Negative feedback regulation as a means to constrain the oncogenic potential of mutant Egfr in NSCLC

Abstract

The discovery of EGFR kinase domain mutations in NSCLC patients who responded to tyrosine kinase inhibitors (TKIs) represented the first example of a targeted therapy for lung cancer. The dependence of these human tumors on sustained mutant receptor expression for survival, together with the discovery that ectopic expression of the receptor resulted in transformation, suggested that these mutations are causal events, and as such would be sufficient to induce tumor formation in the lung. To investigate this, and to further our understanding of how deregulated signaling through the mutant receptor could initiate tumor formation, we generated both a conditional and constitutive knock-in allele of one such mutation, L858R, at the endogenous murine Egfr locus. Expression of mutant Egfr failed to induce lung tumors in these mice; further analysis of the germline mutant mice revealed significant downregulation of the mutant receptor, and this was predominantly at the post-transcriptional level. These data suggest that normal cells can respond to an oncogenic lesion by upregulating negative feedback pathways to counteract the induction of aberrant signaling, and disabling these feedback mechanisms may be an essential component of the progression of EGFR mutant tumors. A multitude of positive and negative feedback loops converge on signaling pathways to ensure the appropriate output in response to a given stimulus. The role of oncogenes is typically thought of in terms of increasing the output of a signaling pathway, and while the contribution of the associated negative feedback loops is no doubt important, they have been afforded little attention. Recent studies have highlighted the existence of negative feedback mechanisms in established tumors and the integral role they play in shaping the signaling network, with a corresponding appreciation for how such feedback loops can profoundly influence therapeutic response. The capacity of negative regulators to modulate the oncogenic potential of a mutant protein in the context of tumor initiation has rarely been examined. Using a doxycyclineinducible system we recapitulate the aforementioned downregulation of mutant Egfr, initially observed in both tissues and MEFs derived from EgfrL858R mutant mice, in an ectopic cell culture expression system. We establish a role for ERK pathway signaling, and specifically DUSP6, in receptor degradation, and solidify the role of the E3 ligase, CULLIN5, in the downregulation of the mutant receptor. The existence of these negative feedback loops may explain the observation that mutation of EGFR is often coincident with gene amplification in NSCLC, and suggest that such amplification may primarily serve as a means to counteract the downregulation. The amplification of oncogenes is a recurring feature of many human tumors, but the contribution of gene amplification to particular stages of tumor development, or the molecular requirements for amplification to occur are unknown. EGFR is mutated and coincidentally amplified in NSCLC, but the relative contribution of mutation and amplification, both to tumor phenotype and therapeutic sensitivity, is not clear. The inability to model amplification in the mouse has contributed significantly to our limited mechanistic understanding of how gene amplification occurs in tumors. Using a yeast endonuclease, -Scel, and an allele that contains target sites for this enzyme engineered telomeric to mutant Egfr on chromosome 11, we attempted to initiate breakage-fusion-bridge (BFB) cycles in the lung, as these are thought to be a precursor to gene amplification. Our inability to elicit tumor formation using this strategy highlights the limitations in our understanding of how amplicons form in human tumors or the particular context required. While it would provide tremendous insight into mutant EGFR tumor development, a model of targeted gene amplification has thus far eluded us, and remains one of the significant challenges facing the mouse modeling community.