Integrated gene expression and mutation profiling of chemoresistance in canine mammary carcinoma cells

Mammary gland tumors (MGTs) are common in female dogs, and are usually not treated with chemotherapy due to the lack of evidence of benefit for the patients. In clinical practice, when a female dog receives neoadjuvant or adjuvant chemotherapy, minimal improvement is achieved. Therefore, chemotherapy is not usually applied for these patients. We hypothesized that some canine MGTs do not respond to chemotherapy due to intrinsic chemoresistance. Chemoresistance can be a challenge and is often associated with overexpression of ABC transporters, such as MDR1 , MRP1 , MRP2 , MRP3 and BCRP , which reduce the intracellular accumulation of chemotherapeutic agents into the cells. This study aimed to induce chemoresistance in canine MGT cells in vitro , identify potential genetic changes linked with resistance and to evaluate the expression of ABC transporter genes ( MDR1 , MRP1 , MRP2 , MRP3 and BCRP ) using RT-qPCR. We also aimed to verify the relation between chemoresistance and single nucleotide polymorphisms (SNPs). Five cell cultures were used (UNESP-CM1, UNESP-CM4, UNESP-CM5, UNESP-CM70 and UNESP-CMR1). In four cell lines, chemoresistance was induced with doxorubicin (DOX) and one cell line was naturally resistant. Single Nucleotide Polymorphism (SNP) analysis via genotyping and RT-qPCR were performed in cell line cultures, before and after chemoresistance induction. Evaluation by RT-qPCR showed that the UNESP-CMR1 cell presented higher expression of MDR1 , BCRP , MRP1 and MRP3 compared to the other cells. Three cells lines treated with DOX had increased expression of MDR1 , MRP1 and MRP2 after treatment. SNP analysis identified 35 potentially altered SNPs, with emphasis on SNPs on chromosomes 17 and 24, associated with the ALK and RALGAPA2 genes. Interestingly, ALK gene presents a tyrosine kinase domain and it is considered a therapeutic target. In conclusion, these preliminary data suggest that the induction of chemoresistance in canine mammary carcinoma cells may be associated with variations in the expression of ABC transporter genes.