Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: involvement of the RB-microRNA axis

Abstract

Background: The HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells.

Methods: Human breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines.

Results: Immunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs.

Conclusions: This study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.

Figure 1

Smurf2 expression is decreased in triple-negative breast cancer tissues. (A) Immunohistochemistry for Smurf2 was conducted using human mammary tissues including benign mammary epithelia, ductal carcinomas in situ (DCIS), ER+/PR + and triple-negative (TN) invasive carcinomas. Upper left panel shows x10 magnification, and the other panels show x20 pictures with x40 magnified views. (B) Percent of Smurf2-positive cells in ER+/PR + (n = 47) and TN (n = 43) breast cancer specimens. To quantify focal loss of Smurf2 expression in carcinoma regions, immunohistochemical labeling of Smurf2 was scored according to the following five categories: 0 (Smurf2⁺ cells in carcinoma tissues: 0-5%), 1+ (6%-25%), 2+ (25%-50%), 3+ (50%-75%), and 4+ (>75%). Data are shown as mean + SEM with the p value for statistical significance.

Figure 2

Smurf2 protein is downregulated in triple-negative breast cancer (TNBC) cell lines without concomitant decreases in Smurf2 mRNA. (A) Immunoblotting for Smurf2 in the following cell lines: MCF-10A, untransformed human mammary epithelial cells; MCF-7 and T47D, mammary carcinomas with expression of the estrogen and progesterone receptors (ER+/PR+); MDA-MB-231, BT549, MDA-MB-436, DU4475 and MDA-MB-468, TNBCs; BT474 and SK-BR-3, mammary carcinomas with HER2 amplification. The bar graph indicates relative levels for Smurf2 protein in the cancer cell lines to that in MCF-10A cells. The density of each Smurf2 signal on immunoblots was normalized by that of α-tubulin. Data are shown as mean + SEM from at least three experiments, and the asterisks indicate statistically significant (p < 0.05) differences from the level in MCF-10A cells. (B) Real time PCR analysis for Smurf2 mRNA. Data are normalized by signals for GAPDH mRNA, and presented as relative expression levels to that in MCF-10A cells. (C) Degradation of Smurf2 protein assessed by treatment with cycloheximide (CHX). MCA-10A, MDA-MB-231 and BT549 cells were treated with 100 μg/ml CHX for the indicated hours, and lysates were analyzed by immunoblotting for Smurf2 and α-tubulin.

Figure 3

Expression levels of miR-15a, miR-15b, miR-16 and miR-128 in breast cancer cell lines. Levels of the indicated microRNAs were determined by real time PCR in the following cell lines: MCF-10A, untransformed human mammary epithelial cells; MCF-7, mammary carcinomas with expression of the estrogen receptor (ER+); MDA-MB-231, BT549, MDA-MB-436, and DU4475, triple-negative mammary carcinomas. Data are normalized by signals for RNU6-2 as controls, and presented as relative expression levels to that in MCF-10A cells as 1.0. The bars indicate S.E.M. from 3–5 experiments, and the asterisk indicates statistically higher (p < 0.05) levels of each miRNA in the particular cell line than in MCF-10A cells.

Figure 4

MicroRNAs such as miR-15, miR-16 and miR-128 are involved in downregulation of Smurf2 protein in triple-negative breast cancer. Human triple-negative breast cancer cell lines, BT549, MDA-MB-436 and DU4475 cells, were transfected with microRNA inhibitors against miR-15a, miR-15b, miR-16 and miR-128, or nonspecific ssRNA as negative control (NC), and cellular levels of Smurf2 protein were determined at 24 h (A, B) or 48 h (C) post-transfection by immunoblotting. The density of each Smurf2 signal on immunoblots was normalized by that of α-tubulin, and presented as relative expression levels to that in negative control (NC) as 1.0. Quantified data on bar graphs show means + SEM from three experiments, and the asterisks indicate statistically significant (p < 0.05) differences from negative control (NC).

Figure 5

The loss of the retinoblastoma tumor suppressor (RB) expression plays a role in Smurf2 downregulation in triple-negative breast cancer (TNBC) cells, via upregulation of miR-15, miR-16 and miR-128. (A) Immunoblotting for RB protein in the indicated TNBC cell lines. (B) Increased expression of Smurf2 by forced expression of RB in RB-null BT549 cells. Cells were transfected with an expression vector for a green fluorescence protein (GFP)-RB fusion protein, a vector for GFP, or an empty vector (pcDNA3.1). Levels of Smurf2 and RB were determined at 42 h post-transfection by immunoblotting. The bar graph indicates relative levels for Smurf2 protein normalized by those for α-tubulin, shown as means + SEM from three experiments. The asterisk indicates statistical significance (p < 0.05). (C) Decreased expression of miR-15a, miR-15b, miR-16 and miR-128 in BT549 cells transfected with the GFP-RB vector. Levels of the indicated microRNAs were determined by real time PCR. Data are normalized by signals for RNU6-2 as controls, and presented as relative expression levels to that in cells transfected with an empty vector as 1.0. The bars indicate S.E.M. from three experiments, and the asterisk indicates statistical significance (p < 0.05).