Designer Monotransregulators Provide a Basis for a Transcriptional Therapy for De Novo Endocrine-Resistant Breast Cancer

Abstract

The main circulating estrogen hormone 17β-estradiol (E2) contributes to the initiation and progression of breast cancer. Estrogen receptors (ERs), as transcription factors, mediate the effects of E2. Ablation of the circulating E2 and/or prevention of ER functions constitute approaches for ER-positive breast cancer treatments. These modalities are, however, ineffective in de novo endocrine-resistant breast neoplasms that do not express ERs. The interaction of E2-ERs with specific DNA sequences, estrogen responsive elements (EREs), of genes constitutes one genomic pathway necessary for cellular alterations. We herein tested the prediction that specific regulation of ERE-driven genes by an engineered monomeric and constitutively active transcription factor, monotransregulator, provides a basis for the treatment of ER-negative breast cancer. Using adenovirus infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that the monotransregulator, but not the ERE-binding defective counterpart, repressed cellular proliferation and motility, and induced apoptosis through expression of genes that required ERE interactions. Similarly, the monotransregulator suppressed the growth of ER-negative BT-549 cells derived from a breast-ductal carcinoma. Moreover, the ERE-binding monotransregulator repressed xenograft tumor growth in a nude mice model. Thus, specific regulation of genes bearing EREs could offer a therapeutic approach for de novo endocrine-resistant breast cancers.

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

“…The engineering of expression vectors bearing ERα, the ERE binding module CDC and the ERE binding defective counterparts ERα EBD and CDC EBD were described previously [ 10 , 12 , 13 , 16 ]. We also described the construction of PV and PV EBD previously [ 10 ].…”

“…To assess the functional protein synthesis, we used immunocytochemistry (ICC), Western blot (WB) and electrophoretic mobility shift assay (EMSA). Transfected or infected cells were processed for WB, EMSA, and ICC as described previously [ 9 , 10 , 13 , 17 – 19 ]. Briefly, transfected or infected cells for 48h were collected and lysed with three cycles of freeze/thaw in a lysis buffer.…”

“…Recombinant adenovirus bearing none or a cDNA was produced as described previously [ 9 , 10 , 13 , 20 ] with the exception that we used the AdenoVator Adenoviral Expression System from Qbiogene Inc. (Carlsbad, CA). The purified viruses were titered using an Adeno-X Rapid Titer Kit (BD Biosciences, Palo Alto, CA, USA) to determine the multiplicity of infection (MOI).…”

“…The total RNA was subjected to quantitative PCR (qPCR) using TaqMan probes (Thermo-Fisher Sci. Inc., Foster City, CA, USA) as described [ 9 , 10 , 13 ]. The relative quantification was performed using the comparative 2 ΔΔC T method [ 21 ].…”

“…The integration of strong activation domains (ADs) from other transcription factors into this ERE binding module generated monotransactivators that robustly induced ERE-driven reporter gene expressions independent of E2 or dimerization [ 12 ]. Moreover, monotransactivators modulated cellular proliferation by activating as well as repressing the endogenous ERE-driven gene expressions in a manner similar to those mediated by E2-ERα [ 13 ].…”