br Introduction Epidermal growth factor receptor EGFR activa

Introduction Epidermal growth factor receptor (EGFR)-activating-mutant non-small cell lung cancer (NSCLC) often initially responds well to EGFR tyrosine kinase inhibitors (TKIs) (Haber et al., 2011); however, the disease almost always recurs about 10–13 months of therapy. Analysis of clinical specimens indicated that TKI-resistant NSCLC harbors multiple acquired resistance mechanisms, including amplification or upregulation of Axl, Her-2, c-Met, Akt, Erk, and nuclear factor κB (NF-κB) signaling, and EGFR second-site mutation T790M (Rotow and Bivona, 2017). To overcome T790M-mediated resistance, third-generation TKIs, e.g., AZD9291 (osimertinib), were developed and showed promising results (Janne et al., 2015), but virtually all tumors eventually develop resistance after about 10 months of treatment (Minari et al., 2016). Likewise, tumors from patients who failed AZD9291 treatment also harbor similar mechanisms underlying disease progression, e.g., EGFR C797S mutation, activation of Akt and MAPK, and amplification of HER-2, MET, or EGFR, which are highly heterogeneous even within an individual patient (Minari et al., 2016, Piotrowska et al., 2015). In addition to acquired resistance, intrinsic resistance, which by definition is a lack of response or an initial response with tumor reprogression <4 months (Park et al., 2014), may also be attributed to heterogeneous resistant mechanisms. Previous in vitro studies indicated that EGFR amplification in EGFR-mutant NSCLC chemical d causes resistance to an irreversible TKI (Ercan et al., 2010). Those findings raise the interesting question of whether and how the expression level of EGFR per se plays a role in resistance to EGFR kinase inhibition. We hypothesized that EGFR-mutant NSCLC is addicted to EGFR via the well-known kinase-mediated downstream signaling (TKI-sensitive) and additional unknown roles of EGFR (TKI-insensitive), and that the TKI-insensitive EGFR pathways, including multiple known resistant mechanisms, contribute to the heterogeneity of TKI resistance. Thus, identification of a common and targetable mediator involved in the TKI-insensitive EGFR pathways may provide a treatment strategy to overcome disease recurrence.
Results
Discussion Diverse upregulation or activation of RTKs as well as EGFR downstream signaling have been shown to induce TKI resistance in EGFR-mutant NSCLC (Camidge et al., 2014, Minari et al., 2016). In several studies, the therapeutic effects in TKI-resistant NSCLC are concurrent with drug-induced downregulation of EGFR, such as heat-shock protein 90 inhibitor (Johnson et al., 2015) and dual targeting of EGFR by afatinib and cetuximab in clinical trials (Janjigian et al., 2014). Nonetheless, whether and how downregulation of EGFR induces TKI-resistant tumor regression is not clear. Moreover, the lack of effective strategies to overcome relapses due to heterogeneity of resistance suggested that different lesions may develop distinct mechanisms of resistance within a tumor or in an individual patient, complicating the treatment strategies (Suda et al., 2016). Here, we report a mechanism by which EGFR-mutant NSCLC escapes the antitumor activity of TKI via the EGFR-mediated resistance through nPKCδ upregulation (Figure 6G). Combination of PKCi and TKI leads significant tumor shrinkage. It has been reported that kinase inhibitor induces the formation of inactive EGFR heterodimers (Anido et al., 2003, Ferrer-Soler et al., 2007). Consistently, we showed that gef induced EGFR heterodimers in TKI-resistant EGFR-mutant NSCLC, in which phosphorylation of EGFR Y845, Y1068, and Y1086 were almost completely suppressed by gef. However, EGFR pY1173 was only partially reduced by gef, and the sustained pY1173 by EGFR heterodimer promoted activation of PLCγ2 and PKCδ. Identification of pY1173 EGFR in TKI resistance is interesting and encouraging to further understand the detailed mechanisms underlying how RTKs mediate PKCδ activation. A systematic study will be required to derive a clear conclusion on this aspect and pursue it in the future.