和侵袭、上皮-间质转化的标志物和典型的TGF-β信号传导。从机制上讲，沉默ALK 4表达会增加I型（TβRI/ALK 5）和II型（TβRII）TGF-β受体水平。基于这些初步研究，我们假设ALK 4的缺失通过降低TGF-β受体内化、增加TGF-β受体水平、经典TGF-β信号传导和TGF-β诱导的EMT促进乳腺癌和胰腺癌的侵袭和转移。这个假设将通过两个目标来解决。具体目标1：确定ALK 4缺失是否通过降低TGF-β受体内化和增加TGF-β受体水平促进TGF-β信号传导和EMT。为此，我们将（a）确定ALK 4是否抑制TGF-β配体结合、受体复合物形成、稳定性和/或TGF-β受体内化;（B）确定Smad（2/3，1/5/8）和/或非Smad（MAPK、PI 3 K、NF-kB）信号传导通路通过ALK 4的缺失而促进;（c）鉴定癌细胞系和人类癌症样本中由ALK 4沉默诱导的TGF-β靶基因，和（d）通过对TGF-β信号传导的这些影响确定ALK 4的丧失是否介导EMT和侵袭。具体目标2：确定ALK 4的缺失是否促进体内癌症转移。为此，我们将（a）确定转移性乳腺癌和胰腺癌细胞中恢复ALK 4表达是否降低其体内转移潜力，以及降低ALK 4表达是否增加乳腺癌和胰腺癌细胞的体内侵袭性和转移潜力;（B）确定ALK 4损失是否通过增加TGF-β受体表达诱导癌症转移;和（c）研究源自ALK 4表达降低的细胞的转移是否对临床开发的TGF-β受体抑制剂敏感。这些研究将确定ALK 4丢失促进癌症进展的机制，建立调节TGF-β信号传导的新机制，并可能将ALK 4丢失鉴定为抗TGF-β方法的预测生物标志物。

 DESCRIPTION (provided by applicant): Activin receptor-like kinase 4 (ALK4) is a type I transforming growth factor- (TGF-) superfamily receptor that mediates signaling for several TGF- superfamily ligands including activin, nodal and GDF1. Inactivating mutations in ALK4 occur in a small percentage of breast and pancreatic cancers. In addition, ALK4 expression is frequently decreased/lost in breast and pancreatic cancer patients, with loss associated with a poor prognosis. ALK4 was also identified as a gene whose disruption cooperates with oncogenic Ras to promote pancreatic cancer progression in a murine model. While these data suggest an important role for loss of ALK4 function in breast and pancreatic cancer progression, the mechanism by which ALK4 contributes to cancer progression is unknown. We have demonstrated that loss of ALK4 expression coincides with metastatic potential in breast and pancreatic models, with silencing of ALK4 expression increasing cancer cell migration and invasion, markers of epithelial-mesenchymal transition, and canonical TGF-β signaling. Mechanistically, silencing ALK4 expression increases type I (TβRI/ALK5) and type II (TβRII) TGF-β receptor levels. Based on these preliminary studies, we hypothesize that loss of ALK4 promotes breast and pancreatic cancer invasion and metastasis by decreasing TGF-β receptor internalization, increasing TGF-β receptor levels, canonical TGF-β signaling and TGF-β-induced EMT. This hypothesis will be addressed by 2 aims. Specific Aim 1: To determine whether loss of ALK4 promotes TGF-β signaling and EMT by decreasing TGF-β receptor internalization and increasing TGF-β receptor levels. In this aim, we will (a) determine whether ALK4 inhibits TGF-β ligand binding, receptor complex formation, stability and/or internalization of TGF-β receptors; (b) determine whether Smad (2/3, 1/5/8) and/or non-Smad (MAPK, PI3K, NF-kB) signaling pathways are promoted by loss of ALK4; (c) identify TGF-β target genes induced by ALK4 silencing in cancer cell lines and human cancer specimens and (d) establish whether loss of ALK4 mediates EMT and invasion through these effects on TGF-β signaling. Specific Aim 2: To determine whether loss of ALK4 promotes cancer metastasis in vivo. In this aim, we will (a) determine whether restoring ALK4 expression in metastatic breast and pancreatic cancer cells decreases their metastatic potential in vivo, and whether decreasing ALK4 expression increases invasiveness and metastatic potential of breast and pancreatic cancer cells in vivo; (b) determine whether ALK4 loss induces cancer metastasis via increasing TGF-β receptor expression; and (c) investigate whether metastasis derived from cells with decreased ALK4 expression are sensitive to clinically developed TGF-β receptor inhibitors. These studies will define mechanisms by which ALK4 loss promotes cancer progression, establish a novel mechanism for regulating TGF-β signaling and potentially identify ALK4 loss as a predictive biomarker for anti-TGF-β approaches.