Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer

• Published in: Cellular oncology (Dordrecht) Vol. 45; no. 5; pp. 873 - 891
• Main Authors: Dey, Pranay, Joshi, Mansi, Mujawar, Aaiyas, Malhotra, Renu, De, Abhijit
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.10.2022; Springer Nature B.V
• Subjects: AKT protein; Biomedical and Life Sciences; Biomedicine; Breast cancer; Cancer Research; Cell activation; Cell survival; Cytotoxicity; Electroporation; ErbB-2 protein; Immunoblotting; Immunofluorescence; Monoclonal antibodies; Niclosamide; Oncology; Original Article; Pathology; Proteins; Stat3 protein; Transfection; Trastuzumab; Tumor cell lines; Post-translational modification; Protein knockdown; Breast cancer; HER2; STAT3; HER3
• ISSN: 2211-3428; 2211-3436
• DOI: 10.1007/s13402-022-00693-6

Purpose In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called ‘TRIM-ing’. Methods To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. Results TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7–8 days. This significantly inhibited BC survival ( p  < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity ( p  < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. Conclusion The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.