Abstract
Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and metabolism. Epidermal growth factor receptor (EGFR) is a transmembrane receptor-tyrosine kinase that is associated with cancer progression, and the EGFR inhibitors erlotinib/tarceva and tyrphostin/AG-1478 are potent anti-cancer therapeutics. Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b(5)-related protein that is up-regulated in tumors and promotes cancer growth. Pgrmc1 and its homologues have been implicated in cell signaling, and we show here that Pgrmc1 increases susceptibility to AG-1478 and erlotinib, increases plasma membrane EGFR levels, and co-precipitates with EGFR. Pgrmc1 co-localizes with EGFR in cytoplasmic vesicles and co-fractionates with EGFR in high density microsomes. The findings have therapeutic potential because a Pgrmc1 small molecule ligand, which inhibits growth in a variety of cancer cell types, de-stabilized EGFR in multiple tumor cell lines. EGFR is one of the most potent receptor-tyrosine kinases driving tumorigenesis, and our data support a role for Pgrmc1 in promoting several cancer phenotypes at least in part by binding EGFR and stabilizing plasma membrane pools of the receptor.
Highlights
19-IRG and by the Kentucky Lung Cancer Research Program. □S The on-line version of this article contains supplemental Figs. 1 and 2. 1 To whom correspondence should be addressed
In the present study we demonstrate an association between Epidermal growth factor receptor (EGFR) and progesterone receptor membrane component 1 (Pgrmc1)
We have found that EGFR and Pgrmc1 co-localize in a microsomal fraction, where Pgrmc1 is found in the lumen
Summary
Tissue Culture and RNAi—Cells were grown in Dulbecco’s modified Eagle’s medium with 10% serum supreme (Lonza, Basel, Switzerland) and antibiotics and were maintained at 37 °C in 5% CO2 in air. Cell surface labeling reactions were performed in triplicate, and 20 l of the biotin-labeled eluate or unbound fraction were analyzed by SDS-PAGE or Western blot. Cell surface proteins were labeled with biotin, purified with avidin-agarose, and analyzed by Western blot. 7.4-fold (Fig. 2A, lane 4, A549/RNAi cells) compared with control cells (Fig. 2A, lane 3), a significant result (p ϭ 0.01, t test) in triplicate labeling reactions Both of the plasma membrane proteins E-cadherin and CXCR4 were not significantly changed in A549/RNAi cells (Fig. 2, C and D), and SDS-PAGE of the avidin-bound proteins revealed few changes in band intensity (Fig. 2E). When Pgrmc was inhibited by siRNA at a different site in the Pgrmc transcript, EGFR membrane levels were de-
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