Canine Melanoma Cell Lines Research Articles

Establishment of Canine Oral Mucosal Melanoma Cell Lines and Their Xenogeneic Animal Models.

Canine oral melanoma is the most prevalent malignant tumor in dogs and has a poor prognosis due to its high aggressiveness and high metastasis and recurrence rates. More research is needed into its treatment and to understand its pathogenic factors. In this study, we isolated a canine oral mucosal melanoma (COMM) cell line designated as COMM6605, which has now been stably passaged for more than 100 generations, with a successful monoclonal assay and a cell multiplication time of 22.2 h. G-banded karyotype analysis of the COMM6605 cell line revealed an abnormal chromosome count ranging from 45 to 74, with the identification of a double-armed chromosome as the characteristic marker chromosome of this cell line. The oral intralingual and dorsal subcutaneous implantation models of BALB/c-nu mice were successfully established; Melan-A (MLANA), S100 beta protein (S100β), PNL2, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) were stably expressed positively in the canine oral tumor sections, tumor cell lines, and tumor sections of tumor-bearing mice. Sublines COMM6605-Luc-EGFP and COMM6605-Cherry were established through lentiviral transfection, with COMM6605-Luc-EGFP co-expressing firefly luciferase (Luc) and enhanced green fluorescent protein (EGFP) and COMM6605-Cherry expressing the Cherry fluorescent protein gene. The COMM6605-Luc-EGFP fluorescent cell subline was injected via the tail vein and caused lung and lymph node metastasis, as detected by mouse live imaging, which can be used as an animal model to simulate the latter steps of hematogenous spread during tumor metastasis. The canine oral melanoma cell line COMM6605 and two sublines isolated and characterized in this study can offer a valuable model for studying mucosal melanoma.

Identification of hypoxia-induced metabolism-associated genes in canine tumours.

Canine tumours including urothelial carcinoma, lung adenocarcinoma, mammary gland tumour, squamous cell carcinoma, and melanoma have been identified as causes of death, but effective therapies are limited due to insufficient knowledge of the molecular mechanisms involved. Within the tumour microenvironment, hypoxia activates hypoxia-inducible factor 1α (HIF1α) in tumour cells. High HIF1α expression correlates with enhanced glycolysis and poorer outcomes in human cancers. However, the molecular mechanisms underlying hypoxic tumour cells remain elusive in dogs. In our study, we investigated upregulated genes in a canine malignant melanoma cell line during hypoxia using RNA-sequencing analysis. Glycolysis and HIF1 signalling pathways were upregulated in hypoxic melanoma cells. HIF1α knockout melanoma cells revealed that the glycolysis marker MCT4 is regulated by HIF1α activation. Hypoxia induces high lactate secretion due to enhanced glycolysis in canine melanoma cells. Furthermore, we examined monocarboxylate transporter 4 (MCT4) expression in malignant melanoma and eight other types of canine tumour tissues using immunohistochemistry (IHC). Membrane-localized MCT4 protein was mostly detected in urothelial carcinoma and lung adenocarcinoma rather than malignant melanoma. We conclude that canine MCT4 protein plays a role in lactic acid efflux from glycolytic cells and may serve as a marker for hypoxia and glycolysis in canine tumours. These findings could inform future therapeutic strategies targeting MCT4.

Repurposing of the Cardiovascular Drug Statin for the Treatment of Cancers: Efficacy of Statin-Dipyridamole Combination Treatment in Melanoma Cell Lines.

Metastatic melanoma has a very poor prognosis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitors, are cholesterol-lowering agents with a potential for cancer treatment. The inhibition of HMGCR by statins, however, induces feedback, which paradoxically upregulates HMGCR expression via sterol regulatory element-binding protein-2 (SREBP2). Dipyridamole, an antiplatelet agent, is known to inhibit SREBP2 upregulation. We aimed to demonstrate the efficacy of statin-dipyridamole combination treatment in both human and spontaneously occurring canine melanoma cell lines. The half maximal inhibitory concentration (IC50) of atorvastatin showed a 68-92% reduction when combined with dipyridamole, compared with that of atorvastatin alone. In some melanoma cell lines, cell proliferation was suppressed to almost zero by the combination treatment (≥3 μM atorvastatin). Finally, the BRAF inhibitor, vemurafenib, further potentiated the effects of the combined statin-dipyridamole treatment in BRAF V600E mutation-bearing human melanoma cell lines. In conclusion, the inexpensive and frequently prescribed statin-dipyridamole combination therapy may lead to new developments in the treatment of melanoma and may potentiate the effects of vemurafenib for the targeted therapy of BRAF V600E-mutation bearing melanoma patients. The concordance between the data from canine and human melanoma cell lines reinforces this possibility.

Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines.

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Comparison of the anticancer effects of various statins on canine oral melanoma cells.

Canine oral melanoma is a highly malignant cancer with a poor prognosis. Statins, commonly used drugs for treating dyslipidemia, exhibit pleiotropic anticancer effects and marked anti-proliferative effects against melanoma cells. The anticancer effects among statins vary; in human cancers, lipophilic statins have shown stronger anticancer effects compared with hydrophilic statins. However, data on the differences in the effects of various statins on canine cancer cells are lacking, hence the optimal statins for treating canine melanoma remain unknown. Therefore, this study aimed to clarify the most effective statin by comparing the anticancer effects of hydrophilic rosuvastatin and lipophilic atorvastatin, simvastatin, fluvastatin and pitavastatin on three canine oral melanoma cell lines. Time-dependent measurement of cell confluence showed that lipophilic statins had a stronger anti-proliferative effect on all cell lines than hydrophilic rosuvastatin. Quantification of lactate dehydrogenase release, an indicator of cytotoxicity, showed that lipophilic statins more effectively induced cell death than hydrophilic rosuvastatin. Lipophilic statins affected both inhibition of cell proliferation and induction of cell death. The anticancer effects of statins on canine oral melanoma cells differed in the following ascending order of IC50 values: pitavastatin < fluvastatin = simvastatin < atorvastatin < rosuvastatin. The required concentration of pitavastatin was approximately 1/20th that of rosuvastatin. Among the statins used in this study, pitavastatin had the highest anticancer effect. Our results suggest lipophilic pitavastatin as the optimal statin for treating canine oral melanoma.

Therapeutic applications of local injection of hsa-miR-634 into canine spontaneous malignant melanoma tumors.

Malignant melanoma (MM) is one of the most common tumors in both dogs and humans. As canine MM (CMM) and human MM (HMM) have similar clinical characteristics, CMM appears to be a good clinical model for HMM. We previously demonstrated that the introduction of a synthetic double-strand-microRNA-634 (miR-634) mimic triggered apoptotic cell death by directly targeting the genes associated with cytoprotective processes in various human cancer cell lines, including those of HMM. This study aimed to investigate the antitumor effects of the local administration of miR-634 on spontaneous CMMs to provide a basis for future applications of miR-634 formulations in HMM treatment. We found that miR-634 administration induced apoptosis in CMM cell lines in vitro via downregulation of Asct2, Nrf2, and survivin expression, similar to the mechanisms in HMM cell lines. Furthermore, intratumoral miR-634 administration induced antitumor effects in four of seven spontaneous CMM cases, with no adverse effects. Local administration of miR-634 to lung metastasis under ultrasound guidance induced tumor shrinkage. These results confirm the antitumor effect of the local administration of miR-634 in spontaneous CMM, a model for spontaneous HMM, thereby providing a novel treatment strategy for HMM.

Expression and Prognostic Evaluation of the Receptor Tyrosine Kinase MET in Canine Malignant Melanoma

The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs.

Regulation of programmed death ligand 1 expression by interferon-γ and tumour necrosis factor-α in canine tumour cell lines.

Expression of programmed death ligand 1 (PD-L1) on tumour cells provides an immune evasion mechanism by inducing suppression of cytotoxic T cells. Various regulatory mechanisms of PD-L1 expression have been described in human tumours, however, little is known in canine tumours. To investigate whether inflammatory signalling is involved in PD-L1 regulation in canine tumours, the effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α treatment were examined in canine malignant melanoma cell lines (CMeC and LMeC) and an osteosarcoma cell line (HMPOS). The protein level of PD-L1 expression was upregulated by IFN-γ and TNF-α stimulation. Upon IFN-γ stimulation, all cell lines showed an increase in expression of PD-L1, signal transducer and activator of transcription (STAT)1, STAT3 and genes regulated by STAT activation. Upregulated expression of these genes was suppressed by the addition of a JAK inhibitor, oclacitinib. Contrastingly, upon TNF-α stimulation, all cell lines exhibited higher gene expression of the nuclear factor kappa B (NF-κB) gene RELA and genes regulated by NF-κB activation, whereas expression of PD-L1 was upregulated in LMeC only. Upregulated expression of these genes was suppressed by the addition of an NF-κB inhibitor, BAY 11-7082. The expression level of cell surface PD-L1 induced by IFN-γ and TNF-α treatment was reduced by oclacitinib and BAY 11-7082, respectively, indicating that upregulation of PD-L1 expression by IFN-γ and TNF-α stimulation is regulated via the JAK-STAT and NF-κB signalling pathways, respectively. These results provide insights into the role of inflammatory signalling in PD-L1 regulation in canine tumours.

Protein phosphatase 6 regulates trametinib sensitivity, a mitogen-activated protein kinase kinase (MEK) inhibitor, by regulating MEK1/2-ERK1/2 signaling in canine melanoma cells.

Melanoma is a highly aggressive and metastatic cancer occurring in both humans and dogs. Canine melanoma accounts for a significant proportion of neoplastic diseases in dogs, and despite standard treatments, overall survival rates remain low. Protein phosphatase 6 (PP6), an evolutionarily conserved serine/threonine protein phosphatase, regulates various biological processes. Additionally, the loss of PP6 function reportedly leads to the development of melanoma in humans. However, there are no reports regarding the role of PP6 in canine cancer cells. We, therefore, conducted a study investigating the role of PP6 in canine melanoma by using four canine melanoma cell lines: CMec1, CMM, KMeC and LMeC. PP6 knockdown increased phosphorylation levels of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) but not Akt. Furthermore, PP6 knockdown decreased sensitivity to trametinib, a MEK inhibitor, but did not alter sensitivity to Akt inhibitor. These findings suggest that PP6 may function as a tumor suppressor in canine melanoma and modulate the response to trametinib treatment. Understanding the role of PP6 in canine melanoma could lead to the development of more effective treatment strategies for this aggressive disease.

G2/M arrest and mitotic slippage induced by fenbendazole in canine melanoma cells.

BackgroundThe use of fenbendazole (FBZ) in terminal cancer patients has recently increased, as anthelminthic drugs, such as FBZ and benzimidazole, exhibit anti‐tubulin effects in tumour cells.ObjectivesThe present study evaluated the in vitro anti‐cancer effects of FBZ in five canine melanoma cell lines originating from the oral cavity (UCDK9M3, UCDK9M4, UCDK9M5, KMeC and LMeC).MethodsFive canine melanoma cell lines were treated with FBZ and analysed with cell viability assay, cell cycle analysis, western blot assay and immunofluorescence staining to identify apoptotic effect, cell cycle arrest, microtubule disruption and mitotic slippage.ResultsCell viability was reduced in all melanoma cell lines in a dose‐dependent manner after FBZ treatment. Through cell cycle analysis, G2/M arrest and mitotic slippage were identified, which showed a time‐dependent change. All treatment concentrations induced increased cleaved PARP signals in western blot analysis compared to the control groups. Immunofluorescence of cells treated for 24 h revealed defects in microtubule structure, multinucleation or macronucleation. With the exception of UCDK9M3, the melanoma cells showed mitotic slippage and post‐slippage death, indicative of mitotic catastrophe.ConclusionsThese results indicate that FBZ exhibits anti‐cancer effects in vitro against canine melanoma cells; however, further in vivo studies regarding the clinical applications of FBZ are required.

Investigation of the effects of mTOR inhibitors rapamycin and everolimus in combination with carboplatin on canine malignant melanoma cells

BackgroundMalignant melanoma in dogs is considered to be largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that contribute to tumour progression despite aggressive local and systemic therapy. Upregulation of the PI3K/AKT/mTOR pathway has been observed in canine malignant melanoma and may represent a potential target for therapy. Rapamycin (sirolimus) and everolimus are commercially available small molecule inhibitors that target mTOR and therefore may have anticancer activity in canine melanoma. It was hypothesized that there is synergism between rapamycin or everolimus and platinum chemotherapy, and that combination drug treatment would inhibit target/downstream proteins involved in cell viability/proliferation and increase cell death in canine melanoma cells. It was further hypothesized that rapamycin or everolimus would impact metabolism by reducing glycolysis in these cells. Four canine melanoma cell lines were treated in vitro with rapamycin and everolimus as sole treatment or combined with carboplatin. Cell viability, apoptosis, target modulation, and glycolytic metabolism were evaluated by crystal violet colourimetric assay, Annexin V/PI flow cytometry, western blotting, and Seahorse bioanalyzer, respectively.ResultsWhen combined with carboplatin chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis was noted at 72 h after treatment compared to the vehicle control. Levels of phosphorylated mTOR were reduced by rapamycin and everolimus in all four cell lines, but activation of the downstream protein p70S6K was not consistently reduced by treatment in two of the cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma cells, indicating reduced cancer cell glycolytic activity.ConclusionsInhibition of mTOR by rapalogs, such as rapamycin and everolimus combined with carboplatin chemotherapy may have activity in canine melanoma. Future mechanistic investigation is warranted, including in vivo assessment of this combination therapy.

Therapeutic potential of the cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine suicide system for canine melanoma.

We tested the efficacy of a yeast cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine (CDU/5-FC) non-viral suicide system on eight established canine melanoma cell lines. Albeit with different degree of sensitivity 5 days after lipofection, this system was significantly efficient killing melanoma cells, being four cell lines highly, two fairly and two not very sensitive to CDU/5-FC (their respective IC50 ranging from 0.20 to 800 μM 5-FC). Considering the relatively low lipofection efficiencies, a very strong bystander effect was verified in the eight cell lines: depending on the cell line, this effect accounted for most of the induced cell death (from 70% to 95%). In our assay conditions, we did not find useful interactions either with the herpes simplex thymidine kinase/ganciclovir suicide system (in sequential or simultaneous modality) or with cisplatin and bleomycin chemotherapeutic drugs. Furthermore, only two cell lines displayed limited useful interactions of the CDU/5-FC either with interferon-β gene transfer or the proteasome inhibitor bortezomib respectively. These results would preclude a wide use of these combinations. However, the fact that all the tested cells were significantly sensitive to the CDU/5-FC system encourages further research as a gene therapy tool for local control of canine melanoma.

Anti-tumor effects of rivoceranib against canine melanoma and mammary gland tumour in vitro and in vivo mouse xenograft models

BackgroundRivoceranib, a novel tyrosine kinase inhibitor, exhibits anti-tumour effects by selectively blocking vascular endothelial growth factor receptor-2 (VEGFR2) in cancer cells. Recently, the therapeutic effects of rivoceranib on solid tumours have been elucidated in human patients. However, the anti-tumour effects of rivoceranib against canine cancer remain unclear. Here, we investigated the anti-tumour effects of rivoceranib using in vitro and in vivo mouse xenograft models.MethodsWe performed cell proliferation, cell cycle, and migration assays to determine the effects of rivoceranib on canine solid tumour cell lines in vitro. Furthermore, apoptosis and angiogenesis in tumour tissues were examined using a TUNEL assay and immunohistochemistry methods with an anti-cluster of differentiation-31 antibody, respectively. Additionally, the expression levels of cyclin-D1 and VEGFR2 activity were determined using western blot analysis.ResultsRivoceranib treatment showed anti-proliferative effects and mediated cell cycle arrest in the canine melanoma cell line (LMeC) and the mammary gland tumour (MGT) cell line (CHMp). In animal experiments, rivoceranib decreased the average volume of LMeC cells compared to that following control treatment, and similar results were observed in CHMp cells. Histologically, rivoceranib induced apoptosis and exerted an anti-angiogenic effect in tumour tissues. It also downregulated the expression of cyclin-D1 and inhibited VEGFR2 activity.ConclusionOur results show that rivoceranib inhibits proliferation and migration of tumour cells. These findings support the potential application of rivoceranib as a novel chemotherapeutic strategy for canine melanoma and MGTs.

H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment.

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.

Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression.

Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-β1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-β1 has been reported in dog cancers. This study aimed to examine whether TGF-β1 is upregulated in canine cancers. We measured TGF-β1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-β1 production was observed in several cell lines, and serum TGF-β1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-β1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4+CD25+Foxp3+ lymphocytes, suggesting that TGF-β1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-β, namely TGF-βRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-βRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-βRII and the Fc region of canine IgG-B. As expected, TGF-βRII-Ig bound to TGF-β1. In the presence of TGF-β1, the treatment with TGF-βRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4+CD25+Foxp3+ lymphocytes. Our results suggest that TGF-βRII-Ig competitively inhibits the immunosuppressive effects of TGF-β1 and thereby activates immune responses. This study demonstrated the potential of TGF-βRII-Ig as a novel biologic for canine melanoma.

Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma.

The cyclin-dependent kinase inhibitors (CKIs) belong to a group of key cell cycle proteins that regulate important cancer drug targets such as the cyclin/CDK complexes. Gene defects in the INK4A/B CKI tumor suppressor locus are frequently associated with human cancers and we have previously identified similar defects in canine models. Many of the cancer-associated genetic alterations, known to play roles in mammary tumor development and progression, appear similar in humans and dogs. The objectives of this study were to characterize expression defects in the INK4 genes, and the encoded p16 family proteins, in spontaneous canine primary mammary tumors (CMT) as well as in canine malignant melanoma (CML) cell lines to further develop these models of spontaneous cancers. Gene expression profiles and characterization of p16 protein were performed by rtPCR assay and immunoblotting followed by an analysis of relevant sequences with bioinformatics. The INK4 gene family were expressed differentially and the genes encoding the tumor suppressor p16, p14, and p15 proteins were often identified as defective in CMT and CML cell lines. The altered expression profiles for INK4 locus encoded tumor suppressor genes was also confirmed by the identification of similar gene defects in primary canine mammary tumor biopsy specimens which were also comparable to defects found in human breast cancer. These data strongly suggest that defects identified in the INK4 locus in canine cell lines are lesions originating in spontaneous canine cancers and are not the product of selection in culture. These findings further validate canine tumor models for use in developing a clear understanding of the gene defects present and may help identify new therapeutic cancer treatments that restore these tumor suppressor pathways based on precision medicine in canine cancers.

DEVELOPMENT OF ADENOVIRAL VECTOR-MEDIATED GENE THERAPY AGAINST CANINE MELANOMA CELL LINES

<h3>Background</h3> Metastatic melanoma is an aggressive type of cancer, challenging to treat in advanced cases. It often presents dysfunctions in the p53 pathway, although the p53 gene itself often remains in its wild type form. Our group has developed adenoviral vectors for the delivery of p14ARF and IFNβ in order to re-establish the functions of p53 and also induce an anti-tumour immune response. In previous in vitro and in vivo studies, in mouse and human cells, we observed that the transduction of both transgenes by adenoviral vectors leads to immunogenic cell death (ICD) events, characterised by calreticulin exposure and release of ATP and HMGB1. Despite our previous results it is imperative to ascertain these effects in more complexes organisms, capable of developing spontaneous tumours and metastases. Therefore, in this project, we aim to adapt and validate this approach in canine melanoma models. <h3>Methods</h3> Here we report the construction of adenoviral vectors expressing canine p14ARF and IFNβ under the control of a p53 responsive promoter (PG). We evaluated PG activity in canine melanoma cell lines (CMCL) through luciferase assay as well as the expression of the therapeutic proteins by immunofluorescence and ELISA assays. Additionally, flow cytometry was used to quantify hypodiploid cells upon viral transduction. <h3>Results</h3> Although the detection of IFNβ was inconclusive we could verify that our canine vectors are able to express p14ARF and also induce a significant amount of hypodiploid cells. <h3>Conclusion</h3> Canine melanoma cell lines BAN and GAB are permissive to RGD-modified serotype 5 adenoviral vectors. Furthermore, we verified the potential of our vectors to induce increased levels of hypodiploid cells 72hs post in vitro treatment. As a perspective of our work, we plan to further investigate the ICD potential of those vectors as well as to perform in situ gene therapy in CMCLs using xenograft models.

JARID1-targeted histone H3 demethylase inhibitors exhibit anti-proliferative activity and overcome cisplatin resistance in canine oral melanoma cell lines.

Histone demethylases are overexpressed or display altered activity in numerous human cancers leading to alterations in cell cycle dynamics, DNA repair kinetics, and therapeutic resistance. Consequently, therapeutic targeting of histone demethylases has become an active and promising area of research in human oncology. However, the role of histone demethylases and the potential efficacy of demethylase inhibition in canine cancers remains largely unknown. In the present work, we addressed this knowledge gap by exploring the therapeutic potential of histone demethylase inhibitors (HDIs) in canine oral melanoma. Using canine melanoma cell lines, we determined that broad spectrum HDIs result in decreased cell survival and prolonged DNA damage repair kinetics. We then showed that JARID1B, a histone H3 demethylase implicated in proliferation-dormancy regulation and drug sensitivity in human cancers, is highly expressed in canine tumour tissues. HDIs targeting JARID1B, and related JARID1 family members, significantly reduced survival fractions in canine melanoma cell lines, but did not appear to modulate DNA damage repair kinetics like broad spectrum HDI treatments. Importantly, we found that the anti-proliferative effects of JARID1-targeted HDIs are preserved in cell lines resistant to platinum-based chemotherapeutics, suggesting that HDIs may serve as a viable therapeutic strategy when faced with oral melanomas that progress despite the use of conventional therapies.

Influence of Canine Macrophage-derived Extracellular Vesicles on Apoptosis in Canine Melanoma and Osteosarcoma Cell Lines.

The purpose of this study was to evaluate the effect of extracellular vesicles derived from canine M1-polarized macrophages (M1EVs) on canine tumor cells, such as D17 (osteosarcoma cells) and LMeC (melanoma cells). Protein expression was determined by western blot analysis. Gene expression was determined by RT-qPCR. In addition, cell apoptosis was analyzed by Annexin V/PI staining. In the case of M1EV, the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β were increased, and nitrate/nitrite levels were also increased. M1EV induced apoptosis of tumor cells by increasing caspase-3 and caspase-7 activation. In addition, M1EVs decreased expression of CCR4, Foxp3 and CTLA-4 in canine peripheral mononuclear cells cocultured with tumor cells. M1EV could be an effective anti-cancer therapeutic approach in melanoma and osteosarcoma and M1EVs can be used as immunomodulators in the tumor microenvironment for cancer treatment.

Efficacy, Tolerability, and Pharmacokinetics of Combined Targeted MEK and Dual mTORC1/2 Inhibition in a Preclinical Model of Mucosal Melanoma.

Melanomas arising in the mucous membranes are a rare and aggressive subtype. New treatment approaches are needed, yet accumulating sufficient evidence to improve patient outcomes is difficult. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell-cycle alteration, synergistically reducing cell survival in canine mucosal melanoma cell lines with varying basal signaling activation levels. Compared with individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary mucosal melanoma xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. The combination did not significantly change plasma sapanisertib pharmacokinetics; however, trametinib area under the curve was increased in the presence of sapanisertib. Targeting Ras/MAPK and PI3K/AKT/mTOR signal transduction pathways appear rational therapies for canine and human mucosal melanoma.