Synergistic effects of various Her inhibitors in combination with IGF-1R, C-MET and Src targeting agents in breast cancer cell lines

Introduction: Overexpression of the receptor tyrosine kinase HER2 has been reported in around 25% of human breast cancers, usually indicating a poor prognosis. As a result, HER2 has become a popular target for therapy. However, despite recent advances in HER2 targeted therapy, many patients still experience primary and secondary resistance to such treatments. It is therefore important to understand the underlying mechanism of resistance and to develop more effective therapeutic interventions for breast cancer. Methods: The sensitivity of a panel of seven breast cancer cell lines to treatment with various types HER-family inhibitors alone, or in combination with a selection of other tyrosine kinase inhibitors (TKIs) or chemotherapeutic agents was determined using the Sulforhodamine B colorimetric assay. Receptor expression, cell-cycle distribution, cell signalling and cell migration were determined using flow cytometry, Western blot and Incucyte Zoom Live-Cell Analysis System respectively. Results: Overall, breast cancer cells were more sensitive to treatment with the irreversible pan-HER family inhibitors, particularly afatinib and neratinib, than treatment with the first-generation reversible inhibitors. Of three HER-2 overexpressing cell lines in this panel, SKBr3 and BT474 were highly sensitive to treatment with HER-family inhibitors (IC50s as low as 3 nM), while MDA-MB-453 was relatively resistant (lowest IC50 = 0.11 μM). When the HER-family inhibitors were combined with other agents such as NVP-AEW541 (an IGF-1R inhibitor), dasatinib (a Src inhibitor) or crizotinib (a c-Met/ALK inhibitor), such combination produced synergistic effects in some of the cell lines examined. Interestingly, co-targeting of Src and HER-family members in MDA-MB-453 cells led to synergistic growth inhibition, suggesting the importance of Src in mediating resistance to HER2-targeting agents. Finally, treatment with the irreversible HER family blockers and dasatinib were also most effective at inhibiting the migration of breast cancer cells. Conclusion: We concluded that the irreversible inhibitors of HER-family members are generally more effective at inhibiting growth, downstream signalling and migration compared with reversible inhibitors, and that combining HER-family inhibitors with other TKIs such as dasatinib may have therapeutic advantages in certain breast cancer subtypes and warrants further investigation

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

Tumour suppressor ​PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway. The ability to directly monitor ​PTEN conformation and function in a rapid, sensitive manner is a key step towards developing anti-cancer drugs aimed at enhancing or restoring ​PTEN-dependent pathways. Here we developed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, capable of detecting signal-dependent ​PTEN conformational changes in live cells. The biosensor retains intrinsic properties of ​PTEN, enabling structure–function and kinetic analyses. BRET shifts, indicating conformational change, were detected following mutations that disrupt intramolecular ​PTEN interactions, promoting plasma membrane targeting and also following physiological ​PTEN activation. Using the biosensor as a reporter, we uncovered ​PTEN activation by several G protein-coupled receptors, previously unknown as ​PTEN regulators. Trastuzumab, used to treat ​ERBB2-overexpressing breast cancers also elicited activation-associated ​PTEN conformational rearrangement. We propose the biosensor can be used to identify pathways regulating ​PTEN or molecules that enhance its anti-tumour activity