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

Abstract

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.

Keywords: CTSA; canine adenocarcinoma; leptin; metastasis; obesity.

Figure 1

Leptin downregulated the cathepsin A (CTSA) gene in canine inflammatory mammary adenocarcinoma (CHMp) cells. (a) CTSA gene expression in CHMp cells treated with leptin and Allo-aca was evaluated using real-time PCR. (b,c) Western blot analysis of time-course CTSA/lysosomal-associated membrane protein 2a (LAMP2a) alteration induced by leptin (b) and Allo-aca (c) treatment. (d) Western blot analysis of chaperone-mediated autophagy (CMA)-related genes. Cells were incubated with leptin and/or Allo-aca for 24 h, and CTSA small interfering ribonucleic acid (siRNA) was transfected 24 h before chemical treatment. All graphs are visualized as mean ± standard error of the mean (SEM) with at least three replicates. The column bars with different alphabetical letters indicate significant difference among groups (p < 0.05). Combi: combination of leptin and Allo-aca; NC: negative control siRNA.

Figure 2

Cell proliferation assay. Quantitative measurement was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Five thousand cells per well were seeded and cultured for 5 days in a 37 °C, 5% CO₂ incubator. (a) Leptin significantly promoted cancer cell proliferation from day 3, whereas Allo-aca inhibited its effect in CHMp cells. (b) Allo-aca treatment on CTSA siRNA-transfected cells did not affect cell proliferation. However, it seemed that the knockdown of CTSA decreased cell proliferation compared to the negative control group. The results were expressed as mean ± SEM. * p < 0.05, *** p < 0.001.

Figure 3

Leptin stimulated epithelial–mesenchymal transition (EMT) in CHMp cells. (a) Matrigel invasion assay and calculated invasion index of CHMp and siRNA-transfected cells. Invaded cells were counted under light microscopy (40×) using ImageJ software. (b) Comparison of EMT-related gene (E-cadherin and vimentin) expression using real-time PCR analysis among experimental groups. (c) Western blot analysis of tumor invasion-related genes. All graphs are visualized as mean ± SEM with at least three replicates. The column bars with different alphabetical letters indicate significant difference among groups (p < 0.05). Combi: combination of leptin and Allo-aca; NC: negative control siRNA.

Figure 4

Leptin induced a higher LAMP2a/LAMP1 ratio in lysosomal membranes. (a) Western blot analysis of lysosomes isolated from CHMp cells targeting CMA-associated lysosomal proteins. The results showed that the LAMP2a/LAMP1 ratio was increased with leptin treatment and Allo-aca acted as a leptin receptor (OBR) antagonist. Then, the samples were subjected to SDS-PAGE. (b) Leptin and Allo-aca-treated CHMp cells and CTSA siRNA were visualized with confocal microscopy with immunofluorescence technique. The colocalization ratio was measured by Fiji software developed by Schindelin and colleagues [31] and analyzed on the basis of Mander’s colocalization coefficient (MCC). The results were in concordance with the lysosome immunoblot experiments. (c) Western blot analysis of the lysosomal mTORC2/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 pathway in lysosomes isolated from CHMp cells. pAKT1(ser473) and rictor were inhibited with leptin treatment, but PHLPP1 was stimulated. All graphs are visualized as mean ± SEM with at least three replicates. The column bars with different alphabetical letters indicate significant difference among groups (p < 0.05). Combi: combination of leptin and Allo-aca; NC: negative control siRNA.

Figure 5

Schematic images describing the summarized results in this study. Leptin downregulated CTSA and, following delayed LAMP2a degradation, resulted in CMA activation and stimulation of LAMP2a multimerization through the mTORC2/PHLPP1/AKT1 pathway.