Characterization of the promoter region of the proto-oncogene c-kit in canine mast cell tumour

G-quadruplex structures at telomeric and promotorial sequences represent promising chemotherapeutic targets. Small molecules can bind and stabilize already formed G-quadruplexes or can induce their formation from G-rich sequences. In order to select efficient and selective G-quadruplex ligands, the conformational equilibria of the nucleic acid need to be characterized. In this work we are interested in the promotorial sequence of c-kit, a proto-oncogene which over expression or gain of function mutations characterizes human cancers, such as gastrointestinal stromal tumors (GIST), melanoma and mastocytosis. In humans, this region contains two G-rich sequences, c-kit1 and c-kit2. The G-quadruplex structures assumed by these two sequences have been extensively studied by means of X-ray crystallography and NMR. The stabilization of these secondary structures resulted in a suppression of gene transcription. In the domestic dog, c-kit over expression or mutations have been found in cutaneous mast cell tumor (MCT), representing the most common (7%-21%) canine skin tumor.. Therefore, we expect that dog could be a robust model to have a better insight into c-kit abnormalities in cancer and into the role of potential c-kit G-quadruplex ligand in anticancer chemotherapy. To support this, the canine upstream promotorial sequence was cloned and sequenced in both healthy and MCT-suffering dogs. Our results identified two putative G-quadruplex sequences in the canine promotorial region. Thus, we compared them with the human ones. In particular, large attention was devoted to clarify the conformational equilibria occurring in physiologically relevant conditions by means of spectroscopic and electrophoretic techniques. This work represents the required preliminary step for a better understanding of MCT biology, progression and treatment as well as to assess dogs as a translational model for the many c-kit related human tumours.