Investigations of nuclear HER family receptors in cancer and resistance to cetuximab therapy.

摘要

The HER family of receptor tyrosine kinases consists of four family members: epidermal growth factor receptor (EGFR/HER1/ErbB1), HER2 (ErbB2/Neu), HER3 (ErbB3), and HER4 (ErbB4). Ligand binding to HER family receptors initiates receptor dimerization, tyrosine kinase activation, and auto/trans phosphorylation of tyrosine residues located on each receptor's C-terminal tail. Classically, HER receptors are localized on the cell surface where they serve to initiate growth and survival signaling pathways.;Advances in HER family biology over the last decade have established that HER receptors can also be localized to intracellular organelles, one of which includes in the nucleus. Inside the nucleus HER family receptors can function as both co-transcription factors and nuclear kinases. These nuclear functions have been linked to three parameters of tumor biology: 1) inverse correlation with overall survival in numerous cancers, 2) resistance to both conventional and molecular targeted therapies, and 3) enhanced tumor growth.;Previous investigations into mechanisms of acquired resistance to the anti-EGFR monoclonal antibody therapeutic cetuximab demonstrated that resistant cells expressed increased levels of nuclear EGFR, which was regulated by Src family kinase (SFK) activity. To better understand how SFKs regulated EGFR nuclear translocation, we aimed to elucidate which SFK member(s) controlled this process as well as the EGFR tyrosine residues that were involved. Analyses of cetuximab resistant (CtxR) cells indicated that the SFKs Yes and Lyn were overexpressed and associated with the EGFR. Genetic ablation of either Yes or Lyn led to a loss of nuclear EGFR expression, and conversely, their overexpression resulted in increased nuclear EGFR expression. Interestingly, the stable overexpression of each SFK in MCF-7 cells resulted in an increase in nuclear EGFR expression, indicating that while Yes and Lyn function in this model other SFKs may regulate nuclear EGFR trafficking in other cell types. Further, site-directed mutagenesis of the SFK dependent phosphorylation sites on the EGFR, tyrosine 845 and tyrosine 1101, indicated that mutation of tyrosine 1101 (and not tyrosine 845) impaired nuclear entry of the EGFR. Collectively, these studies indicated that the SFKs Yes and Lyn could phosphorylate EGFR on tyrosine 1101 to influence its nuclear translocation in a model of acquired resistance to cetuximab.;Based on previous studies indicating that acquired resistance to cetuximab was mediated by nuclear EGFR, we hypothesized that nuclear EGFR may also promote intrinsic resistance to cetuximab in Triple-negative breast cancer (TNBC). To examine this question a battery of TNBC cell lines and human tumors were screened and found to express nuclear EGFR. Genetic ablation of the EGFR demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab therapy. Further, SFKs influenced nuclear EGFR translocation in TNBC cell lines and in vivo TNBC xenografts, where inhibition of SFK activity led to potent reductions in nuclear EGFR expression and increases in membrane localized EGFR. Importantly, the inhibition of nuclear EGFR translocation greatly enhanced TNBC cell line sensitivity so cetuximab, suggesting that nuclear EGFR may be a functional molecular target in TNBC.;Recent studies indicate that all HER receptors can function as co-transcription factors in the nucleus; however, the specific domains on HER receptors that confer transcriptional potential remain poorly defined. To identify putative transactivation domains (TADs) of a HER receptor, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain (CTD) of HER3 contained potent transactivation potential, and further mutational analyses resulted in the identification of a bipartite transactivation domain (TAD). Deletion of this bipartite TAD from the full-length HER3 receptor greatly reduced its ability to regulate the known target gene cyclin D1. These studies revealed that HER3 contains a bipartite TAD necessary for its ability to function as a co-transcription factor in the nucleus, and further suggests that all HER family receptors may contain homologous regions.;Collectively, the studies performed in this thesis indicate that nuclear EGFR translocation is dependent on SFK activation on tyrosine 1101 resulting in intrinsic resistance to cetuximab in TNBC. Further, nuclear HER receptors may be dependent on specific C-terminal TADs to function as co-transcription factors in the nucleus.