Leptin Modulates the Metastasis of Canine Inflammatory Mammary Adenocarcinoma Cells Through Downregulation of Lysosomal Protective Protein Cathepsin A (CTSA)

Canine malignant mammary gland tumors present with a poor prognosis due to metastasis to other organs, such as lung and lymph node metastases. Unlike in human studies where obesity has been shown to increase the risk of breast cancer, this has not been well studied in veterinary science. In our preliminary study, we discovered that leptin downregulated cathepsin A, which is responsible for lysosomal-associated membrane protein 2a (LAMP2a) degradation. LAMP2a is a rate-limiting factor in chaperone-mediated autophagy and is highly active in malignant cancers. Therefore, in this study, alterations in metastatic capacity through cathepsin A by leptin, which are secreted at high levels in the blood of obese patients, were investigated. We used a canine inflammatory mammary gland adenocarcinoma (CHMp) cell line cultured with RPMI-1640 and 10% fetal bovine serum. The samples were then subjected to real-time polymerase chain reaction, Western blot, immunocytochemistry, and lysosome isolation to investigate and visualize the metastasis and chaperone-mediated autophagy-related proteins. Results showed that leptin downregulated cathepsin A expression at both transcript and protein levels, whereas LAMP2a, the rate-limiting factor of chaperone-mediated autophagy, was upregulated by inhibition of LAMP2a degradation. Furthermore, leptin promoted LAMP2a multimerization through the lysosomal mTORC2 (mTOR complex 2)/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 (Serine/threonine-protein kinase 1) pathway. These findings suggest that targeting leptin receptors can alleviate mammary gland cancer cell metastasis in dogs.