The potential role of regulator of G-protein signaling 16 in cell motility mediated by δEF1 family proteins

Abstract

The epithelial-mesenchymal transition (EMT) is associated with tumor progression. We reported previously that expression of the δEF1 family proteins (δEF1/ZEB1 and SIP1/ZEB2), key regulators of the EMT, is positively correlated with EMT phenotypes and aggressiveness of breast cancer. Here, we show that the expression levels of regulator of G-protein signaling 16 (RGS16) are negatively correlated with those of the δEF1 family proteins. On the basis of the results of gain- and loss-of-function analyses, we suggest that δEF1 family proteins promote cell motility of breast cancer cells directly or indirectly through repressing expression of RGS16.

Keywords: epithelial-mesenchymal transition; regulator of G-protein signaling 16; smad-interacting protein 1; δEF1; δEF1 family proteins.

Figure 1

Expression profiles of RGS16 in breast cancer cells. RGS16 mRNA levels were determined by qRT‐PCR in 21 human breast cancer cell lines. The collection of cell lines shown here was reported by Neve et al. 6 and Charafe‐Jauffret et al. 7.

Figure 2

Overexpression of RGS16 affects cell morphology and invasion of breast cancer cells. (A) MDA‐MB231 and BT549 cells were infected with lentiviruses encoding negative control or RGS16, followed by immunoblot analyses with the indicated antibodies. α‐tubulin levels were monitored as a loading control. Results are representative of at least three experiments. (B–D) Cells infected with lentiviruses encoding negative control or RGS16 were counted using a hemocytometer (B), and analyzed by phase‐contrast microscopy (C) and Boyden chamber invasion assays (D). Each value in (B) and (D) represents the mean ± SD of triplicate determinations from a representative experiment. Similar results were obtained at least three independent experiments with cells infected with lentiviruses which were prepared for each experiment. (E) Levels of GTP‐Rho. GTP‐Rho in the cell lysate used in (A) was first separated using an affinity‐based procedure, and the cell lysates were also separated by SDS/PAGE, followed by immunoblot analyses using anti‐Rho antibody. The ratio of GTP‐Rho to total Rho was validated by densitometric analyses and shown at the bottom. Results are representative of four independent experiments, three others of which are shown in Fig. S1B.

Figure 3

RGS16 siRNA affects cell morphology and invasion of breast cancer cells. (A) MCF7 cells were transfected with RGS16 or negative control (NC) siRNAs. The transfected cells were analyzed by qRT‐PCR (A), counted using a hemocytometer (B), observed by phase‐contrast microscopy (C) and subjected to Boyden chamber invasion assay (D). Each value in (A) and (B) and (D) represents the mean ± SD of triplicate determinations from a representative experiment. Similar results were obtained at least three independent experiments with cells transfected with RGS16 or NC siRNAs. Mann–Whitney U‐test, *P < 0.05, **P < 0.01.

Figure 4

Expression of RGS16 is regulated by δEF1 family proteins. (A, B) Transfection with δEF1/SIP1 or negative control (NC) siRNAs and infection with lentiviruses (prepared for each experiment) were performed at least three times. The cells were analyzed by qRT‐PCR to measure expression of δEF1,SIP1, and RGS16 mRNAs. (C) The cells infected with shRNAs against δEF1 and SIP1 (used in A) were subjected to Boyden chamber invasion assays. Bottom panels show RGS16 mRNA levels. Mann–Whitney U test, *P < 0.05 (D) MCF7 cells were infected with the indicated constructs, and then subjected to Boyden chamber invasion assays. Bottom panels show the levels of the indicated tagged proteins. Each value represents the mean ± SD of triplicate determinations from a representative experiment. The results of three independent experiments with triplicates were statistically analyzed with the analysis of variance (ANOVA). The analysis clearly indicated that RGS16 significantly inhibited δEF1‐induced migration with P value of 7.37 × 10⁻⁵ (shown as an asterisk).