Identification and characterization of a novel epidermal growth factor receptor variant [electronic resource]

The epidermal growth factor receptor (EGFR) is essential to multiple physiologic and neoplastic processes via signaling by its tyrosine kinase domain and subsequent activation of transcription factors. We identified a novel splice variant of the EGFR called mini-LEEK (mLEEK) which represents a deletion of exons 2-22 but maintains the open reading frame and generates a novel glycine codon at the junction. This variant deletes the extracytoplasmic domain, transmembrane region, and most of the tyrosine kinase domain of the EGFR. mLEEK is localized to the nucleus in most cells and since it contains a previously characterized strong transactivation domain, we hypothesized that mLEEK functions to regulate transcription. Microarray studies revealed upregulation of several molecular chaperone proteins, including GRP78 (also known as BiP) and GRP94, in response to mLEEK expression. Overexpression of mLEEK resulted in increased transcription from GRP78 and GRP94 luciferase reporter plasmids as well as an increase in endogenous GRP78 and GRP94 protein levels, suggesting a direct effect on transcription. Furthermore, mLEEK utilizes the cis-regulatory ER stress response element (ERSE) found in both the GRP78 and GRP94 promoters to activate transcription as mutation of the ERSE elements in the GRP78 promoter abolishes the activation of transcription by mLEEK. Chromatin immunoprecipitation experiments demonstrate that mLEEK physically interacts in a complex with the ERSE-containing region of the GRP78 promoter. Additionally, mLEEK co-precipitates with ATF6, a transcription factor known to be part of the ERSE-mediated activation of chaperones. Molecular chaperone transcription is induced as a downstream effector of the unfolded protein response (UPR). The UPR is a mechanism that increases the cellular capacity for protein folding. Cellular demands for protein folding increase upon physiological conditions that create endoplasmic reticulum (ER) stress. Interestingly, mLEEK specifically upregulates molecular chaperones required for the UPR and does not activate other arms of the UPR. Splicing of XBP1 precursor mRNA, which is uniquely triggered upon ER stress, was reduced in cells overexpressing mLEEK. Another molecular readout for UPR induction is the activation of CHOP transcription. Cells overexpressing mLEEK demonstrated reduced activation of CHOP transcription in the presence of ER stress inducing agents. Moreover, knockdown of mLEEK results in enhanced sensitivity to ER stress. Collectively, these data suggest that expression of mLEEK and the subsequent upregulation molecular chaperones primes cells to accommodate the increased need for protein folding upon ER stress and prevents induction of the UPR. mLEEK is also essential for cell viability, as mLEEK knockdown lead to reduced viability and increased activation of caspases. We found that mLEEK is present at the RNA and protein level in a high percentage of tumors relative to normal tissue. Preliminary data suggest that mLEEK expression does not lead to transformation of NIH3T3 cells. The potential contribution of mLEEK to tumorigenesis will be revisited in the future using knockdown strategies that were subsequently developed. Other preliminary data have shown that mLEEK is secreted into the media and taken up by neighboring cells, leading to increased GRP94 transcription in recipient cells. Collectively, our work describes the discovery and characterization of a novel EGFR variant. These findings reveal an unexpected function for an EGFR variant and represent a new mechanism for the upregulation of molecular chaperones required for the UPR that could be exploited for therapeutic purposes. Our findings also suggest a potential role for mLEEK in cancer and a novel method of cell communication mediated by a secreted protein. These possibilities await future studies.

Advisor: Wong Albert J. primary advisor. Thesis advisor
Kuo Calvin Jay advisor. Thesis advisor
Mitchell Beverly S. advisor. Thesis advisor
Sweet-Cordero Eric advisor. Thesis advisor