p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Abstract

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer-related signalling pathways. In this context, the deregulated expression of p62 -Sequestosome1 (p62/SQSTM1)a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62-based medicine. In veterinary oncology the role of p62 in cancerrelated pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 nonneoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62-negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour-rejection antigen for an anti-cancer immunotherapy. K E Y W O R D Sautophagy, dog, gene homology, immunohistochemistry, mammary tumours, p62/SQSTM1

p62-DNA-encoding plasmid reverts tumor grade, changes tumor stroma, and enhances anticancer immunity

“…However, in canine spontaneous tumors we cannot establish if an adaptive immune system is essential for the antitumor effect of p62 plasmid. Moreover, we know already that eliciting a specific anti-p62 immunity cannot be the only mechanism for the plasmid anti-cancer activity, since canine cancer cells (unlike human ones) omit p62 during tumor progression [25]. To bring these two facts together, we hypothesized that p62 potentiates the effects of T-cells targeting cancer antigens other than p62 (e.g., acting via immunomodulatory mechanisms).…”

“…Because canine mammary tumors, contrary to human tumors, do not express p62 [25], the plasmid could not act as a classic DNA vaccine encoding p62 as a target tumor-specific antigen. Thus, we hypothesized that p62 DNA enhances the adaptive immune response to tumor antigens other than p62.…”

“…Thus, the mechanism of action of the p62 plasmid needs to be reassessed based on the latest observations. Indeed, although treatment with the p62 - encoding plasmid was reported as therapeutically beneficial in dogs with spontaneous mammary tumors [24], it turned out later that unlike in humans, most aggressive canine breast tumors show very low or nil p62 expression [25]. In other word, eliciting an anti-p62 adaptive immunity cannot be the only effect of p62-encoding plasmid.…”

Immunohistochemical Expression of p62 in Feline Mammary Carcinoma and Non-Neoplastic Mammary Tissue

“…As a secondary antibody, a goat anti-rabbit biotinylated antibody was used along with DAB (diaminobenzidine) as the chromogen. Normal mouse pancreas was used as a positive control [ 24 ], while rabbit IgG-isotype (instead of primary antibody) and a blocking peptide-based protocol were used as a negative control for both. Sections were counterstained in Mayer’s hematoxylin.…”

Increased Expression of Toll-Like Receptor 4 in Skin of Dogs with Discoid Lupus Erythematous (DLE)

“…Immunohistochemical analysis was carried out with a standard ABC-peroxidase method on sections with a thickness of 3 µm as described by Mariotti et al (2019) [13]. Sec-tions were pre-treated with citrate buffer for antigen-retrieval, and the labelling expression of TLR4 was evaluated using a mouse monoclonal antibody anti-TLR4, clone 76B357.1, developed against a portion of 100-200 amino acids of human TLR4 (NP_612564, Novus Biologicals, Milan, Italy) diluted 1:100.…”