Table 3_Focal adhesion kinase promotes ribosome biogenesis to drive advanced thyroid cancer cell growth and survival.pdf

IntroductionAdvanced thyroid cancer, including papillary (PTC) and anaplastic thyroid cancer (ATC), are the leading causes of endocrine cancer deaths. Thus, there is a critical need to identify novel therapeutic targets to improve standard of care. Focal Adhesion Kinase (FAK) is overexpressed and phosphorylated in thyroid cancer and drives thyroid cancer growth, invasion, and metastasis. FAK is a nonreceptor tyrosine kinase that is autophosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling, resulting in the recruitment of SRC proto-oncogene and downstream signaling pathways. FAK is predominately localized at the plasma membrane but has recently been shown to accumulate in the nucleus as well as the nucleolus to drive tumor growth. The nucleolus is a membraneless subnuclear organelle that is involved in ribosomal biogenesis through the transcription, processing, and assembly of ribosomal RNA (rRNA). The role of FAK in ribosome biogenesis is currently unknown.
MethodsNuclear/nucleolar FAK localization and function were studied using genetic and pharmacological approaches. High resolution microscopy was used to study the subcellular localization of FAK. Functional and biochemical assays including transformation and clonogenic assays, polysome profiling, and nascent protein synthesis assays were utilized to assess cell growth and survival. Protein-protein interactions of FAK were determined using a proximity dependent biotinylation (BioID) proteomics approach.
ResultsWe have found that pY397 FAK accumulates in the nucleolus of advanced thyroid cancer cells and that autophosphorylation of FAK at pY397 and FAK kinase activity are important for nucleolar accumulation of FAK. Furthermore, knockdown of nucleophosmin 1 (NPM1), an important structural component of the nucleolus, reduced pY397 FAK nucleolar accumulation. Functionally, we showed that nuclear FAK and FAK kinase activity are necessary for anchorage independent growth. We demonstrated that targeted degradation of FAK results in decreased protein synthesis with a specific decrease in the 60S ribosomal subunit. Using a BioID proteomics approach, we showed that autophosphorylated FAK interacts with a network of nucleolar proteins including nucleolar protein 56 (NOP56) which is a core small ribonucleoprotein (snoRNP) important for 60S ribosome biogenesis. Finally, we found that pY397 FAK co-localizes with NOP56 and that knockdown of NOP56 phenocopies FAK depletion.
ConclusionsOverall, these findings highlight a novel function for FAK in promoting ribosome biogenesis and suggest that nucleolar FAK represents a promising therapeutic target.

引言
高级别甲状腺癌，包括乳头状甲状腺癌（PTC）和间变性甲状腺癌（ATC），是内分泌系统癌症致死的首要原因。因此，亟需发掘全新治疗靶点以优化现有临床诊疗标准。黏着斑激酶（Focal Adhesion Kinase, FAK）在甲状腺癌中呈现过表达与磷酸化状态，可驱动甲状腺癌的增殖、侵袭与转移。FAK属于非受体酪氨酸激酶，在整合素或生长因子受体信号刺激下，会在酪氨酸397位点（Y397）发生自磷酸化，进而招募SRC原癌基因并激活下游信号通路。FAK主要定位于细胞膜，但近期研究显示其亦可在细胞核乃至核仁中聚集，从而推动肿瘤生长。核仁是无膜包被的亚核细胞器，通过核糖体RNA（rRNA）的转录、加工与组装参与核糖体生物发生。目前FAK在核糖体生物发生中的作用仍不明晰。

方法
本研究通过遗传与药理学手段，探究核/核仁定位的FAK的定位特征与功能。采用高分辨率显微镜观察FAK的亚细胞定位。通过转化实验、集落形成实验、多聚核糖体谱分析以及新生蛋白质合成实验等功能与生化检测手段，评估细胞增殖与存活情况。利用邻近依赖生物素标记（BioID）蛋白质组学方法，鉴定FAK的蛋白质相互作用网络。

结果
本研究发现，磷酸化Y397的FAK（pY397 FAK）会在高级别甲状腺癌细胞的核仁中聚集，且FAK在pY397位点的自磷酸化及FAK激酶活性，对FAK的核仁聚集至关重要。进一步实验显示，敲低核仁重要结构组分核仁磷酸蛋白1（NPM1），可减少pY397 FAK的核仁聚集。功能实验表明，核FAK及其激酶活性是细胞非锚定依赖性生长的必要条件。研究证实，靶向降解FAK会降低蛋白质合成速率，并特异性减少60S核糖体亚基的含量。通过BioID蛋白质组学分析，我们发现自磷酸化的FAK可与一系列核仁蛋白形成相互作用网络，其中包括核仁蛋白56（NOP56）——后者是参与60S核糖体生物发生的核心小核糖核蛋白（snoRNP）。最后，我们观察到pY397 FAK与NOP56存在共定位现象，且敲低NOP56的表型与FAK耗竭一致。

结论
综上，本研究揭示了FAK在促进核糖体生物发生中的全新功能，并提示核仁定位的FAK有望成为极具潜力的治疗靶点。