Abstract
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.
Highlights
Increased proliferation is an important characteristic of tumorigenesis
In order to investigate the prognostic significance of the selected E2F target genes in canine melanoma samples, we analyzed RNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor samples of different types of canine melanocytic tumors and we compared different groups of patients, considering the following signs of tumor malignancy and progression: mitotic count, presence of recurrence, metastasis, death due to melanoma, disease free interval (DF), overall survival (OS)
Since no relevant results emerged in our sample population, we considered the diagnosis not influent for the purposes of our study and performed the analysis grouping all tumors (Supplementary Figures 2A–C)
Summary
Increased proliferation is an important characteristic of tumorigenesis. The proliferation rate is routinely estimated immunohistochemically using the Ki67 antibody (MIB-1). The level of Ki67 expression (as Ki67 index) is widely accepted as an indicator of prognosis and used as a prognostic marker in a number of human [1] and canine cancers [2,3,4,5,6]. A protein encoded by the E2F target gene BIRC5, has been suggested as a prognostic marker in several canine tumors, including cutaneous melanoma [17]
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