The strength and duration of NF-κB signaling is tightly controlled at multiple levels under physiologic conditions, but the mechanism underlying constitutive activation of the NF-κB pathway in cancer remains unclear. In this study, we investigated miRNA-mediated regulation of the NF-κB cascade in breast cancer. We report that miR-892b expression was significantly downregulated in human breast cancer specimens and correlated with poor patient survival. Overexpression of miR-892b in breast cancer cells significantly decreased tumor growth, metastatic capacity, and the ability to induce angiogenesis, whereas miR-892b depletion enhanced these properties, in vitro and in vivo. Furthermore, we demonstrate that miR-892b attenuated NF-κB signaling by directly targeting and suppressing multiple mediators of NF-κB, including TRAF2, TAK1, and TAB3, and thus, miR-892b silencing in breast cancer cells sustains NF-κB activity. Moreover, miR-892b downregulation was attributed to aberrant hypermethylation of its promoter. Taken together, our results provide insight into a new mechanism by which NF-κB signaling becomes constitutively activated in breast cancer and suggest a tumor-suppressive role for miR-829b, prompting further investigation into miRNA mimics for cancer therapy. Cancer Res; 76(5); 1101–11. ©2016 AACR.

Since its discovery nearly three decades ago (1), the central roles of the NF-κB pathway in physiologic and pathologic processes, such as immunity, inflammation, and tumorigenesis, have been well established (2, 3). Notably, the NF-κB signaling pathway is constitutively activated in various human cancer types and plays crucial roles in the initiation and progression of a large array of malignancies (2, 4). Therefore, a better understanding of the molecular mechanisms underlying the constitutive activation of the NF-κB signaling pathway in cancer may aid the identification of novel therapeutic targets for cancer.

Ubiquitination- and phosphorylation-mediated signaling transductions are important regulatory mechanisms for the activation of NF-κB (5, 6). The stimulatory factors bind to their respective receptors, leading to the recruitment of multiple receptor-associated factors, including TNF receptor–associated factors (TRAF), which function as a ubiquitin ligase that induces the K-63 polyubiquitination of receptor-interacting protein 1 (RIP1) and other TRAFs, resulting in activation of transforming growth factor β–activated kinase-1 (TAK1)/TAB2/3 complex through association with conserved novel zinc finger domains of TAB2/TAB3. Furthermore, activated TAK1/TAB2/3 complex phosphorylates and activates inhibitor of NF-κB kinase (IKK)-α/β/γ kinase complex, finally leading to nuclear translocation and activation of NF-κB (6, 7).

As an adaptor protein of NF-κB signaling cascades, TRAF2 is amplified and rearranged in 15% of human epithelial cancers and contributes to the constitutively activated NF-κB signaling (8). Importantly, silencing of TRAF2 inhibits NF-κB–induced pancreatic cancer cell proliferation and tumorigenicity and could also significantly decrease the growth of glioblastoma cells, indicating that TRAF2 might represent a potential anticancer target (9, 10). On the other hand, another essential component of the NF-κB pathway, TAK1, is involved in the progression of various cancers through activation of the NF-κB pathway. For instance, overexpression of TAK1 could promote the tumor growth and metastatic capacity of ovarian cancer cells by enhancing NF-κB transactivity (11). However, deletion of the TAK1 gene in skin tumors induces tumor cell apoptosis, and inhibition of TAK1 kinase activity leads to a proapoptotic phenotype and reverts the intrinsic chemoresistance of pancreatic cancer (12, 13), suggesting that targeting TAK1 might be a promising and effective therapeutic approach to counteract tumor progression. In addition, TAB3, the key factor of the TAK1/TAB2/3 complex, is markedly elevated in a variety of cancers (14), and silencing either TAK1 or TAB3 could enhance the rates of doxorubicin-induced apoptosis and the chemosensitivity of hepatocellular carcinoma cells (15). Thus, further exploring the mechanisms of regulation of key components of the NF-κB cascade, such as TRAF2, TAK1, and TAB3, would increase our knowledge of the biologic basis of the constitutive activation of NF-κB in cancer and provide novel insights for tumor therapy.

Herein, we reported that miR-892b was downregulated in breast cancer via methylation of its promoter. Silencing miR-892b sustained the NF-κB activation via upregulation of TRAF2, TAK1, and TAB3 and promoted the aggressiveness of breast cancer in vitro and in vivo. These results demonstrated that miR-892b functions as a tumor-suppressive miRNA in breast cancer and uncovered a novel mechanism for constitutive NF-κB activation in breast cancer.

Ethics statement

Investigation has been conducted in accordance with the ethical standards according to the Declaration of Helsinki and national and international guidelines and has been approved by the authors' Institutional Review Board.

Cells and cell culture

Primary normal mammary epithelial cells (NMEC) were established according to previous report (16). A nontumorigenic epithelial cell line, MCF-10A, was cultured in keratinocyte-serum free medium supplemented with 0.1 ng/mL human recombinant EGF and 20 μg/mL bovine pituitary extract (Invitrogen). The breast cancer cell lines, including ZR-75-30, ZR-75-1, MCF-7, BT-549, BT-474, SKBR3, MDA-MB-415, MDA-MB-435, MDA-MB-468, MDA-MB-231, and MDA-MB-453, were purchased from the ATCC, and cultured in DMEM medium (Gibco) supplemented with 10% FBS (HyClone). All cell lines were authenticated by short tandem repeat fingerprinting at IDEXX RADIL and Services at SYSU Forensic Medicine Lab within 6 months.

Patient information and tissue specimens

This study was conducted on 30 breast cancer tissues and 20 noncancerous breast tissues and a total of 195 paraffin-embedded breast cancer samples, which were histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center between 1999 and 2007. For the clinical materials used in our study, prior patient consent and approval from the Institutional Research Ethics Committee were obtained. In order to ensure the differential expression of miR-892b in normal breast epithelial cells and breast cancer cells, the Veritas Laser Capture Microdissection System (Arcturus Bioscience, Inc.) was used to microdissect at least 1,000 normal lobular and ductal cells in normal breast tissues, as well as ductal or lobular tumor cells in breast cancer tissues were identified by an experienced pathologist. Clinical and clinicopathologic classification and stage were determined according to the American Joint Committee on Cancer criteria. Clinical information on the samples is shown in Supplementary Table S1.

Vectors, retroviral infection, and transfection

The miR-892b antisense (miRZip-892b) plasmid and the vector control (miRZip-V) were purchased from System Biosciences and used according to previous report (17). The 3′-untranslated region (UTR) regions of human TRAF2, TAB3, and TAK1, generated by PCR amplification from genomic DNA, were cloned into the pGL3-luciferase reporter plasmid (Promega). pNF-κB-luc and control plasmids (Clontech) were used to examine NF-κB activity. Transfection of siRNAs (Ribo Biotech) or plasmids was performed using the Lipofectamine 2000 reagent. Stable cell lines expressing miR-892b or miRZip-892b were generated via retroviral infection using HEK293T cells and selected with 0.5 μg/mL puromycin for 10 days.

Xenografted tumor model, hematoxylin and eosin, and IHC staining

BALB/c-nude mice (female, 4–5 weeks of age, 18–20 g) were purchased from the Center of Experimental Animal of Guangzhou University of Chinese Medicine. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University. The BALB/c nude mice were randomly divided into two groups. The indicated cells were inoculated subcutaneously in the mammary fat pad of nude mice. Tumor volume was calculated using the equation (L × W2)/2. Thirty days after tumor implantation, the mice were sacrificed, the mammary tumors were removed and weighed. For the metastasis status analysis, nude mice were intravenously injected with miR-892b–transduced or –silenced cells or control cells via lateral tail veins. The lungs were collected to count surface metastases under a dissecting microscope at 40 days after tumor implantation. Tumors and lungs were fixed in formalin and embedded in paraffin using the routine method. Serial 6.0-μm sections were cut and subjected to hematoxylin and eosin (H&E) stained with Mayer's hematoxylin solution, or IHC analyzed using anti-Ki67, anti-CD31, anti-MMP9, and anti-VEGFC antibodies (Cell Signaling Technology), and anti–NF-κB/p65 antibody (Santa Cruz Biotechnology).

miRNP immunoprecipitation

Cells were cotransfected with HA-Ago1 together with 100 nmol/L miR-892b, followed by HA-Ago1 immunoprecipitation using HA-antibody. Real-time PCR analysis of the immunoprecipitation material was used to test the association of the mRNA of TRAF2, TAB3, TAK1, and GAPDH with the RNA-induced silencing complex.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed by using the LightShift Chemiluminescent EMSA Kit from Pierce Biotechnology according to the manufacturer's instruction. Following DNA probes containing specific binding sites were used: NF-κB: sense, 5′-AGTTGAGGGGACTTTCCCAGGC-3′, antisense, 5′-GCCTGGGAAAGTCCCCTCAAC-3′; OCT-1: sense, 5′-TGTCGAATGCAAAT CACTAGAA-3′, antisense, 5′-TTCTAGTGATTTGCATTCGACA-3′.

5-aza-2′-deoxycytidine treatment

DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-AZA; Sigma) was dissolved in 50% acetic acid at 100 mmol/L. Throughout the experiments, 50% acetic acid was used as the vehicle control. Cells were treated with 5-AZA (5 μmol/L) or vehicle control for 72 hours to achieve demethylation. RNA was extracted and subsequently subjected to expression analysis of miR-892b.

Statistical analysis

Statistical tests for data analysis included the Fisher exact test, log-rank test, χ2 test, and Student two-tailed t test. Bivariate correlations between study variables were calculated by Spearman rank correlation coefficients. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The significance of various variables for survival was analyzed by univariate and multivariate Cox regression analyses. Statistical analyses were performed using the SPSS 11.0 statistical software package. Data represent mean ± SD. P values of 0.05 or less were considered statistically significant.

Silencing miR-892b induces NF-κB activity in breast cancer cells

In an attempt to determine which miRNA species might be involved in NF-κB activation in breast cancer, gene set enrichment analysis (GSEA) in the published breast cancer dataset (NCBI/GEO/GSE19526, n = 100) was performed. As shown in Fig. 1A, the level of miR-892b was inversely correlated with the NF-κB–activated gene signatures (RASHI_NFKB1_TARGETS and V$NFKB_C), suggesting that miR-892b might be involved in deactivation of NF-κB signaling (Fig. 1A). Furthermore, we found that the expression of miR-892b in 2 normal breast tissues and 7 breast cancer tissues was negatively correlated with the DNA-binding activity of NF-κB (r = −0.751; P = 0.003) and the mRNA levels of multiple NF-κB downstream targets (Fig. 1B and C; Supplementary Fig. S1A), which indicate that miR-892b downregulation contributes to NF-κB activity in breast cancer.

Figure 1.

Silencing miR-892b induces NF-κB activity in breast cancer cells. A, GSEA analysis showing that miR-892b expression was inversely correlated with NF-κB target gene signatures (RASHI_NFKB1_TARGETS and V$NFKB_C) in a published breast cancer dataset (NCBI/GEO/GSE19526, n = 100). B, miR-892b levels were inversely associated with NF-κB activity, as determined by EMSA analysis. Bottom, the quantification of the relative NF-κB activity and the relative expression of miR-892b. C, real-time PCR analysis showing that miR-892b levels were inversely associated with the mRNA levels of multiple NF-κB downstream targets, including CCND1, MMP9, and VEGFC, in breast cancer tissues, compared with two normal breast tissues. D, NF-κB luciferase reporter activity analyzed in the indicated cells. E, real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-892b–regulated gene expression. The pseudocolors represent the intensity scale of miR-892b versus vector or miRZip-892b versus miRZip vector generated by a log2 transformation. F, subcellular localization of NF-κB p65 in the indicated cells, as analyzed by an immunofluorescence staining assay. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

Figure 1.

Silencing miR-892b induces NF-κB activity in breast cancer cells. A, GSEA analysis showing that miR-892b expression was inversely correlated with NF-κB target gene signatures (RASHI_NFKB1_TARGETS and V$NFKB_C) in a published breast cancer dataset (NCBI/GEO/GSE19526, n = 100). B, miR-892b levels were inversely associated with NF-κB activity, as determined by EMSA analysis. Bottom, the quantification of the relative NF-κB activity and the relative expression of miR-892b. C, real-time PCR analysis showing that miR-892b levels were inversely associated with the mRNA levels of multiple NF-κB downstream targets, including CCND1, MMP9, and VEGFC, in breast cancer tissues, compared with two normal breast tissues. D, NF-κB luciferase reporter activity analyzed in the indicated cells. E, real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-892b–regulated gene expression. The pseudocolors represent the intensity scale of miR-892b versus vector or miRZip-892b versus miRZip vector generated by a log2 transformation. F, subcellular localization of NF-κB p65 in the indicated cells, as analyzed by an immunofluorescence staining assay. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

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In agreement with results obtained from the clinical samples, a luciferase reporter assay revealed that downregulation of miR-892b in breast cancer cells significantly enhanced, but upregulation of miR-892b reduced, NF-κB–induced luciferase activity and the expression of numerous NF-κB downstream genes (Fig. 1D and E). However, the stimulatory effect of miR-892b downregulation on NF-κB activation was dramatically inhibited upon IκBα-mut (IκBα-dominant–negative mutant) transfection (Fig. 1D). Moreover, immunofluorescence staining and subcellular fractionation assays showed that overexpressing miR-892b led to a significant cytoplasm location of NF-κB p65, whereas silencing miR-892b resulted in stronger nuclear signals for NF-κB (Fig. 1F and Supplementary Fig. S1B). Collectively, these results suggest that miR-892b plays an important role in regulation of NF-κB signaling in breast cancer.

Reduced miR-892b correlates with breast cancer progression and prognosis

Consistent with analyses of multiple miRNA expression profiling datasets, miR-892b was significantly reduced in breast cancer (NCBI/GEO/GSE40525/26659/44899; P < 0.001; Fig. 2A). Our qPCR analysis also showed that miR-892b expression was dramatically reduced in all 11 tested breast cancer cell lines compared with two normal breast epithelial cells and in breast cancer cells (n = 30) compared with the normal breast cells (n = 20; Fig. 2B and C), which was further confirmed by the ISH analysis (Supplementary Fig. S2A). Meanwhile, we found that the expression levels of miR-892a and miR-892c, other two members of miR-892 family, were also significantly reduced in breast cancer (data not shown). Taken together, these results indicate that miR-892b is downregulated in breast cancer.

Figure 2.

Expression of miR-892b inversely correlates with breast cancer progression and poor prognosis. A, expression profiles of miRNAs in primary breast cancer tissues and normal breast tissues (NCBI/GEO/GSE40525/26659/44899; P < 0.001). B and C, real-time PCR analysis of miR-892b expression in NMEC and MCF-10A, and 11 breast cancer cell lines (B), as well as 30 tumor samples compared with 20 nontumor tissues (C). Transcript levels were normalized to U6 expression. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05. D, miR-892b expression in normal breast tissues and breast cancer tumor specimens at clinical stages I–IV; miR-892b expression levels were inversely associated with clinical stages (r = −0.731; P < 0.001). E, Kaplan–Meier curves of breast cancer patients with low- versus high expression of miR-892b (n = 195; P < 0.001, log-rank test).

Figure 2.

Expression of miR-892b inversely correlates with breast cancer progression and poor prognosis. A, expression profiles of miRNAs in primary breast cancer tissues and normal breast tissues (NCBI/GEO/GSE40525/26659/44899; P < 0.001). B and C, real-time PCR analysis of miR-892b expression in NMEC and MCF-10A, and 11 breast cancer cell lines (B), as well as 30 tumor samples compared with 20 nontumor tissues (C). Transcript levels were normalized to U6 expression. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05. D, miR-892b expression in normal breast tissues and breast cancer tumor specimens at clinical stages I–IV; miR-892b expression levels were inversely associated with clinical stages (r = −0.731; P < 0.001). E, Kaplan–Meier curves of breast cancer patients with low- versus high expression of miR-892b (n = 195; P < 0.001, log-rank test).

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Furthermore, statistical analyses revealed that miR-892b expression in 195 archived human breast cancer specimens was inversely correlated with the clinical stage in all four breast cancer subtypes (P < 0.001) and tumor–node–metastasis (TNM) classification (T: P < 0.001; N: P < 0.001; M: P = 0.018) in patients with breast cancer (Fig. 2D; Supplementary Fig. S2B–S2D; Supplementary Tables S1–S3). Notably, we found that patients with higher miR-892b expression had a longer survival time, whereas patients with lower miR-892b expression had a shorter survival time (P < 0.001; Fig. 2E). In addition, univariate and multivariate analyses revealed that miR-892b expression might be an independent prognostic factor in breast cancer (Supplementary Table S4). Taken together, these results suggest a potential correlation between reduced miR-892b and human breast cancer progression.

Downregulation of miR-892b contributes to breast cancer progression in vivo

To further confirm the GSEA results, which suggest the role of reduced miR-892b in the aggressive behavior of breast cancer cells (Fig. 3A and Supplementary Fig. S3A), MDA-MB-231 and ZR-75-30 breast cancer cells stably overexpressing miR-892b or miRZip-892b were established (Supplementary Fig. S3B). As shown in Fig. 3B and Supplementary Fig. S3C–S3G, the tumors formed by miR-892b–silenced breast cancer cells were larger, both in size and weight, than control tumors, whereas the tumors formed by miR-892b–overexpressing cells were smaller and lighter than control tumors. Furthermore, IHC analysis showed that miR-892b–silenced tumors exhibited increased percentages of Ki67-positive cells and microvascular density (MVD), whereas miR-892b–transduced tumors displayed lower Ki67 and decreased MVD (Fig. 3C; Supplementary Fig. S3H). Meanwhile, expressions of MMP9 and VEGFC, the NF-κB downstream targets, were significantly upregulated in tumors formed by miR-892b–silenced cells, but downregulated in tumors formed from miR-892b–transduced cells (Fig. 3C; Supplementary Fig. S3H). Importantly, miR-892b–silenced tumors showed increased nuclear localization of NF-κB p65, whereas NF-κB p65 in miR-892b–overexpressing tumors was predominantly cytoplasmic (Fig. 3C; Supplementary Fig. S3H). Moreover, we found that mice bearing tumors formed by miR-892b–silenced cells displayed prominent lung metastasis, whereas less-visible metastasis was found in mice transplanted with control cells. Conversely, the lung metastatic capability of breast cancer cells was significantly impaired by overexpression of miR-892b (Fig. 3D and E; Supplementary Fig. S3I and S3J). In addition, the Kaplan–Meier analysis revealed that mice implanted with miR-892b–silenced cells survived significantly shorter, whereas mice implanted with miR-892b–transduced cells survived longer, than the control mice (Fig. 3F; Supplementary Fig. S3K). Thus, these data indicate that miR-892b reduction plays a pivotal role in breast cancer progression in vivo.

Figure 3.

Silencing miR-892b contributes to breast cancer progression in vivo. A, a GSEA plot showing that miR-892b expression was inversely correlated with related gene signatures of cell proliferation (FIRESTEIN_PROLIFERATION), migration (WU_CELL_MIGRATION), metastasis (TOMLINS_METASTASIS_UP), and angiogenesis (GALIE_TUMOR_ANGIOGENES) in a published breast cancer dataset (NCBI/GEO/GSE19526, n = 100). B, representative images of the tumors from all the mice in each group. The MDA-MB-231/miR-892b, MDA-MB-231/miRZip-892b, and control cells were injected into the mammary fat pad of the mice (n = 5/group). C, IHC staining displaying that downregulation of miR-892b induced, whereas overexpression of miR-892b inhibited, the aggressive phenotype and the NF-κB activity of breast cancer cells in vivo, as indicated by the nuclear expression of NF-κB, the percentages of Ki67 and CD31, the expression of MMP9 and VEGFC. D, representative brightfield images (left) and quantifications (right) of the lungs. Arrows, surface metastatic nodules. E, lung metastases in the mice confirmed by H&E staining. F, Kaplan–Meier curves of miR-892b–silencing mice (P = 0.02) or miR-892b–overexpressing mice (P = 0.02) compared with the control (log-rank test). Each bar represents the mean ± SD of three independent experiments. * and #, P < 0.05.

Figure 3.

Silencing miR-892b contributes to breast cancer progression in vivo. A, a GSEA plot showing that miR-892b expression was inversely correlated with related gene signatures of cell proliferation (FIRESTEIN_PROLIFERATION), migration (WU_CELL_MIGRATION), metastasis (TOMLINS_METASTASIS_UP), and angiogenesis (GALIE_TUMOR_ANGIOGENES) in a published breast cancer dataset (NCBI/GEO/GSE19526, n = 100). B, representative images of the tumors from all the mice in each group. The MDA-MB-231/miR-892b, MDA-MB-231/miRZip-892b, and control cells were injected into the mammary fat pad of the mice (n = 5/group). C, IHC staining displaying that downregulation of miR-892b induced, whereas overexpression of miR-892b inhibited, the aggressive phenotype and the NF-κB activity of breast cancer cells in vivo, as indicated by the nuclear expression of NF-κB, the percentages of Ki67 and CD31, the expression of MMP9 and VEGFC. D, representative brightfield images (left) and quantifications (right) of the lungs. Arrows, surface metastatic nodules. E, lung metastases in the mice confirmed by H&E staining. F, Kaplan–Meier curves of miR-892b–silencing mice (P = 0.02) or miR-892b–overexpressing mice (P = 0.02) compared with the control (log-rank test). Each bar represents the mean ± SD of three independent experiments. * and #, P < 0.05.

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NF-κB activity is required for miR-892b reduction–induced breast cancer aggressiveness

Consistently, loss-of-function studies showed that silencing miR-892b significantly enhanced the colony formation and anchorage-independent growth abilities of both MDA-MB-231 and ZR-75-30 breast cancer cells, augmented the invasive ability of breast cancer cells, and provoked their ability to induce human umbilical vein endothelial cell (HUVEC) tube formation (Fig. 4A–E). In contrast, miR-892b–transduced cells displayed a reduced capacity for colony formation and anchorage-independent growth ability than control cells. Overexpression of miR-892b also decreased the invasive ability of breast cancer cells and the ability of breast cancer cells to induce HUVEC tubule formation (Supplementary Fig. S4A–S4E). These results suggest that miR-892b functions as a tumor-suppressive miRNA in breast cancer.

Figure 4.

Downregulation of miR-892b promotes aggressiveness of breast cancer cells in vitro. A, representative pictures (left) and quantification (right) of colony number of the indicated cells, as determined by a colony formation assay. B, representative pictures (left) and quantification (right) of colony number of the indicated cells, as determined by an anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. C, representative pictures (left) and quantification (right) of invaded cells, as analyzed using the Transwell matrix penetration assay. D, representative micrographs of the indicated cells cultured in a three-dimensional spheroid invasion assay. E, representative images (left) and quantification (right) of HUVECs cultured on Matrigel-coated plates with conditioned medium from the vector control and miR-892b–downregulated breast cancer cells. F, inhibition of NF-κB activity by SN-50 (50 μg/mL), a specific NF-κB inhibitor, which drastically abrogated the effects of miR-892b reduction on the promotion of colony formation, the anchorage-independent growth and invasive capability of breast cancer cells, and the ability of breast cancer cells to induce HUVEC tubule formation. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

Figure 4.

Downregulation of miR-892b promotes aggressiveness of breast cancer cells in vitro. A, representative pictures (left) and quantification (right) of colony number of the indicated cells, as determined by a colony formation assay. B, representative pictures (left) and quantification (right) of colony number of the indicated cells, as determined by an anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. C, representative pictures (left) and quantification (right) of invaded cells, as analyzed using the Transwell matrix penetration assay. D, representative micrographs of the indicated cells cultured in a three-dimensional spheroid invasion assay. E, representative images (left) and quantification (right) of HUVECs cultured on Matrigel-coated plates with conditioned medium from the vector control and miR-892b–downregulated breast cancer cells. F, inhibition of NF-κB activity by SN-50 (50 μg/mL), a specific NF-κB inhibitor, which drastically abrogated the effects of miR-892b reduction on the promotion of colony formation, the anchorage-independent growth and invasive capability of breast cancer cells, and the ability of breast cancer cells to induce HUVEC tubule formation. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

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In addition, we found that inhibition of NF-κB activity by SN-50, a specific NF-κB inhibitor, drastically abrogated the effects of miR-892b reduction on the promotion of anchorage-independent growth and invasive capability of breast cancer cells, as well as the ability of breast cancer cells to induce HUVEC tubule formation (Fig. 4F), which further support the notion that activation of the NF-κB pathway contributes to the miR-892b reduction–induced breast cancer aggressiveness.

miR-892b directly suppresses multiple key components in NF-κB cascade

Analysis using publicly available algorithms predicted that TRAF2, TRAF6, IKBKE, TAK1, TAB3, and TRAF3, the key components in the NF-κB cascade, might be potential targets of miR-892b (Fig. 5A). However, a miRNP immunoprecipitation assay revealed that miR-892b only specifically associated with 3′-UTRs of TRAF2, TAB3, and TAK1, but not with 3′-UTRs of TRAF6, IKBKE, and TRAF3 (Fig. 5B and C). Furthermore, Western blotting analysis revealed that expression of TRAF2, TAB3, and TAK1 was dramatically decreased in miR-892b–transduced breast cancer cells, but increased in miR-892b–silenced cells (Fig. 5D). Meanwhile, luciferase assays showed that overexpressing miR-892b attenuated, while silencing miR-892b elevated, the reporter activities driven by the 3′-UTRs of TRAF2, TAB3, and TAK1 transcripts (Fig. 5E). In addition, the negative correlation between miR-892b expression and TRAF2, TAB3, and TAK1 expression was further confirmed in clinical specimens (Supplementary Fig. S5A). Moreover, individual silencing of these target genes potently inhibited NF-κB activity in miR-892b–silenced cells (Supplementary Fig. S5B), further demonstrating that TRAF2, TAB3, and TAK1 are functional effectors of miR-892b downregulation-mediated NF-κB activation.

Figure 5.

miR-892b directly suppresses multiple key components of the NF-κB cascade. A, analysis using publicly available algorithms (TargetScan 6.2, miRWalk, and miRanda) predicted that TRAF2, TRAF6, IKBKE, TAK1, TAB3, and TRAF3 might be potential targets of miR-892b. B, RIP assay revealing the selective association between miR-892b and TRAF2, TAB3, or TAK1. C, predicted miR-892b target sequences in the 3′-UTRs of TRAF2, TAB3, and TAK1, determined by real-time PCR. D, Western blot analysis of TRAF2, TAB3, and TAK1 in the indicated cells. β-Actin served as the loading control. E, luciferase activities of reporter containing the 3′-UTRs of TRAF2, TAB3, and TAK1 in miR-892b–transduced cells, miR-892b–silenced cells, control cells, or miR-892b-mutant–transfected cells, respectively. F, Western blotting analysis of the endogenous K63-linked polyubiquitin levels of RIP1 in the indicated cells treated with TNFα (10 ng/mL). G, Western blotting analysis of phospho-IKK-β and total IKK-β expression in the indicated cells treated with TNFα (10 ng/mL). H, Western blotting analysis of IκBα expression in the indicated cells treated with TNFα (10 ng/mL). β-Actin was used as a loading control. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

Figure 5.

miR-892b directly suppresses multiple key components of the NF-κB cascade. A, analysis using publicly available algorithms (TargetScan 6.2, miRWalk, and miRanda) predicted that TRAF2, TRAF6, IKBKE, TAK1, TAB3, and TRAF3 might be potential targets of miR-892b. B, RIP assay revealing the selective association between miR-892b and TRAF2, TAB3, or TAK1. C, predicted miR-892b target sequences in the 3′-UTRs of TRAF2, TAB3, and TAK1, determined by real-time PCR. D, Western blot analysis of TRAF2, TAB3, and TAK1 in the indicated cells. β-Actin served as the loading control. E, luciferase activities of reporter containing the 3′-UTRs of TRAF2, TAB3, and TAK1 in miR-892b–transduced cells, miR-892b–silenced cells, control cells, or miR-892b-mutant–transfected cells, respectively. F, Western blotting analysis of the endogenous K63-linked polyubiquitin levels of RIP1 in the indicated cells treated with TNFα (10 ng/mL). G, Western blotting analysis of phospho-IKK-β and total IKK-β expression in the indicated cells treated with TNFα (10 ng/mL). H, Western blotting analysis of IκBα expression in the indicated cells treated with TNFα (10 ng/mL). β-Actin was used as a loading control. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05.

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miR-892b reduction sustains NF-κB activity

Because TRAF2-mediated K-63 polyubiquitination of RIP1 and TAK1/TAB2/TAB3 complex–mediated phosphorylation of IKK-β contribute to NF-κB activation (18–20), the effects of miR-892b on the ubiquitination status of RIP1 and phosphorylation level of IKK-β were examined. As shown in Fig. 5F and G, the K63-polyubiquitin levels of RIP1 and the expression of phosphorylated IKK-β were dramatically increased in miR-892b–silenced breast cancer cells but decreased in miR-892b–transduced cells. Moreover, we found that the duration of NF-κB activation, as indicated by the decreased IκBα level, the DNA-binding activity of NF-κB, and the mRNA expression of IL1β after TNFα treatment, was dramatically prolonged in miR-892b–silenced cells, but much shortened in miR-892b–transduced cells, compared with the control cells (Fig. 5H and Supplementary Fig. S5C and S5D), which suggest that miR-892b reduction sustains NF-κB activity in breast cancer cells.

miR-892b is downregulated by promoter hypermethylation

Interestingly, analysis using published breast cancer dataset (NCBI/GEO/E-GEOD-69914) showed that the methylation level of miR-892b in primary breast cancer tissues was significantly higher than that in normal breast tissues (P < 0.001; Fig. 6A), suggesting that downregulation of miR-892b in breast cancer might be caused by hypermethylation of miR-892b promoter. Indeed, treatment with the methylase inhibitor 5-aza-dC (5-AZA) significantly restored the miR-892b expression but reduced the expressions levels of TRAF2, TAB3, and TAK1 in breast cancer cells lines, but not in NMBC and MCF-10A cells (Fig. 6B and C). Moreover, the inhibitory effects of 5-AZA treatment on the reporter activities driven by the 3′-UTRs of TRAF2, TAB3, and TAK1 transcripts, NF-κB luciferase activity, and the expression of NF-κB downstream genes in breast cancer cells were significantly abolished by miR-892b inhibition (Fig. 6D and Supplementary Fig. S6A and S6B). Importantly, bisulfite genomic sequencing PCR showed that the CpG island located in the promoter region in breast cancer cells lines and tissues was hypermethylated, but not in NMEC or nontumor tissues (Fig. 6E). Collectively, these results suggest that downregulation of miR-892b in breast cancer is attributed to hypermethylation of its promoter-associated CpG island.

Figure 6.

Downregulation of miR-892b is attributed to hypermethylation of its promoter. A, methylation level of miR-892b in primary breast cancer tissues and normal breast tissues (NCBI/GEO/E-GEOD-69914; P < 0.001). B, miR-892b was upregulated in the breast cancer cell lines following 5-AZA treatment for 72 hours. C, Western blotting analysis of TRAF2, TAB3, and TAK1 in the indicated cells. β-Actin served as the loading control. D, NF-κB luciferase-reporter activity was analyzed in the indicated cells. Error bars represent the mean ± SD of three independent experiments. *, P < 0.05. E, bisulfite sequencing analysis of NMEC, MCF-10A, breast cell lines, nontumor breast tissues, and breast cancer tissues. Three clones of the PCR products from each sample of bisulfite-treated DNA were sequenced. Filled and open circles indicate methylation and nonmethylation, respectively. F, schematic model of how the methylation-mediated silencing of miR-892b sustains NF-κB activation and promotes aggressiveness of breast cancer cells, resulting in progression and poorer clinical outcomes in breast cancer.

Figure 6.

Downregulation of miR-892b is attributed to hypermethylation of its promoter. A, methylation level of miR-892b in primary breast cancer tissues and normal breast tissues (NCBI/GEO/E-GEOD-69914; P < 0.001). B, miR-892b was upregulated in the breast cancer cell lines following 5-AZA treatment for 72 hours. C, Western blotting analysis of TRAF2, TAB3, and TAK1 in the indicated cells. β-Actin served as the loading control. D, NF-κB luciferase-reporter activity was analyzed in the indicated cells. Error bars represent the mean ± SD of three independent experiments. *, P < 0.05. E, bisulfite sequencing analysis of NMEC, MCF-10A, breast cell lines, nontumor breast tissues, and breast cancer tissues. Three clones of the PCR products from each sample of bisulfite-treated DNA were sequenced. Filled and open circles indicate methylation and nonmethylation, respectively. F, schematic model of how the methylation-mediated silencing of miR-892b sustains NF-κB activation and promotes aggressiveness of breast cancer cells, resulting in progression and poorer clinical outcomes in breast cancer.

Close modal

The key findings of the current study provide new insights into the important role of miR-892b reduction in the activation of the NF-κB signaling pathway and promotion of breast cancer progression. We reported that miR-892b was significantly reduced in breast cancer, which resulted from hypermethylation of its promoter, and correlated with better survival of breast cancer patients. Downregulation of miR-892b in breast cancer cells sustained NF-κB activation via upregulation of TRAF2, TAB3, and TAK1 and promoted breast cancer aggressiveness both in vitro and in vivo. Therefore, our results revealed a novel mechanism for constitutive NF-κB activation in breast cancer and demonstrated that miR-892b functions as a tumor-suppressive miRNA in breast cancer (Fig. 6F).

NF-κB signaling plays diverse and important roles in physiologic and pathophysiologic processes, such as cell survival, proliferation, inflammation, and immunity (3). Numerous studies have reported that the NF-κB signaling pathway is frequently activated in a variety of human cancer types and is associated with tumor initiation and progression (2, 4). For example, activation of the NF-κB signaling pathway resulted in promotion of migration, invasion, and apoptotic resistance in glioma cells (21–23). Multiple NF-κB downstream genes, such as VEGF, IL-8, and Ang-2, are transcriptionally upregulated in response to NF-κB activation for the induction of angiogenesis in cancers (24). Meanwhile, activated NF-κB could induce autocrine production of the inflammatory cytokine IL6 and activation of the JAK2/STAT3 signaling, which results in spontaneous lung cancer (25). NF-κB activation has also been demonstrated as essential for normal mammary gland development (26, 27), which suggests that activated NF-κB signaling might be involved in human breast cancers. Consequently, multiple research groups have documented that the NF-κB signaling pathway is constitutively activated in breast cancer and might be a potential prognostic marker to identify primary breast cancers (28, 29). Liu and colleagues reported that NF-κB signaling is required for cell proliferation and colony formation of Her2-derived murine mammary tumor cell lines and contributes to the expansion of breast cancer stem cells and heterotypic signals that enhance tumor-associated macrophages and vasculogenesis (30). Helbig and colleagues found that activation of the NF-κB pathway promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor, CXCR4 (31). The constitutively activated form of NF-κB is frequently found in breast cancer; however, the regulatory mechanism of constitutive activation of NF-κB in breast cancer remains largely unknown. Herein, we found that downregulation of miR-892 significantly increased, but overexpression of miR-892 inhibited, NF-κB activation in breast cancer. Consistently, downregulation of miR-892b could enhance the aggressiveness of breast cancer cells both in vitro and in vivo. However, inhibition of NF-κB signaling by its specific inhibitor dramatically abrogated the simulative effects of miR-892b reduction–mediated breast cancer aggressiveness. Therefore, our results indicated that miR-892b plays important roles in breast cancer progression via regulation of NF-κB signaling.

The network of polyubiquitin-mediated protein–protein interactions plays crucial role in the regulation of NF-κB cascades, such as K63-linked polyubiquitin-mediated activation of TRAFs, IKK, and TAK1, and K48-linked polyubiquitin-mediated proteasomal degradation of NF-κB inhibitor IκB-α and ubiquitin-modification–mediated processing of NF-κB precursors (6, 7). Numerous studies support a central role of polyubiquitination in NF-κB activation by multiple immune and inflammatory pathways. Therefore, identification of new regulator(s) involved in ubiquitination would increase our knowledge of the mechanism of NF-κB activation. As a major component of the NF-κB cascade, TRAF2 functions as an E3 ligase and catalyzes K63-linked polyubiquitination of RIP1 in response to TNFα stimulation. The polyubiquitin chain of RIP1 serves as a platform to recruit and activate both the TAK1/TAB2/TAB3 complex and the IKK complex (19). In the present study, miR-892b was suggested to suppress the expression of TRAF2, leading to inhibition of RIP1 polyubiquitination and inhibition of the IKK complex. Therefore, our results uncover a novel regulatory mechanism of NF-κB activation in breast cancer.

Previous studies have shown NF-κB activation to be predominantly associated with estrogen receptor (ER)–negative breast tumors (32, 33). However, there is an increasing amount of evidence that NF-κB activation occurs in a subset of endocrine-resistant ER-positive tumors, and NF-κB activation in ER-positive tumors is associated with a more aggressive phenotype, which was caused by positive cross-talk between ER and the NF-κB pathway. For example, ER and p65 can cooperate to synergistically regulate expression of specific genes, such as PTGES, BIRC3, and ABCG2, which are important in regulation of invasiveness, survival, and chemoresistance of breast cancer cells (34, 35). Meanwhile, it has been also reported that IKK-α could interact and phosphorylate ER, and then both proteins form a complex with SRC-3, leading to the phosphorylation of histone H3, and additional histone modifications that favor gene expression are recruited to the promoter of cyclin D1 and E2F1 (36, 37). Together, these studies suggest that cross-talk between ER and NF-κB could be an important mechanism in the progression of breast cancer. Therefore, loss of miR-892b in ER-positive breast cancer might promote more aggressive phenotype via activation of NF-κB pathway.

miRNAs, a class of endogenous, small noncoding RNAs of 20 to 22 nucleotides, have recently emerged as important molecules involved in various biologic processes (38, 39). Accumulating evidence indicates that the abnormal expression of miRNAs in cancer could serve as potential markers for cancer progression and prognosis. For example, upregulation of miR-182 is correlated with glioma clinical grade and patient survival (40). Upregulated miR-508 was associated with esophageal squamous cell carcinoma progression and poorer survival (41). However, reduced miR-93 was markedly correlated with advanced clinicopathologic progression and short survival in patients with colon cancer (42), and decreased miR-141 levels were significantly associated with tumor size, TNM stage, lymph node and distant metastasis, and patients' overall survival in pancreatic cancer (43). Importantly, it has been reported that antisense oligonucleotides against oncomiR-21 induced apoptosis and inhibited migration in leukemic cells (44). Silencing of miR-10b in breast cancer markedly suppressed the formation of lung metastases (45). We found previously that inhibition of miR-30e-3p attenuates gliomas' invasion and angiogenesis, both in vitro and in vivo (21). Inspiringly, the first candidate miRNA product for cancer therapy, MRX34 (NCT01829971; ClinicalTrials.gov), which is designed to deliver a mimic of tumor suppressor miR-34, has entered clinical testing in 2013 and is currently being studied in a multicenter, open-label phase I clinical trial. All of these studies suggest the potential value of miRNAs in cancer diagnosis and therapy. Herein, we found that miR-892b was downregulated in breast cancer, and the expression level of miR-892b negatively correlated with breast cancer progression and prognosis. Importantly, upregulation of miR-892b could significantly inhibit NF-κB activity, resulting in the suppression of breast cancer aggressiveness, including tumor growth, invasion, and angiogenesis. Therefore, our results suggest that miR-892b could serve as a potential diagnostic and therapeutic target for breast cancer.

No potential conflicts of interest were disclosed.

Conception and design: L. Jiang, L. Song, J. Li

Development of methodology: L. Jiang, L. Yu, X. Zhang, F. Lei, L. Wang, S. Wu, J. Zhu, G. Wu, A. Liu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Jiang, L. Yu, X. Zhang, F. Lei, L. Wang, X. Liu, S. Wu, J. Zhu, G. Wu, A. Liu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Jiang, L. Yu, X. Zhang, F. Lei, L. Wang, X. Liu, S. Wu, J. Zhu, G. Wu, L. Cao, A. Liu, L. Song

Writing, review, and/or revision of the manuscript: L. Jiang, L. Song, J. Li

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Jiang, X. Zhang, L. Cao

Study supervision: L. Jiang, L. Song, J. Li

This work was supported by the Ministry of Science and Technology of China grant (973 Program, no. 2014CB910604); the Distinguished Young Scholar of Guangdong Province, China (no. 2015A030306033); GDUPS (2012); Natural Science Foundation of China (81325013, 81530082, 81201548, 81201546, and 91529301); the Science and Technology of Guangdong Province (No. 2013B021800096, 2015A030313468, 2014A030313008, and 2014A030313220); the Guangdong special Support program (2014TX01R076); the Guangzhou scholars research projects of Guangzhou municipal colleges and universities (no. 12A009D); Pearl River projects (Young Talents of Science and Technology) in Guangzhou (no. 2013J2200028); and Foundation of Key Laboratory of Gene Engineering of the Ministry of Education.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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