Paulina Fuentes is a pharmaceutical chemist from Valdivia Chile, with close to 10 years trying to contribute to the population from the private and clinical area. Since 2020 she is coursing a Ph.D. in Medical Sciences. She is now working on her doctoral thesis whose main topic of investigation, and challenge, is to contribute to the understanding of breast cancer cell biology and resistance.
The kallikrein-related peptidases (KLKs) are a family of serine proteases that are being investigated as potential cancer biomarkers. KLK4, in particular, has been identified as a promising prognostic marker for various types of cancer, including breast cancer, in which having overexpression of KLK4 has been linked to poor prognosis, though the biological function or precise mechanism in breast cancer progression is still not fully understood. Breast cancer is a heterogeneous disease, with the luminal subtype (positive for estrogen receptor, ER+) being the most prevalent and with the best prognosis. However, more than half of advanced ER+ breast cancers are intrinsically resistant to tamoxifen, and about 40% will acquire resistance during treatment. To contribute to understanding the role of KLK4 in ER+ breast cancer biology, including determining proinflammatory factors’ effects on their expression, we have investigated its role in the modulation of endothelial permeability, in adhesion to extracellular matrix components, and explored the potential contribution of KLK4 to tamoxifen resistance. We also aimed to determine whether KLK4 can be used as a biomarker for luminal breast cancer.
To achieve our goals, we used different in vitro approaches in estrogen-sensitive breast cancer cell lines (MCF-7) or tamoxifen-resistant cells (MCF-7 TAMR) stimulated with recombinant KLK4 (rKLK4; 2, 10, and 20 ng/ml), IL-8 (10, 50, 100, and 150 pg/ml), and/or tamoxifen (1, and 7,6 μM). We then used a variety of techniques, including Western blotting, immunofluorescence, ELISA, an in vitro permeabilization assay, MTT, and an adhesion assay, to investigate the role of KLK4 in breast cancer progression.
Our results suggest that KLK4 may play a pro-tumorigenic role in breast cancer as a promotor of vascular permeability and increased adhesion to collagen type I, fibronectin, and fibrinogen. We also found that tamoxifen treatment increases the levels of endogenous KLK4. The immunolocalization of KLK4 in MCF-7 TAMR cells changed compared to MCF-7 WT cells, from a cytosolic to a more nuclear pattern, an effect that may shed some light on its functional role. We also found that KLK4 may positively regulate an alternative, plasmatic membrane estrogen receptor called GPER-1 and the levels of aromatase, the enzyme involved in estrogen synthesis but not ER. Interestingly, even when KLK signaling is mediated by members of the protease-activated receptors (PAR) family, so far, we have not observed changes in the PAR2 subtype, the most important subtype reported in breast cancer. Additionally, we found that rKLK4 and IL-8 increased KLK4 protein levels and secretion in MCF-7 cells, respectively. Consistent with these findings, we also found increased serum levels of KLK4 in patients suffering from ER+ breast cancer compared to healthy female participants in a pilot case-control study in the Los Rios region in the south of Chile.
In conclusion, while our study suggests that KLK4 may contribute to breast cancer progression, further research is needed to clarify its specific role. Our results are significant because they provide insights into the biological implications of KLK4 in the ER+ breast cancer model, including some responses to tamoxifen treatment.
Acknowledge: This work was supported by FONDECYT 1201635 (PE); ANID Doctorado nacional 21220130 (PF).