p27^Kip1, a double-edged sword in Shh-mediated medulloblastoma

Figures & data

Figure 1 p27^Kip1 is mislocalized in Shh-mediated medulloblastoma. (A) In cerebellar development of post-natal day 7, the peak for CGNP proliferation, p27^Kip1 (green) is expressed in post-mitotic region, external granule layer b (EGLb), molecular layer (ML) and differentiated region inner granule layer (IGL), whereas aberrant Shh signaling leads to p27^Kip1 mislocalization and increased CGNP proliferation throughout the EGL. Proliferation marker PCNA (red) stains CGNPs in proliferating region EGLa. (B) Wild-type and SmoA1 CGNPs were plated, treated with Shh and measured for proliferation by quantification of phospho-Histone H3. (C) While non-tumor cells undergo cell cycle exit and/or differentiation via p27^Kip1 nuclear localization in the IGL, the Shh-induced medulloblastoma maintains misregulated phosphorylation of p27^Kip1 at Ser10 and Thr187, which leads to mislocalization and inhibition of cell cycle regulation. (D) Non-tumors in NeuroD2-SmoA1 mice have nuclear p27^Kip1 (first column), whereas SmoA1 medulloblastoma have cytoplasmic p27^Kip1 (second column) and high proliferation (third and fourth column).

Figure 2 (A–C) p27^Kip1 loss accelerates medulloblastoma incidence in SmoA1 mice. (A) p27^Kip1 loss accelerates medulloblastoma incidence in homozygous and hemizygous mice for SmoA1 transgene. (B and C) SmoA1 mice heterozygous for p27^Kip1 have a dramatic decreased survival latency compared to homozygous and nullizygous for p27^Kip1.

Figure 2D p27^Kip1 loss accelerates medulloblastoma incidence in SmoA1 mice. (D) Western blot analysis of cell cycle indicators in NeuroD2-SmoA1 medulloblastomas arising in wild-type, p27^+/− and p27^−/− mice.

Figure 3 Cyclin D/Cdk4/6 assembly is dependent on p27^Kip1. (A) Immunoprecipitation shows p27^Kip1 interacts with cyclin D1, Cdk4/6 in SmoA1 medulloblastoma. (B) Immunoprecipitation shows decreased cyclin D1/Cdk6 interaction as a result of p27^Kip1 loss. Tumor proliferation benefits as cyclin E/p27^Kip1 is decreased, suggesting p27^Kip1 is haploinsufficient as a tumor suppressor.

Figure 4 p27^Kip1 plays a role in cell motility in SmoA1 medulloblastoma. (A) Schematic diagram of p27^Kip1 regulating cell motility in tumor cells. (B) Immunofluorescence shows p27^Kip1 is inversely correlated with stabilized cell motility, as evidenced by p27^Kip1 (green), active phosphorylated-LIMK to prevent cell motility (red, left panel) and inactive phosphorylation of LIMK substrate and actin-depolymerization protein cofilin (red, right panel) in SmoA1 medulloblastoma. (C) Subcellular fractionation of SmoA1 medulloblastoma shows p27^Kip1 and cell motility regulator RhoA are localized mainly in the cytoplasm. GAPDH serves as cytosolic control and c-jun as nuclear control. (D) Immunoprecipitation of p27^Kip1 reveals interaction with RhoA. This data suggests p27^Kip1 is needed to positively regulate cell motility in SmoA1 medulloblastoma.