Yang Chun-Song, Melhuish Tiffany A, Spencer Adam, Ni Li, Hao Yi, Jividen Kasey, Harris Thurl E, Snow Chelsi, Frierson Henry F, Wotton David, Paschal Bryce M
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia.
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia.
Prostate. 2017 Nov;77(15):1452-1467. doi: 10.1002/pros.23400. Epub 2017 Sep 6.
Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells.
PKN kinase activity was measured by incorporation of P into protein substrates. Phosphorylation of the turn-motif (TM) in PKN proteins by mTOR was analyzed using the TORC2-specific inhibitor torin and a PKN1 phospho-TM-specific antibody. Amino acid substitutions in the TM of PKN were engineered and assayed for effects on kinase activity. Cell motility-related functions and PKN localization was analyzed by depletion approaches and immunofluorescence microscopy, respectively. The contribution of PKN proteins to prostate tumorigenesis was characterized in several mouse models that express PKN transgenes. The requirement for PKN activity in prostate cancer initiated by loss of phosphatase and tensin homolog deleted on chromosome 10 (Pten), and the potential redundancy between PKN isoforms, was analyzed by prostate-specific deletion of Pkn1, Pkn2, and Pten.
PKN1 and PKN2 contribute to motility pathways in human prostate cancer cells. PKN1 and PKN2 kinase activity is regulated by TORC2-dependent phosphorylation of the TM, which together with published data indicates that PKN proteins receive multiple PI-3 kinase-dependent inputs. Transgenic expression of active AKT and PKN1 is not sufficient for progression beyond PIN. Moreover, Pkn1 is not required for tumorigenesis initiated by loss of Pten. Triple knockout of Pten, Pkn1, and Pkn2 in mouse prostate results in squamous cell carcinoma, an uncommon but therapy-resistant form of prostate cancer.
磷酸肌醇-3（PI-3）激酶信号传导在癌症中具有广泛作用。PI-3激酶信号传导的关键效应器之一是AKT，一种在多种癌症中促进生长和存活的激酶。前列腺癌的基因工程小鼠模型表明，AKT信号传导足以诱导前列腺上皮内瘤变（PIN），但不足以进展为腺癌。这与前列腺特异性缺失Pten的小鼠的表型形成对比，在该模型中，过度的PI-3激酶信号传导会诱导PIN和局部浸润性癌。我们推测，其他PI-3激酶效应激酶通过提供与AKT生物学互补的活性来促进前列腺癌进展。我们聚焦于PKN激酶家族成员，它们在响应PI-3激酶信号传导时被激活，在前列腺癌中显示表达变化，并参与癌细胞的细胞运动途径。
通过将磷掺入蛋白质底物来测量PKN激酶活性。使用TORC2特异性抑制剂托瑞米芬和PKN1磷酸化转基序（TM）特异性抗体分析mTOR对PKN蛋白中TM的磷酸化。对PKN的TM进行氨基酸替换设计，并检测其对激酶活性的影响。分别通过缺失方法和免疫荧光显微镜分析细胞运动相关功能和PKN定位。在几种表达PKN转基因的小鼠模型中表征PKN蛋白对前列腺肿瘤发生的作用。通过前列腺特异性缺失Pkn1、Pkn2和Pten，分析在由10号染色体上缺失的磷酸酶和张力蛋白同源物（Pten）缺失引发的前列腺癌中PKN活性的需求，以及PKN亚型之间的潜在冗余性。
PKN1和PKN2参与人前列腺癌细胞的运动途径。PKN1和PKN2激酶活性受TM的TORC2依赖性磷酸化调节，这与已发表的数据一起表明PKN蛋白接受多种PI-3激酶依赖性输入。活性AKT和PKN1的转基因表达不足以进展到PIN之外。此外，Pten缺失引发的肿瘤发生不需要Pkn1。小鼠前列腺中Pten、Pkn1和Pkn2的三重敲除导致鳞状细胞癌，这是一种不常见但对治疗耐药的前列腺癌形式。
