Flávia Alessandra Oliveira Rebouças ¹, Adália Oliveira-Lopes², Denise Damasceno Guerreiro¹, Belarmino Lopes Neto³, Ivan bustamente Filho⁴, Ana Cristina Monteiro⁵, Arlindo de Alencar Araripe Noronha Moura¹

Canine mammary tumors (CMT) are recognized as effective models for comparative oncology studies due to their biological and clinical similarity to human breast cancer (HNC). This study aimed to characterize the proteomic profile of spontaneous CMTs as compared to normal mammary tissues. Mammary tumors (n=5) and non-tumor mammary tissues (n=5) were collected from euthanized female dogs at the Zoonoses Control Center of Fortaleza, CE, Brazil, following institutional ethical guidelines. For histopathology analysis, CMT samples were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. Tumors were classified according to the WHO system, including benign mixed tumors (n=2), inflammatory carcinoma (n=1), and complex adenomas (n=2). Proteins were extracted from CMT and control tissue samples using lysis buffer [urea, NaCl, Tris, sodium orthovanadate, beta-glycerophosphate, protease inhibitors], followed by sonication and centrifugation (14,000 x g, 15 min., 4°C). Proteins (60 μg) were digested with trypsin, and the resulting peptides were desalted and analyzed by label-free data-dependent acquisition mass spectrometry (Synapt XS, Waters®, USA). Protein identification was carried out using MassLynx and Progenesis software against the UniProt database for Canis lupus familiaris, with statistical analysis using ANOVA (p < 0.05; fold change ≥ 1.5). Functional enrichment and interaction networks were assessed using Metascape and STRING (score > 0.9). miRBase and miRNet 2.0 databases were used for in silico prediction of miRNA regulation of genes associated with differentially expressed proteins. Homo sapiens data was used due to incomplete miRNA information for the canine species. A total of 259 proteins were identified, with 192 differentially expressed (DE); among them, 189 were upregulated and 3 downregulated in CMTs as compared to normal tissues. Key upregulated proteins in CMT included ALDOA, TXNDC5, PRDX family members (PRDX1, PRDX2, PRDX4, PRDX6), HSP70 (HSPA5, HSPA8), and HSP90 (HSP90AA1, HSP90AB1, HSP90B1), which were involved in glycolysis, oxidative stress response, neutrophil degranulation, protein folding, and tumor metabolism. Proteins related to the extracellular matrix, such as PCOLCE, ACAN, TNN, and P4HA1, were more abundant in tumor samples, with strong interaction with collagens (COL1A1, COL3A1), integrins, and adhesion molecules. In silico analysis revealed that genes of DE proteins were regulated by miRNAs linked to tumorigenesis, DNA repair, hypoxia response, and hormone signaling. Notably, PCOLCE was targeted by miR-124-3p and miR-155-5p, while P4HA1 was associated with miRNAs related to metastasis and estrogen regulation. Finally, P4HA1 was overexpressed in CMT tumor tissues compared to the control group, and its high expression was significantly associated with lower overall survival in human breast cancer patients (p = 0.00069; TCGA dataset), reinforcing its prognostic relevance. In addition, CFH and TNN were identified for the first time as associated with CMTs, suggesting novel roles in canine tumor biology. These findings emphasize the tumor microenvironment as a source of potential biomarkers and therapeutic targets, supporting the relevance of CMTs in translational oncology and opening avenues for the development of comparative diagnostic and therapeutic strategies.