Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance

Abstract

Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS.DOI: http://dx.doi.org/10.7554/eLife.13446.001

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein

“…To confirm PTHrP's autocrine/paracrine effect in Ocy454 cells, we assessed cAMP levels (with maximal phosphodiesterase inhibition) without adding exogenous PTHrP. In this way cAMP is generated only by autocrine receptor‐linked adenylyl cyclase activation by cell‐derived ligand(s) . Although Pthlh knockdown cells had higher Pth1r mRNA levels than vector control (Fig.…”

“…Because extracting sufficient protein from osteocytes purified from bone would not be feasible, we used overexpression of the murine Pthlh gene in the osteocyte cell line Ocy454 to characterize the PTHrP that they secrete. The biological assay measures PTHrP species containing intact N‐terminal protein (at least 34 residues), with a sensitivity of 0.01nM to 0.1nM . This showed PTHrP activity was retained in conditioned medium in cation exchange chromatography eluates (Fig.…”

“…shRNA with sequence 5′‐CCG‐GCC‐AAT‐TAT‐TCC‐TGT‐CAC‐TGT‐TCT‐CGA‐GAA‐CAG‐TGA‐CAG‐GAA‐TAA‐TTG‐GTT‐TTT‐TG‐3′ was used to knock down Pthlh in Ocy454 cells. This shRNA was synthesized by Sigma–Aldrich (St. Louis, MO, USA) and cloned into a lentiviral vector PLKO, as we reported . Undifferentiated Ocy454 cells were infected with virus and selected with puromycin (5 μg/mL), then cultured at permissive temperature (33°C) before transfer to 37°C for differentiation.…”

Murine models of osteosarcoma: A piece of the translational puzzle

“…We then used a reductionist system to address the requirement for the cAMP pathway, centered on PTHrP and CREB1 during the initiation of OS. Using primary long bone osteoblasts that could be engineered to become p53 deficient, we observed that these cells reproducibly underwent a hyper‐proliferative transformation at approximately 12‐14 days after deletion of p53 . The loss of p53 transcriptional activity was accompanied by activation of a cAMP gene signature.…”

Molecular genetics of osteosarcoma

“…Recently, Walia et al demonstrated that normal osteoblasts survive depletion of both parathyroid hormone-related protein (PTHrP) and CREB1, whereas osteoblasts lacking p53 in OS depend on the continuous activation of the PTHrP-cAMP-CREB1 pathway. In the absence of PTHrP or CREB1, OS cells undergo proliferation arrest and apoptosis[111]. PTHrP and PTH are crucial regulators of bone formation and remodeling, and administration of high-dose human PTH increased incidence of OS in rats [112, 113].…”