Calcitonin Receptor is a Novel Biomarker for NF-κB-Mediated Cellular Stress in Triple-Negative Breast Cancer Cells

Background: Cellular stress can arise from external and internal stimuli and induce mechanisms that either maintain survival or activate cell death pathways. Nuclear factor kappa B (NF-κB) is a key transcription factor that mediates a range of responses to cellular stress, including senescence, DNA repair, and inflammation in breast cancer; however, there are currently limited biomarkers that identify its activation. CalRexinTM, a monoclonal antibody that targets the human calcitonin receptor, was investigated as a novel biomarker for cell stress associated with NF-κB activation. Methods: Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with lipopolysaccharide (LPS) to induce NF-κB-mediated cellular stress or treated with the cytotoxin staurosporine to induce apoptosis as a positive control. CalRexinTM and Annexin V positivity were investigated by flow cytometry and immunofluorescence. Results: Flow cytometry analysis revealed a dose- and time-dependent increase in the mean fluorescence intensity of CalRexinTM following LPS treatment of breast cancer cells. The mean fluorescence intensity of Annexin V, as a marker of apoptosis was not increased by LPS treatment and the cells remained viable. Immunofluorescence imaging demonstrated CalRexinTM positivity in LPS-stimulated cells accompanied by nuclear translocation of NF-κB. Conclusion: Increased expression of calcitonin receptor, identified using the novel CalRexinTM antibody, has potential use as a biomarker for NF-κB-mediated cellular stress in breast cancer cells.