RE-1-silencing transcription factor shows tumor-suppressor functions and negatively regulates the oncogenic TAC1 in breast cancer cells

Abstract

Breast cancer remains the most prevalent cancer among women in the United States. Substance P, a peptide derived from the TAC1 gene, mediates oncogenic properties in breast and other cancers. TAC1 expression facilitates the entry of breast cancer cells into bone marrow. The transcriptional repressor element 1-silencing transcription factor (REST) has been implicated in both oncogenic and tumor-suppressor functions. REST binds to the 5' untranslated region of the TAC1 promoter and suppresses its expression. This study investigated a role for REST in TAC1 induction in breast cancer. Western blots and real-time PCR indicated that REST expression in breast cancer cells was inversely proportional to the cells' aggressiveness, for both cell lines and primary breast cancer cells. REST knockdown in low-metastatic T47D cells and nontumorigenic MCF12A cells resulted in increases in TAC1 induction, proliferation, and migration. These parameters were negatively affected by ectopic expression of REST in highly aggressive MDA-MB-231 cells. Together, these findings show a central role for REST in the oncogenic function of TAC1 and suggest a tumor-suppressor role for REST in breast cancer.

Fig. 1.

REST expression in BCCs. (A) Real-time PCR was performed for REST mRNA. MDA-MB-231 was arbitrarily assigned a value of 1 and the results of T47D and MCF12A presented as the mean ± SD (n = 5) fold change of MDA-MB-231. (B) Western blots were performed for REST with whole-cell extracts. (C) Band densities were normalized with those for β-actin. *, P < 0.05 vs. MDA-MB-231 and MCF12A; **, P < 0.05 vs. T47D.

Fig. 2.

TAC1 expression in REST knockdown BCCs. REST knockdown BCCs (wt) were verified by Western blot (A) and real-time PCR (B). Controls included mutant (mt) siRNA and nontransfectants. mRNA levels for REST (wt) were normalized to 1 for mean fold change ± SD (n = 5). (C) Transfectants in A were cotransfected with pGL3-TAC1-1.2 and pβ-gal. Luciferase activities were normalized with β-gal activities and then presented as the mean ± SD (n = 5). (D) Levels of TAC1 mRNA in cells from A were determined by real-time PCR. The levels in nontransfectants were arbitrarily assigned values of 1 and the experimental levels presented as mean fold change ± SD (n = 5). *, P < 0.05 vs. siRNA (mt) and untransfected cells.

Fig. 3.

Growth curves for REST knockdown BCCs. MDA-MB-231 (A), T47D (B), and MCF12A (C), untransfected or stably transfected with REST siRNA (wt or mt), were analyzed for growth properties. Cells were seeded at 50/mL and viable cells counted daily up to Day 5. The total number of cells are presented as mean ± SD (n = 5). Extracts from transfectants were analyzed for P-AKT in Western blots (D). *, P < 0.05 vs. siRNA (mt) and untransfected cells.

Fig. 4.

Migration of REST knockdown and effects of TAC1. (A) Fluorescence-labeled cells (10⁴) were placed in the inner chamber, and migrated cells were determined from a standard curve, established with BCCs vs. fluorescence intensities. Results are presented as mean ± SD (n = 5). (B) MCF12A was induced to express TAC1 by knockdown of REST. The role of TAC1 peptides were studied in cell proliferation studies by culturing untransfected and mutant (mt) and wild-type (wt) REST siRNA transfectants in the presence or absence of antagonists (10 nM): NK1 (CP-96,345) and NK2 (SR 48968). (C) The cells from B were studied in migration assays. *, P < 0.05 vs. untransfected and REST siRNA (mt); **, P < 0.05 vs. REST siRNA (wt) with antagonists.

Fig. 5.

Ectopic expression of REST in MDA-MB-231. REST was stably expressed in MDA-MB-231. Controls were untransfected or stable transfectants with vector. Efficient expression was determined by Western blots for REST with whole-cell extracts and normalized for β-actin (A) and real-time PCR for REST (B). Parallel studies were done for TAC1 mRNA by real-time PCR (C). The untransfected cells were assigned a value of 1 for fold change (mean ± SD; n = 5) of the other experimental points. *, P < 0.05 vs. the other experimental points.

Fig. 6.

REST expression on the growth and migration of MDA-MB-231. REST was expressed as for Fig. 5 and then analyzed for proliferation (A) and migration (B). Inset in A shows Western blots for Bcl2 and normalization for β-actin. The results represent mean ± SD (n = 5). *, P < 0.05 vs. untransfected and vector transfectants.

Fig. 7.

REST expression in primary breast biopsies. Real-time PCR and Western blots for REST mRNA (A) and protein (B), respectively, in breast tissues at varying stages of BC. mRNA levels for nonmalignant cells were assigned values of 1 and the results presented as mean ± SD (n = 5). Western blots were normalized for β-actin, and the results are shown as normalized band densities. *, P < 0.05 vs. nonmalignant tissues.