Departments of Urology (R.T., A.Morc., M.T., S.-Q.X., M.S., K.G.L., D.H.B., L.G.G., A.Morr.), Biology of Prostate Cancer Program (L.G.G., A.Morr.), and Pathology, Anatomy, and Cell Biology (S.B., R.V.I.) and Cancer Cell Biology and Signaling Program (R.V.I.), Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; Department of Health Sciences (A.Morc., M.S., A.B.), Endocrinology, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy; and CRS Development of Biomolecular Therapies (M.T., K.S.), Experimental Oncology Laboratory, Rizzoli Orthopedic Institute, 40136 Bologna, Italy.
Endocrinology. 2015 Jan;156(1):58-70. doi: 10.1210/en.2014-1590.
The growth factor progranulin is as an important regulator of transformation in several cellular systems. We have previously demonstrated that progranulin acts as an autocrine growth factor and stimulates motility, proliferation, and anchorage-independent growth of castration-resistant prostate cancer cells, supporting the hypothesis that progranulin may play a critical role in prostate cancer progression. However, the mechanisms regulating progranulin action in castration-resistant prostate cancer cells have not been characterized. Sortilin, a single-pass type I transmembrane protein of the vacuolar protein sorting 10 family, binds progranulin in neurons and negatively regulates progranulin signaling by mediating progranulin targeting for lysosomal degradation. However, whether sortilin is expressed in prostate cancer cells and plays any role in regulating progranulin action has not been established. Here, we show that sortilin is expressed at very low levels in castration-resistant PC3 and DU145 cells. Significantly, enhancing sortilin expression in PC3 and DU145 cells severely diminishes progranulin levels and inhibits motility, invasion, proliferation, and anchorage-independent growth. In addition, sortilin overexpression negatively modulates Akt (protein kinase B, PKB) stability. These results are recapitulated by depleting endogenous progranulin in PC3 and DU145 cells. On the contrary, targeting sortilin by short hairpin RNA approaches enhances progranulin levels and promotes motility, invasion, and anchorage-independent growth. We dissected the mechanisms of sortilin action and demonstrated that sortilin promotes progranulin endocytosis through a clathrin-dependent pathway, sorting into early endosomes and subsequent lysosomal degradation. Collectively, these results point out a critical role for sortilin in regulating progranulin action in castration-resistant prostate cancer cells, suggesting that sortilin loss may contribute to prostate cancer progression.
生长因子颗粒蛋白前体(progranulin)是几种细胞系统转化的重要调节剂。我们之前已经证明,颗粒蛋白前体作为自分泌生长因子,刺激去势抵抗性前列腺癌细胞的运动性、增殖和锚定非依赖性生长,支持颗粒蛋白前体可能在前列腺癌进展中发挥关键作用的假说。然而,调节去势抵抗性前列腺癌细胞中颗粒蛋白前体作用的机制尚未得到表征。分选连接蛋白(sortilin)是液泡蛋白分选 10 家族的单次跨膜 I 型蛋白,在神经元中与颗粒蛋白前体结合,并通过介导颗粒蛋白前体靶向溶酶体降解来负调控颗粒蛋白前体信号。然而,分选连接蛋白是否在前列腺癌细胞中表达并在调节颗粒蛋白前体作用中发挥任何作用尚未确定。在这里,我们表明分选连接蛋白在去势抵抗性 PC3 和 DU145 细胞中表达水平非常低。值得注意的是,增强 PC3 和 DU145 细胞中的分选连接蛋白表达严重降低颗粒蛋白前体水平,并抑制运动性、侵袭性、增殖和锚定非依赖性生长。此外,分选连接蛋白过表达负调控 Akt(蛋白激酶 B,PKB)稳定性。这些结果通过耗尽 PC3 和 DU145 细胞中的内源性颗粒蛋白前体得到了再现。相反,通过短发夹 RNA 方法靶向分选连接蛋白可提高颗粒蛋白前体水平并促进运动性、侵袭性和锚定非依赖性生长。我们剖析了分选连接蛋白的作用机制,并证明分选连接蛋白通过网格蛋白依赖性途径促进颗粒蛋白前体内吞作用,分选到早期内体并随后进行溶酶体降解。总之,这些结果指出了分选连接蛋白在调节去势抵抗性前列腺癌细胞中颗粒蛋白前体作用中的关键作用,表明分选连接蛋白缺失可能有助于前列腺癌的进展。