Wnt/β-Catenin Expression Does Not Correlate with Serum Alkaline Phosphatase Concentration in Canine Osteosarcoma Patients

Abstract

Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/β-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/β-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in β-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator.

Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, β-Catenin, P53, Caspase 3) in canine appendicular osteosarcoma

“…In accordance with that, no mutation has been recently identified in canine β-catenin exon 3, similarly to human OSA [19], even if a limited number of cases were examinated, and a mainly cytoplasmic, but rare nuclear localization of the protein was detected [51]. Furthermore, no differences in nuclear β-catenin immunohistochemical expression were observed in samples obtained from two populations of canine OSA patients with normal or high level of serum alkaline phosphatase concentration [52], known to have negative prognostic value in canine osteosarcoma patients [53]. These results seem to be in agreement with recent investigations in human OSA [26], where no β-catenin nuclear expression was observed in 90% of human high grade OSA biopsies examinated and inhibition of the Wnt/β-catenin pathway has been demonstrated to be important in osteoblast neoplastic transformation [26].…”

β‐Catenin transcriptional activity is minimal in canine osteosarcoma and its targeted inhibition results in minimal changes to cell line behaviour

“…Often, the presence of cytoplasmic and/or nuclear localization of β ‐catenin is used as surrogate for pathway activation. While both humans and canines exhibit frequent cytoplasmic β ‐catenin staining, there is minimal nuclear β ‐catenin staining, with 10% (5/52) of human OS samples and 2% (1/56) to 10% (3/30) of canine OS samples displaying positive nuclear β ‐catenin . Contrary to these low numbers, Bongiovanni et al observed 47% (8/17) of canine OS samples to display nuclear staining; however, in nuclear‐positive samples, less than 10% of nuclei/cells within the sample were positive, indicating that these positive nuclei were very weakly positive.…”

“…Treatment options and survival rates for both human and canine OS have essentially stagnated the last 15–20 years, prompting the investigation of various molecules and signalling pathways that could be targeted for therapy in hopes of improving outcomes . Wnt signalling is a pathway of interest for its role in osteoblast differentiation, its dysregulation in numerous cancer types, and the frequency of cytoplasmic accumulation in canine and human OS . While in epithelial‐based cancers – such as hepatocellular, colorectal and mammary carcinomas – β ‐catenin activation is considered a pro‐oncogenic alteration, the role of β ‐catenin in mesenchymal‐based tumours such as melanoma and OS is less well‐defined .…”

“…Wnt signalling is a pathway of interest for its role in osteoblast differentiation, its dysregulation in numerous cancer types, and the frequency of cytoplasmic accumulation in canine and human OS . While in epithelial‐based cancers – such as hepatocellular, colorectal and mammary carcinomas – β ‐catenin activation is considered a pro‐oncogenic alteration, the role of β ‐catenin in mesenchymal‐based tumours such as melanoma and OS is less well‐defined . Some authors have reported that the Wnt/ β ‐catenin pathway is active in OS, as indicated by nuclear and cytoplasmic β ‐catenin staining in human OS cells, increased cytoplasmic and/or nuclear β ‐catenin expression in tissue from xenogeneic murine pulmonary metastasis, and decreased tumourigenesis by inhibition of Wnt receptors; while others have reported that activation of the Wnt/ β ‐catenin pathway is selected against in OS development and that inhibition of Wnt/ β ‐catenin signalling transforms human mesenchymal stem cells (MSCs) into sarcoma cells in vitro .…”

The Prognostic Significance of CD44V6, CDH11, andβ-Catenin Expression in Patients with Osteosarcoma

“…Several studies have revealed that positive cytoplasmic β -catenin expression was associated with the development of metastasis both in vivo and in vitro [9, 52–54]. Although the deregulation of β -catenin is thought to play an important role in oncogenesis of osteosarcomas, the role of Wnt signaling in osteosarcomas remains controversial [18, 22, 53, 55, 56]. Recently, a study has indicated that β -catenin may interact with other proteins, such as NF-kappa B, during oncogenesis [57].…”