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

Surgical specimens were obtained from breast cancer patients (47 ER+/PR + and 43 TN) who had mastectomy or lumpectomy at Louisiana State University Health Sciences Center, Shreveport, LA, during the period between 2002 and 2010. This study was reviewed and approved in advance by the Institutional Review Boards of the Louisiana State University Health Sciences Center and the Feinberg School of Medicine, Northwestern University. All necessary consent was obtained from every patient involved in the study, including consent for participation in the study and publication of data. The patients’ ages ranged from 27 to 96 years, and their mean age was 54.2 years. Tumor stages were classified according to the seventh edition of the TNM (tumor-node-metastasis) classification of breast carcinomas published by American Joint Committee on Cancer. The clinicopathological parameters of the patient cohorts are shown in Table 1 and Additional file 1: Table S1.

Immunohistochemical staining of paraffin-embedded human tissues was performed by the standard avidin-biotin peroxidase complex method. Paraffin sections were labeled and dried in 60°C oven for at least 4 hour, cooled, deparaffinized, and incubated in antigen retrieval solution (#H-3300; Vector Laboratories, Burlingame, CA). For antigen retrieval, slides were heated and cooled in antigen retrieval solution for 25 and 20 minutes, respectively. Slides were then rinsed 4–5 times in distilled water; once in 0.3% peroxide in 50% methanol for 30 minutes, and 3 times for 5 minutes in wash buffer. Subsequently, slides were processed using the BioGenex i6000 Automated Staining System. Blocking was conducted by soaking slides in 10% goat serum in phosphate-buffered saline (PBS), for 15 minutes, in 5% casein block in PBS for 10 minutes, and in 10% goat serum in PBS for 1 minute. Slides were then incubated with the primary antibody for Smurf2 (Y-21: sc-130878, Santa Cruze Biotechnology, CA) at a dilution of 1:100 in Dako antibody diluent (Dako, Glostrup, Denmark) for 1 hour, followed by 5 times rinse with wash buffer. Samples were then incubated with the secondary (MultiLink-BioGenex Super Sensitive Link-Label IHC Detection System #QP900-9 L; BioGenex, Fremont, CA) for 20 minutes, rinsed 3 times in wash buffer, and labeled with a horseradish peroxidase solution (BioGenex Super Sensitive Link-Label IHC Detection System #QP900-9 L) for 15 minutes. Following triple washes, 3,3-Diaminobenzidine (#K3466; DakoCytomation Liquid DAB Substrate Chromogen System) was applied to samples for 5 minutes. Samples were then rinsed 3 times, stained with hematoxylin (#S3301; DakoCytomation Automation Hematoxylin) for 2 minutes, and rinsed 3 times again in wash buffer. Slides were then rinsed with distilled water for 4 minutes, and dehydrated sequentially with ethanol and xylene. A negative control to each section was prepared by using normal rabbit serum instead of the primary antibody. While benign mammary epithelia and ductal carcinomas in situ (DCIS) displayed uniform strong staining for Smurf2, invasive carcinomas often exhibited focal patterns of Smurf2 staining (see Figure 1). To comparatively examine decreased expression of Smurf2 in invasive carcinomas, percentages of Smurf2-positive cells in carcinoma regions were scored as follows: 0 (Smurf2⁺ cells in carcinoma tissues: 0-5%), 1+ (6%-25%), 2+ (25%-50%), 3+ (50%-75%), and 4+ (>75%).

Human non-transformed mammary epithelial MCF-10A cells, and 9 human breast cancer cell lines, MCF-9, T47D, MDA-MB-231, BT549, MDA-MB-436, DU4475, MDA-MB-468, BT474 and SK-BR-3, were obtained from American Tissue Culture Collection (ATCC), and cultured under standard conditions recommended by ATCC. Fetal bovine sera and calf sera were obtained from HyClone/Thermo Fisher Scientific (Logan, UT), and media, antibiotics and other chemicals were purchased from Corning Cellgro (Manassas, VA) and GiBCO/Invitrogen (Carlsbad, CA). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO).

Immunoblotting was performed as previously described [10]. Anti-Smurf2 antibody was obtained from Upstate Biotechnology (07–249, Lake Placid, NY), and anti-RB and anti-α-Tubulin antibodies were from BD Pharmingen (554136, BD Biosciences, San Jose, CA) and Sigma-Aldrich (T6199), respectively. Target proteins were visualized by enhanced chemiluminescence (Thermo Scientific, Rockford, lL). The band intensities were quantified by densitometry using the Photoshop and Image J software and normalized to those of their respective control bands.

Total RNA samples were collected using the Trizol reagent (Invitrogen, Carlsbad, CA). Levels of Smurf2 mRNA were quantified in comparison with those of GAPDH mRNAs, using the Power SYBR® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) and the Applied Biosystems 7900. Levels of miR-15a, miR-15b, miR-16 and miR-128 were measured by quantitative RT-PCR, using miScript PCR system including pre-designed miRNA-specific primers (Qiagen, Valencia, CA) and the Applied Biosystems 7900. RNU6-2 was used as the reference endogenous control, and 2^-ΔΔCt method [11] was used to analyze the relative miRNA expression.

Cells were transfected with Ambion® Anti-miR™ miRNA Inhibitors specifically against miR-15a, miR-15b, miR-16 and miR-128 (Ambion/Invitrogen, Carlsbad, CA), using the Lipofectamine® RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The expression vector for green fluorescence protein (GFP) fused with full length retinoblastoma protein (RB) and pEGFP-C3 for GFP expression were obtained from Addgene (Cambridge, MA). Plasmid transfection was conducted with the Lipofectamine® 2000 reagent from Invitrogen, according to the manufacturer’s protocol.

To determine whether the expression of Smurf2 protein was altered in breast cancer tissues, surgical specimens from 90 breast cancer patients (47 with ER+/PR + cancers and 43 with TNBCs, see Table 1 and Additional file 1: Table S1) were analyzed by immunohistochemistry for Smurf2. Regions of benign mammary epithelia and DCIS showed robust Smurf2 staining both in the cytoplasm and nucleus (Figure 1A, upper panels). In samples with invasive carcinomas, Smurf2 staining was found decreased focally or sometimes diffusely, and the downregulation of Smuf2 was significantly more obvious in TNBCs compared to ER+/PR + cancers (Figure 1A lower panels, Figure 1B). The median of the Smurf2 staining scores in TNBCs was 2 (25%-50% of tumor cells were Smurf2-positive), while that in ER+/PR + cancers was 3 (50%-75% Smurf2-positive). Higher tumor grades and Ki67 scores were observed in the TN group, compared with the ER+/PR + group. Lower Smurf2 staining scores were associated with higher tumor grades (p = 0.0004) and higher Ki67 scores (p = 0.011), but not with stages or p53 staining scores (Additional file 1: Table S1). We then examined human breast cancer cell lines and non-transformed mammary epithelial MCF-10A cells by immunoblotting for Smurf2 (Figure 2A). Levels of Smurf2 protein in ER+/PR + cancer cells (MCF-7 and T47D) and those in HER2+/ER+/PR + BT474 cells and HER2+/ER-/PR- SK-BR-3 cells were comparable with Smurf2 levels in MCF-10A cells. In sharp contrast, Smurf2 protein levels in 4 of 5 TNBC cell lines, BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells, were significantly lower than those in MCF-10A and the ER+/PR + cell lines. Only MDA-MB-231 cells showed high levels of Smurf2 expression. To determine whether Smurf2 downregulation in the TNBC cell lines resulted from transcriptional repression, Smurf2 mRNA levels were measured by real time PCR (Figure 2B). In the four cell lines that exhibited lower levels of Smurf2 protein, no decreases in the mRNA levels were observed, relative to that in MCF-10A cells, suggesting that Smurf2 is downregulated at the posttranscriptional level in those TNBC cell lines. In contrast, MDA-MB-231 cells exhibited remarkably higher Smurf2 mRNA levels, indicating that Smurf2 is transcriptionally upregulated only in this particular cell line. To further examine whether protein degradation plays a dominant role in determining the steady-state level of Smurf2 protein, we examined the stability of Smurf2 in MCF-10A, MDA-MB-231, and BT549 cells, using the translation inhibitor cycloheximide (Figure 2C). According to the decay of Smurf2 levels in the presence of cycloheximide, the half-life of Smurf2 in MCF-10A cells was determined to be about 8 hours. Interestingly, the half-life of Smurf2 in MDA-MB-231 cells was less than 3 hours, suggesting that Smurf2 protein is rather more unstable in this cell line that overexpresses its mRNA. On the other hand, Smurf2 protein was more stable in BT549 cells, displaying a half-life of more than 12 hours. Taken together, these data indicated that the expression of Smurf2 protein is downregulated frequently in human TNBC tissues, and similar downregulation was observed in four of the five TNBC cell lines examined here. MDA-MB-231 cells exceptionally showed transcriptional upregulation of Smurf2, which appeared to be counteracted by enhanced degradation of the protein.

Deregulation of microRNAs (miRNAs) has been implicated to the biology of breast cancer such as estrogen signaling, migration and metastasis [12, 13]. We hypothesized that some miRNAs were involved in the post-transcriptional downregulation of Smurf2 in TNBC, and used multiple online databases such as TargetScan and PicTar to identify miRNAs that potentially bind to Smurf2 mRNAs. The analysis led us to candidates such as miR-128 (binding to Smurf2 3′UTR, 5′-CACUGUGA-3′) and the miR-15 family miRNAs including miR-15a, miR-15b and miR-16 (binding to Smurf2 3′UTR, 5′-GCUGCUA-3′). The miR-15 family and miR-128 have been implicated for the regulatory network in breast cancer initiating cells [14, 15]. Thus, we measured the expression of miR-15a, miR-15b, miR-16 and miR-128b in the breast cancer cell lines (Figure 3). DU4475 cells showed increased expression of miR-15b, miR-16 and miR-128, relative to their expression in MCF-10A cells. BT549 cells exhibited increased expression of miR-15a, miR-15b and miR-16. MDA-MB-436 cells had increased expression of miR-15b, miR-16, and miR-128. Thus, these TNBC cell lines that exhibited Smurf2 downregulation had a tendency to express higher levels of these miRNAs. In contrast, MDA-MB-231 cells, which had high levels of Smurf2 mRNA and protein, showed no major change in the expression of these miRNAs, except for a decrease in miR-15a. Also in MCF-7 cells, the levels of miR-15a, miR-15b and miR16 were low, whereas the expression of miR-128 was modestly higher. To further delineate the role of the miRNAs in Smurf2 downregulation observed in BT549, MDA-MB-436 and DU4475 cells, cells were transfected with miRNA inhibitors (antagomirs) against miR-15a, miR-15b, miR-16 or miR-128 (Figure 4). Treatment with these antagomirs resulted in substantial increases in Smurf2 protein levels in the TNBC cell lines, suggesting the involvement of these miRNAs in downregulating Smurf2 in TNBC.

A recent study demonstrated that miR-15 and miR-16 are direct targets of the E2F transcription factors [16]. A number of TNBCs have inactivating mutations of the retinoblastoma tumor suppressor gene (RB) [17], which lead to hyperactivation of E2F [18]. Therefore, we hypothesized that RB inactivation could result in elevated expression of the miR-15 family and possibly miR-128, which contributed to the downregulation of Smurf2. Immunoblotting for RB demonstrated that all four TNBC cell lines that exhibited Smurf2 downregulation had no detectable expression of RB (Figure 5A). In contrast, MDA-MB-231 cells, which expressed high levels of Smurf2, showed robust RB expression comparable to that in MCF-7 and T47D cells. This RB expression patterns are consistent with the genotypes of the RB gene in these cell lines as summarized in [1]. To further examine the role of RB in the regulation of Smurf2, we transfected BT549 cells with an expression vector for the full-length RB protein fused with green fluorescence protein (GFP) (Figure 5B). Forced expression of GFP-RB resulted in a significant increase in cellular levels of Smurf2 protein, accompanied by substantial decreases in the expression of miR-15a, miR-15b, miR-16 and miR-128b (Figure 5C). These results indicate that forced expression of RB in TNBC cells with RB mutations could restore levels of Smurf2 protein expression, suggesting the significance of the RB-miRNA pathway in the control of Smurf2 in TNBC.