p53 Regulates Bone Differentiation and Osteosarcoma Formation

Osteosarcoma (OS) is the second leading cause of cancer-related death in children and young adults. p53 plays critical roles in suppressing OS in both human and mouse. Bone marrow-derived mesenchymal stem cells (BMSCs) are thought to be one of the cells-of-origin of OS.  When p53 is intact, BMSCs undergo normal tri-lineage differentiation (adipogenesis, chondrogenesis, and osteogenesis). However, when p53 is lost, as in many OS cells, BMSCs have enhanced osteogenesis and may generate osteosarcoma. Therefore

Osteosarcoma (OS) is the second leading cause of cancer-related death in children and young adults. p53 plays critical roles in suppressing OS in both human and mouse. Bone marrow-derived mesenchymal stem cells (BMSCs) are thought to be one of the cells-of-origin of OS. When p53 is intact, BMSCs undergo normal tri-lineage differentiation (adipogenesis, chondrogenesis, and osteogenesis). However, when p53 is lost, as in many OS cells, BMSCs have enhanced osteogenesis and may generate osteosarcoma. Therefore, studying the roles of p53 in BMSCs will provide new insights into the OS suppressive function of p53.

Osteosarcoma is an uncommon cancer that usually begins in the large bones of the arm or leg, but is the second leading cause of cancer-related death in children and young adults. The tumor suppressor protein, p53, appears to be an important player in osteosarcomagenesis in part because these cancers are one of the most common to develop in patients with Li-Fraumeni syndrome, which is caused by an inherited mutation in p53. However, the precise role of p53 in osteosarcoma development has not been established. To begin investigating its importance to the formation of normal bone and osteosarcomas, Jing Huang, Ph.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues, isolated bone marrow-derived mesenchymal stem cells (BMSCs) from p53 wild type (WT) and knock out (KO) mice using a recently validated approach. Because BMSCs are one of the cells-of-origin of osteosarcoma, they serve as a useful model system. BMSCs contain a subset of multipotent stem cells that can differentiate into several cell types, including osteoblasts, and are important mediators of bone homeostasis.

While p53 WT and KO BMSCs had similar morphology and surface markers, the researchers consistently isolated twice as many cells from the KO mouse bone marrow. Using RNA deep sequencing, they also observed that the WT and KO BMSCs had similar mRNA levels of key regulators of osteogenesis (Runx2), chondrogenesis (Sox9), and adipogenesis (PPARγ). Interestingly, they were unable to grow clones from WT BMSCs because the cells senesced or lost their differentiation ability after isolation. These results indicate that loss of p53 allows BMSCs to grow for more passages and still retain multipotent markers.

The scientists then examined the differentiation capacity of the BMSCs. They found no difference in adipogenesis or chondrogenesis, but KO BMSCs differentiated into osteoblasts much faster. Staining for alkaline phosphatase, an early marker of osteogenesis, revealed that KO BMSCs expressed higher levels of the marker even before differentiation, suggesting the cells have a higher tendency to develop into osteoblasts. However, the KO BMSCs were not committed to the osteogenic lineage because single clones could still differentiate into all three lineages. When the researchers implanted WT or KO BMSCs into immune compromised mice, the KO transplants were larger with more mature bone volume than the WT transplants. The in vitro and in vivo data indicate that p53 loss pushes BMSCs toward pre-osteoblast differentiation.

The investigators next asked what mechanism p53 uses to negatively regulate osteogenesis. They first looked at p53’s usual roles in regulating the cell cycle, cell confluence, and apoptosis. Surprisingly, none could explain the difference in osteogenic differentiation between the WT and KO BMSCs, suggesting it is likely a mechanism novel to BMSCs. In previous studies, the scientists found that p53 controlled the expression of stem cell master regulators. Hypothesizing that the same might occur in BMSCs, they looked at protein levels of several master regulators and found higher Runx2 levels in the KO cells with no difference in Sox9 or PPARγ. Using various approaches, the researchers confirmed that Runx2 mediated the enhanced osteogenic differentiation of KO BMSCs. However, Runx2 mRNA levels were the same in WT and KO BMSCs, suggesting that p53 does not directly regulate the transcription of Runx2. The investigators verified this by chromatin immunoprecipitation and deep sequencing (ChIP-seq), which did not detect binding of p53 to the Runx2 gene locus. This led them to examine post-transcriptional mechanisms. Because they found no difference in Runx2 protein turnover rates, the scientists evaluated potential p53-regulated microRNAs. Using the ChIP-seq data, they identified miR34a and miR34b/c, which have the same seed sequence, and found higher levels of these microRNAs in the WT BMSCs. The researchers transfected the microRNAs into KO BMSCs, and each decreased Runx2 protein levels. A miR34 decoy sequestered the microRNAs in WT BMSCs, increasing Runx2 levels. The investigators also identified three miR34 family target sites in the Runx2 untranslated region.

To determine whether the results from mouse BMSCs might apply to human cells, the scientists examined human osteosarcoma cell lines. They found an inverse correlation between p53 expression and Runx2 expression. Likewise, cells with p53 loss had enhanced osteogenic differentiation. The researchers then examined global gene expression in the WT and KO BMSCs, identifying genes increased at least two-fold and decreased at least two-fold in the WT cells. As expected, p53 signaling was enriched in the upregulated genes. The investigators then compared the altered genes to human BMSCs and osteosarcoma tissues using gene set enrichment analysis. The up-regulated genes were associated with normal human BMSCs, while the down-regulated genes were associated with osteosarcoma, suggesting p53 controls the expression of genes important for osteosarcomagenesis. These results also show that osteogenic differentiation and osteosarcomagenesis are closely related and are regulated via the p53/miR34/Runx2 axis.

Summary Posted: 01/2015

Reference

He Y, de Castro LF, Shin MH, Dubois W, Yang HH, Jiang S, Mishra PJ, Ren L, Gou H, Lal A, Khanna C, Merlino G, Lee M, Robey PG, Huang J. p53 Loss Increases the Osteogenic Differentiation of BMSCs. Stem Cells. December 18, 2014 PubMed Link