Immunohistochemical evaluation of LOX, LOXL2 and HIF1A expression in canine mammary tumors

Author(s)

Jessika Daniel

• Document type: Master's Dissertation
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2023-11-16

Examining board members

Ricardo de Francisco Strefezzi; Rodrigo dos Santos Horta; Adelina Maria Gaspar Gama Quaresma

• Advisor: Ricardo de Francisco Strefezzi

Abstract

Mammary tumors account for about 50% of neoplasms in female dogs, and even the conventionally accepted prognosis indicators cannot adequately predict the clinical behavior of these tumors. This fact highlights the need to identify more effective prognostic markers. The LOX family proteins play a role in cancer progression, associated with tumor invasion, metastasis, and unfavorable prognosis in various tumor types. HIF1A is related to aggressiveness, progression, and resistance to breast cancer therapy and controls the expression pathways of Lysyl oxidases. The objectives of this research were to characterize the immunohistochemical expression of the LOX, LOXL2, and HIF1A proteins in mammary neoplasms of bitches for prognostic evaluation, and to investigate the effects of hypoxia on the expressions of LOX and HIF1A in human and canine mammary carcinoma cell lines, as well as the effect of these proteins on cell migration and proliferation. Ninety-five samples of mammary carcinomas from 91 female dogs with a minimum clinical follow-up of 180 days were analyzed. Tumor samples were subjected to immunohistochemistry for the detection of LOX, LOXL2, and HIF1A. The reactions were quantified in five images using a 40x objective, obtained at random fields or hot spots. The results were compared with histological types, mortality due to the disease, and post-surgical survival time. For in vitro experiments, a human and a canine mammary adenocarcinoma cell lines (MDA-MB-231 and CIPp, respectively), and a metastatic cell line derived from a solid carcinoma from a dog (CIPm) were used. Hypoxia was simulated by applying cobalt dichloride (CoCl2) and glucose oxidase/catalase. No statistically significant differences were found for LOX, LOXL2, and HIF1A between the High and Low Malignancy histological groups when comparing the presence/absence of labeling in epithelial cells, the percentage of labeled epithelial cells, and the intensity of labeling. All censored cases in the survival analysis were positive for LOXL2 in epithelial cells, while 33.3% of the dogs that died due to the tumor were negative. Only 12.5% of High Malignancy tumors had positive fibroblasts for HIF1A, compared to 61.4% of Low Malignancy group (p=0.0180). In the first 24 hours of hypoxia, the CIPm lineage did not show significant reduction in proliferation and, 8 hours after treatment, cells treated with 100 µM of CoCl2 showed a significant increase in migration compared with the control group. Our results suggest that lower HIF1A expression in intratumoral fibroblasts and lower LOXL2 expression are indicators of a higher chance of death due to mammary carcinomas. CoCl2-induced hypoxia triggers a significant reduction in cell proliferation and, under hypoxic conditions, CIPm cells increase their migratory activity. (AU)

• FAPESP's process: 20/12789-1 - Immunohistochemical evaluation of LOX, LOXL2 and HIF1-alpha expression in canine mammary tumors
• Grantee: Jessika Daniel Urel
• Support Opportunities: Scholarships in Brazil - Master