RT-PCR-based tyrosine kinase display profiling of canine melanoma: IGF-1 receptor as a potential therapeutic target

Canine malignant melanoma (CMM) resembles human malignant melanoma in terms of metastatic behavior, refractoriness to standard therapy, and tumor antigen expression but it is largely unknown how CMM resembles human melanoma with regard to molecular pathogenesis and cellular signaling. No attempt has been made to systematically define the repertoire of tyrosine kinases (TKs) expressed in CMM. This study used a reverse transcription-PCR display technique to evaluate the expression of multiple TKs in the 17CM98 CMM cell line. RT-PCR was performed using degenerate primers coding for highly conserved regions flanking the kinase domains of many TKs and the repertoire of TKs expressed was determined using standard molecular cloning techniques. Sequencing 46 clones yielded canine homologs of insulin-like growth factor-1 receptor (IGF-1R) (50%), JAK1 (17%), PDGFR-a (11%), FGFR1 (9%), Axl (7%), Abl (4%), and PTK2 (2%). Interestingly, IGF-1R, JAK1, and Axl were detected in human melanoma using similar techniques, supporting the cross-species validity of this assay. Given the abundance of IGF-1R clones, we determined the biological effect of rhIGF-1 in 17CM98 cells. IGF-1 stimulated cell proliferation and vascular endothelial growth factor production in 17CM98, and addition of the IGF-1R inhibitor ADW742 abrogated IGF-1-induced phenotypic changes. Expression of IGF-1R mRNA was detected in five of five additional CMM cell cultures, and IGF-1R protein was detected in five of six primary tumors evaluated, suggesting that IGF-1R expression may be common in CMM and may provide a novel target for future therapy. In conclusion, this study suggests that similar TKs are expressed in human and canine melanoma, and shows potential antitumor effects of IGF-1R inhibition in CMM

Pilot study of safety and feasibility of DNA microseeding for treatment of spontaneous canine melanoma

Abstract Spontaneous canine malignant melanoma provides an excellent pre‐clinical model to study DNA vaccines for melanoma immunotherapy. A USDA‐approved xenogeneic human tyrosinase (huTYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of huTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti‐huTYR ELISA was used to test pre‐ and post‐treatment sera. Biopsies of treated skin were obtained for detection of huTYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. huTYR‐expressing cells were observed in biopsies of huTYR DNA microseeding sites. Increased humoral anti‐huTYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy

Comparative gene expression profiling identifies common molecular signatures of NF-κB activation in canine and human diffuse large B cell lymphoma (DLBCL)

We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-κB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature-derived NF-κB target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-κB/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-κB-targeted therapeutics for human lymphoma. To validate this, tissue arrays were generated for canine and human NHL and immunohistochemistry was employed to assess NF-κB activation status. In addition, human and canine B-cell lymphoma lines were assessed for NF-κB activity and the effects of NF-κB inhibition

Hierarchical clustering of canine and human datasets using exclusively the expression levels of the NF-κB target genes (probesets).

<p>Hierarchical clustering of the canine (A) and human (B) datasets using exclusively the expression levels of the NF-κB target gene set. The samples are arranged in the columns (blue squares denote healthy and red squares denote DLBCL) and the probesets are in the rows. The dendrograms are drawn using Euclidean distances with average linkage method. (A) In the canine dataset (GSE30881), the 199 NF-κB target probesets separate the dataset into three top-level clusters. While the first and the third clusters have exclusively of DLBCL samples, the second cluster has all the healthy samples with two DLBCL samples. (B) In the human dataset (GSE12195), the 259 NF-κB target probesets separate the dataset into two top-level clusters. The first cluster has 12 samples that include all the healthy samples and two DLBCL samples, while the second cluster is solely of 43 DLBCL samples. The numbers above each column refer to sample identification numbers.</p

Comparison of enrichment of KEGG pathways in the co-expressed clusters of canine DLBCL and human DLBCL.

<p>The highly connected gene clusters identified in the co-expression networks of canine DLBCL and human DLBCL were analysed for enrichment of KEGG pathways using DAVID functional annotation tool. The results from the analysis of each cluster are compiled and the <i>p-values</i> of the enrichment score computed by Fisher's exact test are represented graphically as coloured icons.</p

The effect of doxorubicin and IKK inhibitor VII on the activation of NF-κB in human B cell lymphoma cell lines and canine B cell lymphoma cell lines.

<p>Nuclear protein from human B (RL, JM1, Pfeiffer) cell lymphoma lines as well as canine B (3132) cell lymphoma line treated with doxorubicin (A) and/or IKK inhibitor VII (B), were extracted. EMSAs were performed on these samples using non-radioactive DIG-labelled NF-κB consensus oligonucleotide probes in binding reactions. Samples were then subjected to electrophoresis in DNA retardation gels, before transfer onto nylon membranes and chemiluminescence detection of the DIG-labels. Bands are indicated by arrows and annotated. ‘+’ and ‘−’ refer to components being added or omitted respectively, to standard gel shift binding reactions. Expression of classical pathway NF-κB subunits in 3132 nuclear extracts when 3132 cells are treated with doxorubicin and/or IKK inhibitor VII at IC₅₀ doses was detected by western blotting of whole cell lysates (B).</p

Comparison of differentially expressed probesets in canine and human DLBCLs.

<p>Venn diagrams comparing the NF-κB target genes in the differentially expressed probesets (between DLBCL and healthy) of canine DLBCL and human DLBCL and comparison of the number of the differentially expressed probesets of canine DLBCL and human DLBCL. The differentially expressed probesets in the datasets were identified by one-way ANOVA (DLBCL Vs. healthy) of the expression values; selecting probesets with log₂ fold change over 2 with FDR adjusted <i>p-value</i> less than 0.05. (A) 25 NF-κB target probsets (17 NF-κB target genes out of the 120 genes) present in the differentially expressed probesets of canine DLBCL. (B) 101 NF-κB target probsets (54 NF-κB target genes out of the 120 genes) present in the differentially expressed probesets of human DLBCL. (C) Comparison of canine array probesets converted to orthologous human array probesets. (D) Comparison of human array probesets converted to orthologous canine array probesets.</p

NF-κB target genes in the differentially expressed gene set of the canine DLBCL.

<p>NF-κB target genes in the differentially expressed gene set of the canine DLBCL.</p

Top 20 Gene Ontology (BP) Enrichment in the differentially expressed probesets in canine DLBCL and human DLBCL (ranking based on FDR in the DAVID functional annotation chart).

<p>Top 20 Gene Ontology (BP) Enrichment in the differentially expressed probesets in canine DLBCL and human DLBCL (ranking based on FDR in the DAVID functional annotation chart).</p