The canonical Wnt/β-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/β-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)–competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/β-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.

Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. However, the link between G-proteins and β-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and β-catenin using G-alpha subunit genetic knockout and knockdown approaches. The Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a β-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active β-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of β-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total β-catenin than wild-type cells. The stimulation of active β-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq. Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active β-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced β-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of β-catenin is context dependent.

Many Pasteurella multocida strains are carried as commensals, while some cause disease in animals and humans. Some type D strains cause atrophic rhinitis in pigs, where the causative agent is known to be the Pasteurella multocida toxin (PMT). PMT activates three families of G-proteins-G q/11 , G 12/13 , and G i/o -leading to cellular mitogenesis and other sequelae. The effects of PMT on whole animals in vivo have been investigated previously, but only at the level of organ-specific pathogenesis. We report here the first study to screen all the organs targeted by the toxin by using the QE antibody that recognizes only PMT-modified G-proteins. Under our experimental conditions, short-term treatment of PMT is shown to have multiple in vivo targets, demonstrating G-alpha protein modification, stimulation of proliferation markers and expression of active β-catenin in a tissue-and cell-specific manner. This highlights the usefulness of PMT as an important tool for dissecting the specific roles of different G-alpha proteins in vivo.

Osteosarcoma (OS) is the most common primary malignancy of the skeleton, occurring primarily in children and adolescents. Metastatic disease is the single most important prognostic factor with survival dropping to 15-20% in patients with lung metastases. We have recently demonstrated that high FGFR1 signaling in an oncogene-induced murine model of OS as well as in human OS samples contributes to the pathogenesis and metastasis of OS. Significantly, xenograft studies showed that genetic or pharmacological inhibition of FGFR1 decreased lung metastasis. We have identified the mTOR signaling pathway as a cooperating pathway with FGFR signaling in OS cells. Stimulation of murine and human OS cell lines with FGF2 showed an increase in both MAPK and pAkt/mTOR signaling and mTOR target genes that was inhibited by the FGFR inhibitor, PD173074. Colony growth in soft agar showed that inhibition of mTOR blocked FGF2-stimulated colony growth of OS cells and shRNA silencing of mTOR prevented FGF2-stimulated cell migration in wound/scratch assays. To confirm pathway cooperativity in vivo, we performed xenograft studies. Silencing of mTOR in FGFR1-expressing OS cells reduced tumor formation on a chick chorioallantoic membrane, with reduced pHistone-H3 expression. Orthotopic injection of OS cells demonstrated that small molecule inhibition of both FGFR1 and mTOR signaling (AZD4547 and AZD8055, respectively), showed greater inhibition of size and diameter of lung metastatic nodules than each inhibitor alone. This was associated with a decrease in the number of Ki67+ cells in lung metastases of FGFR/mTOR inhibitor-treated mice, whereas no changes in activated caspase-3 were detected. In a complementary in vivo model, tail vein injections of OS cells showed that combined FGFR/mTOR inhibition reduced cell seeding in the lungs, suggesting that FGFR/mTOR signaling are driving lung colonization independently of the primary tumor. Finally, preliminary studies indicate that long term growth of OS cells in the lung following tail vein injection may reveal a potential for OS cells to acquire chemoresistance to mTOR inhibition. The mechanisms are not yet known but are independent of the primary tumour. Taken together, our data suggest that combinatorial treatment significantly decreased lung metastasis of OS cells from primary xenografts. Ongoing analysis of activated FGFR and mTOR pathways in patient-derived tissue microarrays will enable patient stratification that will be useful in the clinical setting to exploit this as a potential anti-metastatic therapy in OS. Citation Format: Arshiya Banu, Carolina Zandueta, Fernando Lecanda, Agamemnon E. Grigoriadis. Regulation of osteosarcoma cell metastasis by FGFR and mTOR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 416.

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