ABSTRACT
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which requires heterodimerization with the Ah receptor nuclear translocator (Arnt) for function. Arnt is also a dimerization partner of the hypoxia inducible factor 1alpha (HIF-1alpha) for the hypoxia signaling. Additionally, Arnt is found to be a potent coactivator of the estrogen receptor (ER) signaling. Thus we examined whether the presence of an increased amount of AhR may suppress both the HIF-1alpha and ER signaling pathways by sequestering Arnt. We tested our hypothesis using a human AhR construct C Delta553 which is capable of heterodimerizing with Arnt in the absence of a ligand. Transient transfection studies using a corresponding luciferase reporter plasmid in MCF-7 cells showed that C Delta553 effectively suppressed the AhR, HIF-1alpha, and ER signaling pathways. Reverse transcription/real-time QPCR data showed that C Delta553 blocked the up-regulation of the target genes controlled by AhR (CYP1A1), HIF-1alpha (VEGF, aldolase C, and LDH-A), and ER (GREB1, pS2, and c-myc) in MCF-7 cells. Since both HIF-1alpha and ER are highly active in the ER-positive breast cancer, C Delta553 has the potential to be developed as a protein drug to treat breast cancer by blocking these two signaling pathways.
Subject(s)
Breast Neoplasms/therapy , Hypoxia/prevention & control , Receptors, Aryl Hydrocarbon/therapeutic use , Receptors, Estrogen/metabolism , Adenocarcinoma/therapy , Basic Helix-Loop-Helix Transcription Factors , Gene Deletion , Genes, Reporter/drug effects , Genetic Therapy/methods , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Luciferases/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Signal Transduction/genetics , Transfection , Tumor Cells, CulturedABSTRACT
The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.
