William C. Kisseberth

Expression of stem cell factor receptor (c-kit) by the malignant mast cells from spontaneous canine mast cell tumours

Stem cell factor receptor (SCFR, c-kit), normally expressed on haematopoietic and mast cells, plays a regulatory role in cellular growth and differentiation. Dysregulated expression of SCFR may contribute to neoplastic transformation. We investigated expression of SCFR on malignant canine mast cells obtained directly from spontaneous canine mast cell neoplasms, in an attempt to determine whether these undifferentiated cells maintained expression of this growth-promoting cytokine receptor. Malignant mast cells (histological grade 2) from skin tumours or lymph node metastases were collected from canine patients, and SCFRs were detected by flow cytometric analysis of these cells. All of the tumours bound mouse and canine recombinant stem cell factor (SCF), indicating that the cells not only expressed SCFRs, but that the receptors possessed the functional property of ligand binding. Immunoglobulin Fc receptors for canine IgE were identified on these cells by flow cytometry, a further indication that the cells analysed were mast cells and retained some differentiated features. Immunohistochemical analysis of formalin-fixed, paraffin wax-embedded mast cell tumour biopsies confirmed expression of SCFRs by malignant cells from each tumour. The relative binding of SCF to suspensions of tumour cells, as assessed by flow cytometry, correlated with the intensity of immunolabelling for SCFR in sections of the same tumours, suggesting variability in SCFR expression between tumours. Agarose gel electrophoresis of the products of SCFR reverse transcription-polymerase chain reaction derived from each tumour had the molecular weight predicted for canine SCFR cDNA on the basis of the mouse and human counterparts. This further confirmed SCFR expression by malignant canine mast cells. Taken together, these results show that a membrane receptor capable of triggering cell growth is expressed by malignant canine mast cells, suggesting a role for this receptor in the aetiology of canine mast cell cancer. This relatively common malignancy of the dog would seem to present an opportunity for the investigation of the potential role of the SCF/SCFR pathway in the development of spontaneous malignancies of mast cells.

Characterization of STAT3 activation and expression in canine and human osteosarcoma

BackgroundDysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.MethodsTo examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.ResultsOur data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.ConclusionThese data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

BackgroundCanine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible.ResultsThrough genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors.ConclusionsMapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease.

Oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

BackgroundWe have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.MethodsRT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.ResultsOur data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.ConclusionsThese data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.

Canine Osteosarcoma: A Naturally Occurring Disease to Inform Pediatric Oncology

Osteosarcoma (OSA) is the most common form of malignant bone cancer in children and dogs, although the disease occurs in dogs approximately 10 times more frequently than in people. Multidrug chemotherapy and aggressive surgical techniques have improved survival; however, new therapies for OSA are critical, as little improvement in survival times has been achieved in either dogs or people over the past 15 years, even with significant efforts directed at the incorporation of novel therapeutic approaches. Both clinical and molecular evidence suggests that human and canine OSA share many key features, including tumor location, presence of microscopic metastatic disease at diagnosis, development of chemotherapy-resistant metastases, and altered expression/activation of several proteins (e.g. Met, ezrin, phosphatase and tensin homolog, signal transducer and activator of transcription 3), and p53 mutations, among others. Additionally, canine and pediatric OSA exhibit overlapping transcriptional profiles and shared DNA copy number aberrations, supporting the notion that these diseases are similar at the molecular level. This review will discuss the similarities between pediatric and canine OSA with regard to histology, biologic behavior, and molecular genetic alterations that indicate canine OSA is a relevant, spontaneous, large animal model of the pediatric disease and outline how the study of naturally occurring OSA in dogs will offer additional insights into the biology and future treatment of this disease in both children and dogs.

AR-42, a novel HDAC inhibitor, exhibits biologic activity against malignant mast cell lines via down-regulation of constitutively activated Kit

Histone hypoacetylation occurs in many cancers and inhibition of histone deacetylation is a promising approach to modulate these epigenetic changes. Our laboratory previously demonstrated that the histone deacetylase inhibitors (HDACis) vorinostat and AR-42 reduced the viability of a canine malignant mast cell line. The purpose of this study was to further investigate the mechanisms of pan-HDAC inhibition in normal and malignant mast cells. Mouse and canine malignant mast cell lines expressing various Kit mutations, normal canine mast cells, and primary canine malignant mast cells were treated with AR-42 (a novel HDACi) and effects on cell viability, cycling, and signaling were evaluated. Treatment with AR-42 induced growth inhibition, cell- cycle arrest, apoptosis, and activation of caspases-3/7. AR-42 promoted hyperacetylation of H3, H4, and alpha-tubulin, and up-regulation of p21. Down-regulation of Kit occurred after AR-42 treatment via inhibition of Kit transcription. Disassociation between Kit and heat shock protein 90 (HSP90) and up-regulation of HSP70 were observed after AR-42 treatment, suggesting potential loss of HSP90 chaperone function. Lastly, AR-42 down-regulated the expression of p-Akt, total Akt, phosphorylated STAT3/5 (pSTAT3/5), and total STAT3/5. In summary, AR-42 exhibits in vitro and ex vivo biologic activity against malignant mast cells, representing a promising therapeutic approach for malignant mast cell disease.

Molecular Profiling Reveals Prognostically Significant Subtypes of Canine Lymphoma

We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.

Salivary gland neoplasia in the dog and cat: survival times and prognostic factors

Twenty-four dogs and 30 cats with histopathologically confirmed salivary gland neoplasia were retrospectively reviewed in a multi-institutional study. The predominant presenting complaint for animals with salivary gland neoplasia was that of a mass being noted by the owner; other common complaints included halitosis, dysphagia, and exophthalmia. Siamese cats were overrepresented, indicating a possible breed predisposition. The most common histopathological type was simple adenocarcinoma. Cats had more advanced disease at diagnosis than did dogs, and clinical staging was prognostic in dogs. The median survival times for dogs and cats were 550 days and 516 days, respectively.

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

BackgroundCurcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.MethodsHuman and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.ResultsOur data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.ConclusionsThese data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo.

Preclinical evaluation of the novel, orally bioavailable Selective Inhibitor of Nuclear Export (SINE) KPT-335 in spontaneous canine cancer: Results of a phase i study

Background The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability. Methods and Findings Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2–42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35–256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35–354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities. Conclusions This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer.