Fatty acid synthase as a potential therapeutic target in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy in both feline and human patients. Recent work has demonstrated aberrant expression of fatty acid synthase (FASN) and an increased capacity for lipogenesis in human OSCC and other cancers. In human OSCC, inhibition of FASN decreased cell viability and growth in vitro, and diminished tumour growth and metastasis in murine preclinical models. This study aimed to characterize FASN as a therapeutic target in feline OSCC. Immunohistochemistry revealed high FASN expression in primary feline OSCC tumours, and FASN expression was detected in OSCC cell lines (3 feline and 3 human) by immunoblotting and quantitative real-time-polymerase chain reaction (qRT-PCR). Orlistat, a FASN inhibitor, substantially reduced cell viability in both feline and human OSCC lines, although feline cell lines consistently displayed higher sensitivity to the drug. FASN mRNA expression among cell lines mirrored sensitivity to orlistat, with feline cell lines expressing higher levels of FASN. Consistent with this observation, diminished sensitivity to orlistat treatment and decreased FASN mRNA expression were observed in feline OSCC cells following incubation under hypoxic conditions. Treatment with orlistat did not potentiate sensitivity to carboplatin in the cell lines investigated; instead, combinations of the 2 drugs resulted in additive to antagonistic effects. Our results suggest that FASN inhibition is a viable therapeutic target for feline OSCC. Furthermore, cats may serve as a spontaneous large animal model for human oral cancer, although differences in the regulation of lipogenesis between these 2 species require further investigation.

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers.

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Identification of drug-resistant subpopulations in canine hemangiosarcoma.

Canine hemangiosarcoma is a rapidly progressive disease that is poorly responsive to conventional chemotherapy. Despite numerous attempts to advance treatment options and improve outcomes, drug resistance remains a hurdle to successful therapy. To address this problem, we used recently characterized progenitor cell populations derived from canine hemangiosarcoma cell lines and grown as non-adherent spheres to identify potential drug resistance mechanisms as well as drug-resistant cell populations. Cells from sphere-forming cultures displayed enhanced resistance to chemotherapy drugs, expansion of dye-excluding side populations and altered ATP-binding cassette (ABC) transporter expression. Invasion studies demonstrated variability between cell lines as well as between sphere and monolayer cell populations. Collectively, our results suggest that sphere cell populations contain distinct subpopulations of drug-resistant cells that utilize multiple mechanisms to evade cytotoxic drugs. Our approach represents a new tool for the study of drug resistance in hemangiosarcoma, which could alter approaches for treating this disease.

Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma.

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain.

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs.

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.

GD3 ganglioside antibody augments tumoricidal capacity of canine blood mononuclear cells by induction of interleukin 12.

Monoclonal antibody R24 recognizes ganglioside GD3 expressed on the cell surfaces of some tumor cells and on a subset of human T lymphocytes. Binding of R24 to these lymphocytes induces proliferation, cytokine production, and activation of intracellular signaling pathways. In the current report, we investigated expression of gangliosides by canine mononuclear immune cells and studied the ability of antiganglioside antibody to activate these cells using tumor cell killing as a measure of activation. A subset of canine monocytes, but not lymphocytes, was found to express gangliosides GD3 and GD2 as determined by the binding of monoclonal antibodies R24 and 14.G2a, respectively. Only R24 augmented the tumoricidal potential of fresh canine peripheral blood mononuclear cells (PBMCs) against tumor cell lines that did not express surface gangliosides GD3 or GD2. The augmenting effect of R24 on PBMC-mediated tumor cytotoxicity required cooperation between monocytes and lymphocytes because there was no enhancement of cytotoxicity mediated by R24 combined with either monocytes or lymphocytes individually. The enhancing effect of R24 on canine PBMC-mediated tumor cytotoxicity was blocked by anti-interleukin (IL)-12 neutralizing antibody, suggesting that R24 binding to monocytes triggered IL-12 release, contributing to the observed tumor killing effects. Reverse transcription-PCR confirmed that the binding of R24 to canine monocytes induced transcription of mRNA for canine IL-12. These data indicate that monocytes can be activated for tumoricidal responses through a membrane structure associated with ganglioside GD3 triggered by the binding of R24 and that the mechanism for enhanced cytotoxicity is due to the production and secretion of IL-12.