Focal Adhesion Kinase (FAK) is an important regulator of tumor cell proliferation, survival and metastasis. As such it has become a therapeutic target of interest in cancer. Previous studies suggested that use of FAK tyrosine kinase inhibitors (TKIs) blocks osteolysis in in vivo models of bone metastasis. However, from these studies it was not clear whether FAK TKIs blocked bone degradation by osteoclasts or also promoted bone formation by osteoblasts. In this study we evaluated whether use of the FAK TKI PF-562,271 affected the differentiation of pre-osteoblasts, or activity of mature differentiated osteoblasts. MC3T3-E1 pre-osteoblastic cells were treated with various doses of PF-562,271 following 3 or 10 days of differentiation which led to the inhibition of alkaline phosphatase (ALP) expression and reduced viable cell numbers in a dose-dependent manner. MC3T3-E1 cells which had been differentiated for 21 days prior to treatment with PF-562,271 showed a dose dependent decrease in mineralization as assessed by Alizarin Red staining, with concomitant decreased expression of ALP which is known to facilitate the bone mineralization activity of osteoblasts, however mRNA levels of the transcription factors RUNX2 and osterix which are important for osteoblast maturation and mineralization appeared unaffected at this time point. We speculated that this may be due to altered function of RUNX2 protein due to inhibitory phosphorylation by GSK3β. We found treatment with PF-562,271 resulted in increased GSK3β activity as measured by reduced levels of phospho-Ser9-GSK3β which would result in phosphorylation and inhibition of RUNX2. Treatment of 21 day differentiated MC3T3-E1 cells with PF-562,271 in combination with GSK3β inhibitors partially restored mineralization however this was not statistically significant. As we observed that FAK TKI also resulted in suppression of Akt, which is known to alter osterix protein stability downstream of RUNX2, we examined protein levels by western blot and found a dose-dependent decrease in osterix in FAK TKI treated differentiated MC3T3-E1 cells which is likely responsible for the reduced mineralization observed. Taken together our results suggest that use of FAK TKIs as therapeutics in the bone metastatic setting may block new bone formation as an off-target effect and thereby exacerbate the defective bone regulation that is characteristic of the bone metastatic environment.

粘着斑激酶（FAK）是肿瘤细胞增殖、存活和转移的重要调节因子。因此，它已成为癌症的治疗靶点。先前的研究表明，使用 FAK 酪氨酸激酶抑制剂 (TKI) 可阻止体内骨溶解骨转移模型。然而，从这些研究中，尚不清楚 FAK TKI 是阻断破骨细胞的骨降解还是促进成骨细胞的骨形成。在这项研究中，我们评估了使用 FAK TKI PF-562,271 是否影响前成骨细胞的分化或成熟分化的成骨细胞的活性。MC3T3-E1 前成骨细胞在分化 3 或 10 天后用不同剂量的 PF-562,271 处理，导致碱性磷酸酶 (ALP) 表达受到抑制并以剂量依赖性方式减少活细胞数量。用 PF-562,271 处理前已分化 21 天的 MC3T3-E1 细胞显示出矿化的剂量依赖性降低，通过茜素红染色评估，伴随着 ALP 的表达降低，已知这有助于促进成骨细胞的骨矿化活性，但是对于成骨细胞成熟和矿化很重要的转录因子 RUNX2 和 osterix 的 mRNA 水平在此时似乎不受影响。我们推测这可能是由于 GSK3β 抑制磷酸化导致 RUNX2 蛋白功能改变。我们发现用 PF-562,271 治疗会导致 GSK3β 活性增加，这是通过降低磷酸化 Ser9-GSK3β 水平来衡量的，这会导致磷酸化和 RUNX2 的抑制。用 PF-562,271 与 GSK3β 抑制剂联合处理 21 天分化的 MC3T3-E1 细胞可部分恢复矿化，但这没有统计学意义。正如我们观察到 FAK TKI 也导致 Akt 抑制，已知会改变 RUNX2 下游的 osterix 蛋白稳定性，我们通过蛋白质印迹检查了蛋白质水平，发现 FAK TKI 处理的分化 MC3T3-E1 细胞中 osterix 的剂量依赖性降低，这可能是观察到矿化减少的原因。综上所述，我们的研究结果表明，在骨转移环境中使用 FAK TKI 作为治疗剂可能会阻止新骨形成作为脱靶效应，从而加剧骨转移环境中特有的骨调节缺陷。