The combination of anthracyclin-based and non-anthracyclin-based chemotherapies with trastuzumab, a HER2-targeted therapeutic antibody, led to disease-free survival rates at 5 years of 81–84% compared with 75% without trastuzumab in HER2-positive early-stage breast cancer 3. Owing to the development of small molecules and therapeutic antibodies against this target, the treatment of HER2-amplified breast cancer made great progress. ![]() ![]() The amplification of HER2 in breast cancer leads to severe alterations in growth and proliferation signaling, e.g., mitogen-activated protein kinase (MAPK) signaling, resulting in a more aggressive and invasive growth of the tumor 1, 2. The human EGFR-related receptor 2 (HER2) oncogene/oncoprotein represents a perfect example of such a treatable target. The identification of targetable signal nodes and protein–protein interactions is of utmost interest for the development of novel drugs for the treatment of cancer and other diseases such as neurodegenerative diseases. The results clearly reveal the importance of knowing the exact mechanisms of the inhibitors affecting receptor tyrosine kinases in order to develop more efficient anti-HER2-targeted therapies. Selective inhibition of HER2 induced great alterations of mitochondria-associated interactions of PTPIP51, which ultimately led to the most-effective reduction of cell viability of SK-BR3 cells of all tested TKIs. Exclusively inhibiting HER2/ErbB2 by Mubritinib did not affect the interaction of PTPIP51 with the MAPK signaling. Inhibition of both EGFR and HER2/ErbB2R shifted PTPIP51 into the MAPK pathway, but left the mitochondria-associated interactome of PTPIP51 unattended. This study is aimed to elucidate the effects of four different TKIs on the interactome of PTPIP51, namely with the receptors EGFR and HER2, 14-3-3/Raf1 (MAPK pathway), its regulating enzymes, and the mitochondria-associated interaction partners in HER2 breast cancer cell lines (SK-BR3 and BT474) by using the Duolink proximity ligation assay, immunoblotting and knockdown of PTPIP51. The MAPK pathway is one of the most frequently overactivated pathways in HER2-amplified breast cancer cells. In earlier studies, we showed the involvement of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in the mitogen-activated protein kinase (MAPK) pathway. Upcoming resistances against the HER2-targeted therapy make a better understanding of the receptor associated downstream pathways an absolute need. Recently developed tyrosine kinase inhibitors and therapeutic antibodies targeting the HER2 receptor improved the overall survival time compared with sole radio- and chemotherapy. About 20–30% of these tumors exhibit an amplification of the HER2/ErbB2 receptor, which is coupled to a more aggressive and invasive growth of the cancer cells. Quantification of the number of PLA signals was done using the Duolink image tool.Breast cancer is the most common female cancerous disease and the second most cause of cancer death in women. k Quantification of the PLA signals in hippocampal CA1 and cortex for the association between mature γ-secretase and MAO-B. j Quantification of the PLA signals in hippocampal CA1 and cortex for the PS1/MAO-B association. Insets are enlarged parts of the original images to clearly visualize the PLA signals (note that the small size of PLA signals makes it difficult to see them on printouts). ![]() f– i PLA in mouse cortex (with the same antibody and GTB combinations as in b–e). b– e PLA in mouse hippocampal CA1 to visualize the association between PS1 and MAO-B (using a PS1 and an MAO-B antibody) and between active γ-secretase and MAO-B (using GTB and an MAO-B antibody) as well as negative controls (only MAO-B antibody and competition in the presence of L-685,458). a Cross-section of a mouse brain showing the regions chosen for PLA imaging: cortex ( blue squares) and hippocampal CA1 ( red squares). Female adult mouse brains were fixed in formalin, and 10-μm sections were cut using a cryostat and stored at −20 ☌ prior to PLA assay. Proximity ligation assay ( PLA) to visualize the PS1/MAO-B and γ-secretase/MAO-B interactions in mouse brain.
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