WWOX interacts with SMAD3 and modulates its transcriptional activity in breast cells

WWOX expression is lost during tumor progression in many human malignancies including breast cancer. To understand the effects of loss of WWOX expression we analyzed the consequences of its silencing in normal human breast cells (MCF10F). WWOX silencing led to the formation of larger cell colonies, increased cell motility and decreased cell attachment. WWOX silenced cells demonstrated deregulated expression on genes involved in cell cycle, DNA damage response and cell motility. We detected an enrichment of targets activated by the SMAD3 transcription factor. Most notably expression of ANGPTL4, FST, PTHLH and SERPINE1 were all significantly increased upon WWOX silencing. Upregulation of these genes can be reversed by re-expressing WWOX in the previously silenced cells thus suggesting an inverse correlation between WWOX protein expression and SMAD3 transcriptional activity. Importantly, we demonstrate that WWOX physically interacts with SMAD3 protein via WW domain 1, that WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter (3TP-LUX). Furthermore, WWOX expression leads to intracellular redistribution of SMAD3 protein levels redirecting protein availability from the nuclear to the cytoplasmic compartment. Interestingly, meta-analysis of gene expression breast cancer datasets indicate that WWOX and ANGPTL4 expression, encoding a secreted protein of key relevance in breast cancer lung metastatic cells, are inversely correlated and the WWOXlo/ANGPTL4hi cluster of tumors are enriched in triple-negative and basal-like sub-types. In summary, we demonstrate that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer. We compared two independent shRNAs: shWWOX-A and shWWOX-B with 3 biological replicates each one, targeting different regions of the WWOX transcript as a means of ruling out any potential off-target effects.

在包括乳腺癌在内的多种人类恶性肿瘤的进展过程中，WWOX的表达会发生丢失。为阐明WWOX表达缺失所产生的生物学效应，我们针对正常人类乳腺细胞系MCF10F中WWOX的沉默效应展开了分析。
WWOX沉默可诱导形成更大的细胞集落，增强细胞迁移能力，并降低细胞黏附性。WWOX沉默的细胞中，参与细胞周期、DNA损伤应答以及细胞迁移的基因表达出现失调。我们检测到SMAD3转录因子激活的靶基因存在富集现象，其中最为显著的是ANGPTL4、FST、PTHLH与SERPINE1的表达在WWOX沉默后均显著上调。通过在此前沉默了WWOX的细胞中重新表达WWOX，上述基因的上调效应可被逆转，这表明WWOX蛋白表达与SMAD3转录活性之间存在负相关关系。
重要的是，我们证实WWOX可通过第1个WW结构域与SMAD3蛋白发生直接的物理相互作用；WWOX的表达可显著降低SMAD3在ANGPTL4与SERPINE1启动子区域的结合占有率，并大幅抑制TGFβ响应报告基因3TP-LUX的激活。此外，WWOX的表达会导致SMAD3蛋白在细胞内发生重新分布，将其从细胞核内重新分配至细胞质区域。
值得注意的是，对乳腺癌基因表达数据集的荟萃分析结果表明，WWOX与ANGPTL4的表达呈负相关（ANGPTL4编码的分泌蛋白与乳腺癌肺转移细胞的关键病理过程密切相关）；而WWOX低表达/ANGPTL4高表达的肿瘤亚群，在三阴性乳腺癌和基底样亚型中富集。
综上，我们证实WWOX可通过直接结合WW结构域并将SMAD3蛋白潜在隔离于细胞质的方式，调控乳腺细胞中的SMAD3信号通路。鉴于WWOX的表达丢失随乳腺癌进展而加剧，且WWOX可作为SMAD3转录活性的抑制剂，上述发现至少可以部分解释TGFβ信号通路在晚期乳腺癌中表现出的矛盾的促肿瘤发生效应。
为排除潜在的脱靶效应，我们采用了两种独立的短发夹RNA（short hairpin RNA, shRNA）：shWWOX-A与shWWOX-B，二者分别靶向WWOX转录本的不同区域，且每组均设置3次生物学重复。