The Role of EGF Variant mLEEK and Grp78 in Breast Cancer

Institution: Stanford University
Investigator(s): Albert Wong, M.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0080 Award: $241,380
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2009)

The involvement of “molecular chaperones” in the pathogenesis of breast cancer and other cancers is now well documented. In this context “chaperones” are internal cellular proteins that assist in the non-covalent folding/unfolding and the assembly/disassembly of other complex protein structures. However, chaperones do not occur in these structures when they are performing their normal biological functions. Much evidence points to chaperones as playing a critical role in tumor development as well as resistance to chemotherapeutics and radiation therapy. A protein called Grp78 has been identified as key chaperone to help breast cancer cells resist stress, which causes cell proteins to become unfolded. We have now discovered a protein that regulates Grp78. It is a variant of a molecule clearly implicated in breast cancer, the EGF receptor. This EGF receptor variant, called mLEEK, increases Grp78 levels within cells.

The goals of this project are to find evidence that mLEEK is involved in primary breast cancer and to then investigate its contribution to tumor development in cell culture and animal model studies. First, the presence of mLEEK and subtypes will assayed by immunohistochemistry (antibody staining) and its correlation with levels of Grp78 will also be evaluated. Secondly, we will modulate mLEEK using a shRNA (RNA interference) approach. We will determine how this affects Grp78, and if it decreases tumor cell growth and enhances apoptosis, especially in the presence of chemotherapeutics. Finally, to confirm these observations, we will create cell lines that overexpress mLEEK and see if this increases tumor growth and decreases apoptosis.

This work will further elucidate a key protein chaperone pathway that has been shown to be linked to breast tumor development and therapeutic resistance. This will set the stage for the development of a monoclonal antibody inhibitor which can then be clinically tested.

Final Report (2012)

Note: This text was published in Oncogene (2012A) 31(24):2953-67.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein linked to numerous physiological processes through ligand stimulation of its intrinsic kinase activity leading to the induction of signaling cascades (Yarden and Sliwkowski, 2001). Frequent overexpression or alterations of the EGFR in cancer contribute to pathogenesis through effects on cell growth, survival and motility (Yarden and Sliwkowski, 2001). The most common variant, EGFRvIII, contains a deletion of exons 2–7 in the extracellular domain and is frequently found in glioblastoma (GBM) (Wong et al., 1992). Other structural variants have been described, including EGFRvIVa and EGFRvIVb, which contain deletions of segments downstream of the tyrosine kinase (TK) domain and confer oncogenic potential in vivo (Pines et al., 2010). In addition, point mutations in the TK domain contribute to drug sensitivity in non-small-cell lung cancer (Lynch et al., 2004; Paez et al., 2004). Novel signaling mechanisms have also been identified in cancer cells where the receptor has a direct association with the sodium/glucose transporter 1 (Weihua et al., 2008), or translocates to the nucleus and associates with transcription factors (Lin et al., 2001).

In this study, we identified an in-frame splice variant of the EGFR called mini-LEEK (mLEEK) that is more broadly expressed than the EGFR and is overexpressed in several cancers. Unlike previously characterized EGFR variants, mLEEK lacks the extracytoplasmic, transmembrane and tyrosine kinase domains. mLEEK localizes in the nucleus and functions as a transcription factor to regulate target genes involved in the cellular response to endoplasmic reticulum (ER) stress, including the master regulator of the unfolded protein response (UPR) pathways, molecular chaperone GRP78/Bip. We demonstrated that mLEEK regulates GRP78 transcription through direct interaction with a cis-regulatory element within the gene promoter. Several UPR pathways were interrogated and mLEEK expression was found to attenuate the induction of all pathways upon ER stress. Conversely, knockdown of mLEEK resulted in caspase-mediated cell death and sensitization to ER stress. These findings indicate that mLEEK levels determine cellular responses to unfavorable conditions that cause ER stress. This information, along with the overexpression of mLEEK in tumors, suggests unique strategies for therapeutic intervention. Furthermore, the identification of mLEEK expands the known mechanisms by which the EGFR gene contributes to oncogenesis and represents the first link between two previously disparate areas in cancer cell biology: EGFR signaling and the UPR.

The identification of mLEEK has revealed a new means for controlling the expression of molecular chaperones important for the UPR and the protection of cells from ER stress. The current understanding is that ER-resident sensors, such as IRE1, respond to stress by initiating signaling pathways that increases transcription of chaperones. However, the upregulation of chaperones by mLEEK is extrinsic to any changes within the ER. An ER-independent mechanism by which cells can increase chaperone production has not been identified previously. Many investigators have suggested that manipulating the UPR would be of therapeutic benefit for a wide variety of diseases (Lawson et al., 1998; Boyce et al., 2005; Kim et al., 2005; Kudo et al., 2008; Mu et al., 2008). Thus far, research has centered on chemical approaches to controlling chaperones. In the case of cancer, it has been shown that inhibition of chaperones might be used to prolong survival and increase chemosensitivity (Jamora et al., 1996; Fu and Lee, 2006; Pyrko et al., 2007). The discovery of mLEEK suggests new avenues for developing drugs that act upon the UPR. Interference with mLEEK expression and function is a promising strategy to block production of chaperones and thus tumor growth and survival.

A novel epidermal growth factor receptor variant lacking multiple domains directly activates transcription and is overexpressed in tumors.
Index Medicus: Oncogene
Authors: Piccione EC, Lieu TJ, Gentile CF, Williams TR, Connolly AJ, Godwin AK, Koong AC, Wong AJ
Yr: 2012 Vol: 31 Nbr: 24 Abs: Pg:2953-67