Pathogenic HER3 dimerization domain mutations create a structural bias towards un-conventional EGFR-HER3 signalling axis in breast cancer.

Among the EGFR family of receptors, HER3 is considered as a pseudo-kinase which primarily interacts with HER2 in presence of heregulin-1ß. We identified two hotspot mutations i.e. G284R and D297Y and one double mutant HER2-S310F/HER3-G284R in breast cancer patients. Long term MDS (7.5 µs) revealed that HER3-D297Y and HER2-S310F:HER3-G284R do not allow the interaction with HER2 as these mutations cause dramatic conformational changes in its flanking regions. This results in formation of an unstable HER2-WT:HER3-D297Y heterodimer, thereby abrogating the downstream signalling by AKT. We found that His228 and Ser300 of HER3-D297Y form stable interactions with Glu245 and Tyr270 of EGFR-WT, in the presence of either EGF or heregulin-1ß. Applying TRIM-ing mediated direct knockdown of endogenous EGFR protein, specificity of the unconventional EGFR:HER3-D297Y interaction was validated. Due to this unusual ligand mediated interaction, cancer cells were found susceptible to EGFR targeted therapeutics i.e. Gefitinib and Erlotinib. Further, in TCGA analysis, BC patients harbouring HER3-D297Y mutation showed increased p-EGFR levels as compared to the patients harbouring HER3-WT and HER3-G284R mutations. For the first time, this comprehensive study showed the importance of specific hotspot mutations in HER3 dimerization domain can defy the Trastuzumab therapy, rather cells become susceptible to the EGFR inhibitors.

Recent advancements in targeted protein knockdown technologies-emerging paradigms for targeted therapy.

A generalized therapeutic strategy for various disease conditions, including cancer, is to deplete or inactivate harmful protein targets. Various forms of protein or gene silencing molecules, e.g., small molecule inhibitors, RNA interference (RNAi), and microRNAs (miRNAs) have been used against druggable targets. Over the past few years, targeted protein degradation (TPD) approaches have been developed for direct degradation of candidate proteins. Among the TPD approaches, proteolysis targeting chimeras (PROTACs) have emerged as one of the most promising approaches for the selective elimination of proteins via the ubiquitin-proteasome system. Other than PROTACs, TPD methods with potential therapeutic use include intrabody-mediated protein knockdown and tripartite motif-21 (TRIM-21) mediated TRIM-Away. In this review, protein knockdown approaches, their modes of action, and their advantages over conventional gene knockdown approaches are summarized. In cancers, disease-associated protein functions are often executed by specific post-translational modifications (PTMs). The role of TRIM-Away is highlighted in the direct knockdown of PTM forms of target proteins. Moreover, the application challenges and the prospective clinical use of TPD approaches in various diseases are also discussed.

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

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.

In Silico Identification of Potential Phosphorylation in the Cytoplasmic Domain of Epithelial Cell Adhesion Molecule.

The epithelial cell adhesion molecule (EpCAM) is a transmembrane cell adhesion glycoprotein, which primarily contributes to stemness, proliferation, and metastasis properties of tumor cells. Regulated intramembrane proteolysis by ADAM proteases and γ-secretase cleaves EpCAM into an ∼27 kDa soluble extracellular and an ∼4 kDa cytoplasmic domain (EpICD). After the EpICD fragment is released inside the cell, the formation of a nuclear signaling complex with the FHL2 molecule is critical for exerting its regulatory role. Trop-2, a homologous protein of EpCAM, undergoes phosphorylation in its cytoplasmic domain (Trop-IC). The phosphorylation of Trop-2 is reported to be crucial for its function. This led us to ask the fundamental question if EpCAM does undergo similar post-translational modification(PTM) like its homologous protein to carry out its diverse biological function. Here, we identify a putative phosphorylation site at Tyr297 located in the cytoplasmic domain of EpCAM. Molecular dynamic simulation (MDS) of 90 ns was carried out to understand the biological/functional relevance of the putative phosphorylation. It was observed that this phosphorylation stabilizes the α-helical structure of the EpICD. Though Tyr297 does not affect the γ-secretase mediated cleavage of EpCAM, it affects the binding of EpICD to FHL2. Docking analysis revealed that phosphorylation mediated structural stability of EpICD positively impacts its binding affinity with FHL2, which was further validated using 100 ns MDS. Phosphorylated EpICD forms higher numbers of hydrogen bonds, salt bridges, and other non-bonded interactions with FHL2, leading to enhanced interactions. This in silico study reveals a potential PTM in the EpICD, providing the basis for future research in understanding the mechanism behind the diverse biological function of EpCAM.

Targeting stem cells in the realm of drug-resistant breast cancer.

Since its first documentation, breast cancer (BC) has been a conundrum that ails millions of women every year. This cancer has been well studied by researchers all over the world, which has improved the patient outcome significantly. There are many diagnostic markers to identify the disease, but early detection and then subclassification of this cancer remain dubious. Even after the correct diagnosis, more than half the patients come back with a more aggressive and metastatic tumor. The underpinning mechanism that governs the resistance includes over-amplification of receptors, mutations in key gene targets, and activation of different signaling. A plethora of drugs have been devised that have shown promising results in clinical settings. However, in recent times, the role played by cancer stem cells in disease progression and their interaction in mediating the resistance to cellular insults have come into the limelight. As breast cancer stem cells (BCSCs) are dormant in nature, it is highly likely that they fail to directly respond to the cytotoxic drugs which are meant for ablating rapidly proliferating cells. Furthermore, the absence of well-characterized, drug-able surface markers to date, has limited the application of targeted therapies in complete eradication of the disease. In this review, our intent is to discuss versatile therapeutics in practice followed by discussing the upcoming therapy strategies in the pipeline for BC. Furthermore, we focus on the roles played by BCSCs in mediating the resistance, and therefore, the aspects of new therapeutics against BCSCs under development that may ease the burden in future has also been discussed.