Osteosarcoma (OSA) represents the most common primary bone tumour of both dog [1] and childhood/adolescence [2]. Numerous histological and biological features have been shown to be shared by canine and human OSA to date [3–8], even if further studies are warranted to define more precisely similarities and differences of OSA in the two species, so as to really consider the dog as a valuable spontaneous tumour model for human OSA. Even if several pathways have been investigated in canine OSA so far, few molecules have been identified as prognostic tools or potential therapeutic targets [5, 9–18]. Leading on from this, there is still the need to investigate molecular pathways with specific influence over canine OSA progression to facilitate earlier prognosis and treatment.

The goal of the present study was to evaluate a panel of molecules involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA. Among these molecules, of particular significance, the role of β-catenin, and the related canonical Wnt pathway, has been largely investigated in human OSA [19–26], but its exact role during neoplastic transformation of osteoblast and OSA progression still remains a debated item. While several studies indicated an important contribution of the activation of the Wnt pathway in OSA development [19–25, 27, 28], Cai et al. [26] have recently argued the opposite, suggesting that the loss of Wnt pathway activity, which is required for osteoblast differentiation, may contribute to OSA development. Consequently, in contrast to other tumors, β-catenin might not play an oncogenic role in OSA cells [26, 29].

Survivin has been recently proposed as a prognostic marker in human [30–35] and canine [36] OSA, but contrasting results have been observed concerning the relation between its subcellular localization and a favourable or poor prognosis. One of the most important aspects of the study of survivin expression in cancer is related to its high cancer specificity and consequent potential role as therapeutic target [37, 38]. Survivin has been identified as a target gene of the β-catenin pathway [39], and it acts as a cell cycle regulator and apoptosis inhibitor, even if different isoforms with different functions of the molecule have been described [40].

Loss of p53 tumour suppressor gene functions have been frequently reported in OSA, and mutated p53 has been reported in both human and canine OSA [41]. An interaction between p53 and β-catenin pathway has been proposed to play a key role in osteoblast differentiation and maintenance of bone tissue homeostasis [42]. Furthermore, activation of the Wnt/β-catenin signalling has been shown to cause p53 accumulation [43].

The aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of survivin, β-catenin, caspase 3 (inactive [procaspase 3] and active forms), and p53 in canine OSA samples, to reveal specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS). The attempt is to contribute to a better understanding of both the pathogenesis of canine OSA and the role of the investigated molecules, in particular establishing their role as prognostic biomarkers and/or potential therapeutic targets. Interestingly, in this study, the expression of nuclear survivin was mainly found in less malignant OSA cases, and β-catenin did not seem to play a major role in oncogenic transformation of canine osteoblasts, while nuclear p53 was confirmed to represent a negative prognostic marker.

The present study demonstrated and confirmed the expression of several important molecules in canine OSA, showing a potential prognostic value of some of them, that could also be considered as possible candidates for a therapeutic target.

Even though the importance of the Wnt/β-catenin pathway in bone development and homeostasis is well defined [46, 47], the role of β-catenin in the development and progression of bone tumours is not completely known [48]. Since OSA formation is probably the result of an altered bone regulation, it seems likely that deregulation of the Wnt signalling could be associated with the pathogenesis of OSA. The presence of intense β-catenin-positive nuclei in normal osteoblasts observed in the present study would confirm the fundamental role of the Wnt/β-catenin pathway in the maintenance of these cells. Indeed, nuclear β-catenin expression has been considered a hallmark of activation of the Wnt/β-catenin pathway [26, 49] or mutations of β-catenin or molecules of its degradation complex [50]. Rare or completely absent positive nuclei were observed in the most malignant cases examinated herein, even if no significant association with the OS was observed, as well as in neoplastic emboli, suggesting that neither mutations nor activation of the Wnt/β-catenin pathway would occur in these cells. 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]. These observations seemed to confirm the hypothesis that inactivation of the Wnt/β-catenin pathway could be essential in OSA progression, differently to what is usually observed in other cancers [54], opening to new potential therapeutic strategies. Specific small molecule compounds that activate Wnt/β-catenin signaling in a highly cell-type specific manner (human OSA U2OS cell line) have recently been identified [55], that could induce the activation of the Wnt/β-catenin pathway and provide new therapeutic opportunities. Interestingly, high β-catenin cytoplasmic expression rate was significantly related to the development of metastasis. High levels of cytoplasmic protein, in absence of nuclear staining, could be related to a reduced membranous β-catenin, reported to be associated to invasion and metastatic potential in human OSA [21], however, an inhibition of β-catenin nuclear translocation may also be hypothesized.

Similarly to what has previously been observed in human [30, 31] and canine [36] OSA, both cytoplasmic and nuclear survivin expression pattern was observed in canine OSA. Mechanisms controlling survivin nuclear and/or cytoplasmic localization in tumour cells are often a debated question, since the item could acquire a different prognostic significance depending on the tumour type [56]. Our results seem to be in contrast with a recent study in which a correlation between survivin immunohistochemical expression and histological grade and mitotic index has been reported in canine OSA tissue samples [36]. Similarly, divergent results have been published concerning the relation between survivin expression and malignancy of human OSA [30, 32–34]. Disagreements could be related to the presence of several survivin isoforms, with different, even opposite, functions, and this might indicate different activities of survivin within the cell. In particular, two survivin variants potentially act in an opposite way to the other three variants, with a potential proapoptotic function: Survivin-2α may attenuate the anti-apoptotic activity of full length survivin [57], while survivin-deltaEx3 has been shown to have apoptotic functions [58]. Noteworthy, the polyclonal antibody used in the present study recognizes all survivin splice variants, and decreased nuclear survivin expression score was found among more malignant grade III cases of canine OSA, as well as higher nuclear survivin expression appeared to be related to a longer post-treatment OS, similarly to that observed by Trieb et al. [30] in the human counterpart. A significant association was revealed between nuclear survivin expression and the activation of caspase 3, most probably indicative of apoptotic pathway activation, further supporting the hypothesis that nuclear survivin expression could be considered a positive prognostic marker in canine OSA. Indeed, despite a diffuse cytoplasmic staining of its inactive form (procaspase 3) observed in all the cases examinated, a progressive decreasing of the active caspase 3 mean values was observed in OSA from grade I to III.

Differently to primary tumours, a frequent nuclear survivin immunostaining was observed among neoplastic emboli, suggesting a different behaviour of the protein in OSA metastatic spread. Therefore, further investigations on survivin expression in metastatic OSA are warranted, in order to understand its functions and role in the response to postoperative chemotherapy, since adjuvant treatment is mainly aimed at controlling the metastatic disease, and survivin has been frequently associated to resistance to chemotherapy [59, 60]. Recently, this has been demonstrated also in canine OSA cell lines (Abrams and D17), where after survivin inhibition an increase of chemosensitivity to both doxorubicin and carboplatin has been observed [36]. Survivin has been proposed as a valuable target for anticancer therapy [37], and several molecules with a direct or indirect effect on survivin expression have been identified to date, some of which have been tested in clinical trials with encouraging results [61]. Decreasing survivin expression through STAT3 [5, 62] or Hsp90 [63] inhibition in canine OSA cell lines has been recently proposed as a possible new therapeutic approach.

There is a strong evidence of the involvement of p53 mutations in the development of canine OSA [41, 64, 65], as well as in the human counterpart [66, 67]. In the present study, highest levels of expression of nuclear p53 were significantly associated with a shorter post surgical OS, and appeared to be related to the development of metastasis, confirming the importance of p53 as a negative prognostic marker in canine OSA, in accordance to previous published data regarding canine OSA, suggesting that, similarly to human OSA [68, 69], alterations in p53 functions are associated with highly aggressive tumour behaviour [70, 71]. Deregulation of the p53-survivin subsystem has been proposed to play an important role in several tumour types [72], since, WTp53 is able to repress survivin expression at both the mRNA and protein level [40]. In spite of this, our results suggests that different mechanisms could regulate survivin expression in canine OSA.

Data obtained from the present immunohistochemical study suggest survivin as a potential positive prognostic marker, rather than a marker of poor prognosis, even if its presence in metastatic emboli suggests its possible involvement in the malignant progression of canine OSA. Further investigations, in particular on the expression and roles of its different isoforms, should be performed in order to clarify if survivin could represent a valid biomarker in this kind of tumour.

Lastly, the present data suggest that β-catenin, a key molecule for normal osteoblast differentiation, could not play a key role in the neoplastic transformation of canine osteoblasts, as recently suggested in human OSA. Further studies should be done in order to confirm this hypothesis.