Frank Sander B, Berger Penny L, Ljungman Mats, Miranti Cindy K
Laboratory of Integrin Signaling and Tumorigenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
Genetics Program, Michigan State University, East Lansing, MI 48824, USA.
J Cell Sci. 2017 Jun 1;130(11):1952-1964. doi: 10.1242/jcs.197152. Epub 2017 Apr 26.
Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.
前列腺癌中许多失调的信号通路也参与上皮分化。为了更好地理解前列腺肿瘤的起始，我们试图研究正常基底细胞向管腔细胞分化所需的特定基因和机制。利用人前列腺基底上皮细胞和一种分化模型，我们测试了以下假设：p38丝裂原活化蛋白激酶（MAPK）家族（以下简称p38-MAPK）通过MYC对NOTCH3的调控是管腔分化所必需的。抑制（SB202190和BIRB796）或敲低p38α（也称为MAPK14）和/或p38δ（也称为MAPK13）会阻止正常分化。此外，用γ-分泌酶抑制剂（RO4929097）处理或敲低NOTCH1和/或NOTCH3会极大地损害分化并导致管腔细胞死亡。通过MKK6（CA）组成型激活p38-MAPK可增加NOTCH3（而非NOTCH1）的mRNA和蛋白质水平，而在MYC抑制（10058-F4和JQ1）或敲低后这种增加会减弱。此外，我们通过增强子（e）RNA检测（BruUV-seq）和荧光素酶报告基因检测相结合的方法验证了两个增强子元件。最后，我们发现NOTCH3 mRNA半衰期在分化过程中或急性p38-MAPK激活后会增加。这些结果揭示了p38-MAPK、MYC和NOTCH信号之间的新联系，证明了NOTCH3调控的两种机制，并为NOTCH3参与前列腺管腔细胞分化提供了证据。
