BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms.

The etiopathogenesis of feline thyrotoxicosis is unknown. The transmembrane part (gene codons 480-640) of the thyrotropin receptor (TSHR) gene of hyperthyroid cats has already been investigated for the presence of stimulating mutations. No mutations were found in this part of the TSHR gene. We have investigated the TSHR gene codons 66-530 for gene mutations in 10 hyperthyroid cats and in 1 euthyroid cat. This part of the TSHR gene encodes the transmembrane part as well as most of the extracellular part of the receptor. The G(s alpha) gene of these cats was also sequenced and subjected to mutational analysis. Although our study revealed a polymorphism in the TSHR gene, no association was found with tumor formation. In 4 of 10 cats with hyperthyroidism a G(s alpha) gene mutation was found. This work suggests that mutations in the extracellular or transmembrane part of the TSHR gene are not likely the cause of feline hyperthyroidism. Mutations in the G(s alpha) gene, however, may play a role in the etiopathogenesis of this disease.

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.

Mammary tumors are the most common form of neoplasia in the bitch. Female dogs are protected when they are spayed before the first estrus cycle, but this effect readily disappears and is already absent when dogs are spayed after the second heat. As the ovaries are removed during spaying, ovarian steroids are assumed to play an essential role in tumor development. The sensitivity toward tumor development is already present during early life, which may be caused by early mutations in stem cells during the first estrus cycles. Later on in life, tumors arise that are mostly steroid-receptor positive, although a small subset of tumors overexpressing human epidermal growth factor 2 (HER2) and some lacking estrogen receptor, progesterone receptor (PR), and HER2 (triple negative) are present, as is the situation in humans. Progesterone (P4), acting through PR, is the major steroid involved in outgrowth of mammary tissue. PRs are expressed in two forms, the progesterone receptor A (PRA) and progesterone receptor B (PRB) isoforms derived from splice variants from a single gene. The dog and the whole family of canids have only a functional PRA isoform, whereas the PRB isoform, if expressed at all, is devoid of intrinsic biological activity. In human breast cancer, overexpression of the PRA isoform is related to more aggressive carcinomas making the dog a unique model to study PRA-related mammary cancer. Administration of P4 to adult dogs results in local mammary expression of growth hormone (GH) and wing less-type mouse mammary tumor virus integration site family 4 (Wnt4). Both proteins play a role in activation of mammary stem cells. In this review, we summarize what is known on P4, GH, and Wnt signaling in canine mammary cancer, how the family of HER receptors could interact with this signaling, and what this means for comparative and translational oncological aspects of human breast cancer development.

No abstract

The present paper reports the effects of allopurinol in a raptor hyperuricaemic model. The study was performed as a follow-up to previous experiments wherein allopurinol was used in doses of 100 and 50 mg/kg, and was proved to be toxic at these higher dose rates. To investigate whether 25 mg/kg (semel in die) s.i.d. allopurinol is a safe and effective dose in Red-tailed Hawks (Buteo jamaicensis) to reduce plasma uric acid concentrations, experimental studies were performed using the physiologically occurring postprandial hyperuricaemia. Preprandial and postprandial plasma concentrations of xanthine, hypoxanthine, allopurinol, oxypurinol and uric acid were established by high-performance liquid chromatography at various time intervals after receiving allopurinol (25 mg/kg SID) or placebo. No significant differences were observed between the experimental and the control group. These results indicate that this dose is safe to administer; however, this dose failed to cause a significant effect on plasma uric acid concentrations. Because of the low therapeutic ratio of allopurinol in Red-tailed Hawks, follow-up studies have concentrated on an alternative for the treatment of hyperuricaemia, namely urate oxidase.

Common disorders of water homeostasis leading to polyuria include a variety of endocrine, metabolic, and renal disturbances. After exclusion of most of these conditions, the diagnostic dilemma of differentiating between central diabetes insipidus, primary polydipsia, and nephrogenic diabetes insipidus may remain. Here, we report on 18 young dogs with polyuria that had been present in most cases since the dogs were puppies. The conditions were categorized according to the plasma vasopressin (VP) response to hypertonicity. The VP response to osmotic stimulation was tested by IV infusion of 20% NaCl for 2 hours. The VP response in all dogs was abnormal. Three categories could be distinguished: an exaggerated response (n = 3), a subnormal response (n = 4), and a nonlinear response with high plasma VP concentrations unrelated to increases in plasma osmolality (n = 11). The VP response to hypertonicity did not consistently distinguish among different clinical entities. In the 9 dogs with variations in urine osmolality compatible with primary polydipsia, exaggerated, subnormal, and nonlinear responses were observed. Examination of the present data questions the generally accepted notion that VP measurements during hypertonic saline infusion are the "gold standard" for the diagnostic interpretation of causes of polydipsia and polyuria. Studies of the peripheral reflection in plasma of the pulsatile VP release in healthy and polyuric individuals, with and without osmotic provocation, should be performed.