# The Presence of SFRP1 Reduces the High Risk of Metastasis in RANKL-Expressing Canine Mammary Cancers

## TL;DR

This study shows that SFRP1 can reduce the risk of metastasis in canine mammary cancers that express RANKL, a protein linked to cancer progression.

## Contribution

The study identifies SFRP1 as a potential suppressor of RANKL-driven metastasis in canine mammary cancer.

## Key findings

- RANKL expression is strongly associated with increased metastasis in canine mammary cancers.
- SFRP1 mRNA is negatively associated with metastasis status, and its presence reduces the metastatic risk in RANKL-positive cancers.
- Vascular-invasive cells in canine mammary tumors are consistently RANKL-positive.

## Abstract

Canine mammary cancer is a very common disease affecting about a quarter of unspayed females. Many of these cancers are malignant and metastasise, causing the death of the animal. Accurate estimation of prognosis is often difficult, in part because the criteria used for human breast cancer are not easily applied to the dog. We found that receptor activator of nuclear factor kappa-Β ligand (RANKL), a protein necessary for, among other processes, bone formation and normal mammary gland development, was strongly expressed by metastatic tumours, and could be used as a biomarker for the progression of the disease. In addition, we found that when secreted frizzled-related protein 1 (SFRP1), a protein that can bind to RANKL and inhibit its signalling pathway, was present at the same time, the risk of metastasis was reduced. SFRP1 may therefore play a role in reducing cancer metastasis risk in dogs, at least in part, by suppressing RANKL signalling.

Canine mammary cancers (CMCs) are one of the most prevalent types of neoplasm in dogs, are frequently malignant, and display high tumour heterogeneity, making evaluating prognosis and predicting successful treatment outcomes difficult. In a previous pilot study, overexpression of the Wnt pathway-associated protein SFRP1 was found to correlate with negative metastasis status in CMCs at both mRNA and protein levels. To establish SFRP1 as a potential biomarker for CMC progression, additional verification of these results in an independent dataset is required, as well as an investigation as to whether SFRP1 expression in CMCs is associated with altered Wnt- or RANKL signalling pathways. In an independent verification cohort of 122 cases of archival CMC FFPE material, expression of SFRP1 was assessed by RT-qPCR and immunohistochemistry. The same tumours were further assessed for RANKL, phosphoROCK2, and NFkB-p65 protein expression. Our data verified that SFRP1 mRNA (p = 0.025) was negatively associated with metastasis status; however, differences in protein expression did not reach statistical significance (p = 0.139). Neither did SFRP1 significantly correlate with expression of any of the other proteins tested. Instead, a strong association was found for RANKL positivity with increased metastasis status (p < 0.001). Co-expression of SFRP1 significantly lowered the higher risk of metastatic spread when compared to RANKLpos/SFRP1neg CMCs (p = 0.033). Noticeably, all vascular-invasive cell clusters observed in tissue section vessels stained positive for RANKL. Our study identified RANKL expression as a strong marker for cancer progression with a strong link to vascular-invasive cells. However, SFRP1 expression may potentially suppress the pro-metastatic nature of RANKLpos CMCs.