Ubiquitin-conjugating enzyme complex Uev1A-Ubc13 promotes breast cancer metastasis through nuclear factor-кB mediated matrix metalloproteinase-1 gene regulation

Introduction UEV1A encodes a ubiquitin-conjugating enzyme variant (Ubc13), which is required for Ubc13-catalyzed Lys63-linked polyubiquitination of target proteins and nuclear factor κB (NF-кB) activation. Previous reports have correlated the level of UEV1A expression with tumorigenesis; however, the detailed molecular events leading to tumors particularly breast cancer and metastasis are unclear. This study is to investigate roles of different UEV1 splicing variants, and its close homolog MMS2, in promoting tumorigenesis and metastasis in breast cancer cells. Methods We experimentally manipulated the UEV1 and MMS2 levels in MDA-MB-231 breast cancer cells and monitored their effects on cell invasion and migration, as well as tumor formation and metastasis in xenograft mice. The underlying molecular mechanisms leading to metastasis were also examined. Results It was found that overexpression of UEV1A alone, but not UEV1C or MMS2, is sufficient to induce cell invasion in vitro and metastasis in vivo. This process is mediated by NF-κB activation and requires functional Ubc13. Our experimental data establish that among NF-κB target genes, UEV1A-regulated matrix metalloproteinase-1 (MMP1) expression plays a critical role in cell invasion and metastasis. Interestingly, experimental depletion of UEV1 in MDA-MB-231 cells reduces MMP1 expression and prevents tumor formation and metastasis in a xenograft mouse model, while overexpression of MMP1 overrides the metastasis effects in UEV1-depleted cells. Conclusions These results identify UEV1A as a potential therapeutic target in the treatment of metastasic breast cancers.

Ubc13-Uev1A is involved in NF-κB activation [10,17,18] and inhibits stress-induced apoptosis in HepG2 cells [19]. Very recently, it was reported that a small-molecule inhibitor of Ubc13-Uev1A interaction can inhibit proliferation and survival of diffuse large Bcell lymphoma cells [20]. These observations collectively establish a close correlation between UEV1 expression and tumorigenic potential; however, whether UEV1 plays a role in promoting tumorigenesis or progression and how this is accomplished remains to be elucidated.
In this study we demonstrate that in MDA-MB-231 breast cancer cells, the UEV1A transcript level is moderately elevated compared to normal breast cells. Overexpression of UEV1A alone in MDA-MB-231 cells is sufficient to activate NF-кB, which in turn upregulates the MMP1 expression to enhance breast cancer cell metastasis. More importantly, experimental depletion of Uev1 in MDA-MB-231 cells reduces MMP1 expression and reduces their ability to grow tumors and metastasize in a xenograft mouse model. These observations provide the experimental and theoretical cornerstone for therapeutic targeting of Uev1A in the treatment of metastatic breast cancers.

Luciferase reporter assay
Cells were seeded in 24-well plates at a density of 1 × 10 5 . After 24 hr, the cells were transfected using X-tremeGENE HP DNA Transfection Reagent (Roche, Indianapolis, IN, USA). Briefly, luciferase reporter gene constructs (400 ng), pcDNA-Uevs plasmids (400 ng) and the pRL-SV40 Renilla luciferase construct (5 ng) (for normalization) were co-transfected into the wells. Cell extracts were prepared 48 hr after transfection and the luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega, Madison, WI, USA).

Immunoprecipitation
We immunoprecipitated 1 mg of protein samples in a total volume of 1 ml with 2 μg of antibody and 20 μl of Protein-A beads (for rabbit polyclonal antibodies) or Protein-G beads (for mouse monoclonal antibodies). The samples were rotated at 4°C overnight. The beads were washed 4 times with 1 ml of cold NP40 lysis buffer containing protease inhibitors. The beads were then boiled for 10 minutes in the presence of 25 μl 2 × sample buffer and the released proteins fractionated by SDS-PAGE in 12% or 15% gels. Proteins were detected by immunoblotting as described above.

Cell invasion and migration assays
In vitro invasion assays were conducted using Transwells (Costar, Cambridge, MA, USA) with 8-μm-pore-size polycarbonate membrane filters in 24-well culture plates. The upper surface of the filter was coated with Matrigel (Becton Dickinson, Bedford, MA, USA) in a volume of 12.5 μl per filter. The Matrigel was dried and reconstituted at 37°C into a solid gel on the filter surface. The lower surface of the filter was coated in fibronectin (20 μg/ml), vitronectin (10 μg/ml), collagen IV (50 μg/ml), or 10% (BSA)-DMEM as chemoattractants. After starving in BSAfree DMEM overnight, 2 × 10 4 cells were seeded in the upper chamber. The cells were allowed to invade for 48 hr. Cells that invaded the lower surface of the filter were counted in five random fields under a light-microscope at high magnification. Experiments were conducted at least in triplicate. The in vitro cell migration ability was detected by wound healing assay or transwell assay without Matrigel coating. The wound healing assay was performed by seeding 2 × 10 5 cells onto 96-well plates. Confluent monolayers were wounded using a pipette tip. After 48 hr the cell migration distance was measured under a light-microscope at high magnification in at least three random fields. The transwell assay without Matrigel was conducted using Transwells with 8-μm-pore-size polycarbonate membrane filters in 24-well culture plates. The lower surface of the filter was coated with 10% BSA-DMEM as chemoattractants. After starving in a BSA-free DMEM medium overnight, 5 × 10 4 cells were seeded in the upper chamber. Cells were allowed to invade for 6 to 36 hr and those migrated to the lower surface of the filter were counted in five random fields under a light-microscope at high magnification. Experiments were conducted at least in triplicate.

Metastasis assay in a xenograft mouse model
The experimental mouse work followed the animal care protocol CNUAREB-2012002 approved by the Capital Normal University Animal Research Ethics Board and was conducted at the Peking University Health Science Center, China. For the tumorigenesis assays, 5 × 10 6 breast cancer cells were injected subcutaneously into the lateral flanks of 4-to 5-week-old BALB/c female nude mice. The palpable tumor diameters were measured once per week. Tumor length (L) and width (W) were measured with a caliper, and the volume (V) was calculated by the following equation: The mice were sacrificed 6 weeks after cell injection. For experimental metastasis assays by intravenous (i.v.) injection, 2 × 10 5 breast cancer cells were injected into the tail veins of the 4-to 5-week-old female BALB/c nude mice. Endpoint assays were conducted 5 weeks after injection. Metastatic lung nodules 0.5 mm in diameter were counted. Analysis of variance (ANOVA) was used for statistical analyses. To ensure representative sampling of lung tumor nodules, four sections were made per lung at various depths along the coronal plane of the lung. The nodules per lung (four sections) were counted under a light-microscope.

Histopathology
Formalin-fixed lungs were paraffin-embedded, and tissue sections derived from tumor nodules or other tissues were stained with H&E to evaluate the morphology and invasiveness of breast cancer cells. Metastatic tumor nodules were counted throughout the entire lung section at all three depths under a light-microscope. Anti-Uev1A (LN1), anti-MMP1 (sc-30069) and NF-κB p65 (sc-372) primary antibodies from Santa Cruz were used for immunohistochemistry (IHC). TissueFocus™ breast cancer tissue microarrays (CT565863) for IHC were obtained from Origene (Beijing, China). Microscopic images were captured by a SPOT digital camera mounted in a light-microscope.

Preparation of nuclear fraction
HeLa cells were treated with 40 ng/ml TNF-α for 2 hr. Cells were washed, scraped with PBS, and centrifuged at 3,000 rpm at 4°C. The pellet was suspended in 10 mM Tris (pH 8.0) with 1.5 mM MgCl 2 , 1 mM dithiothreitol, and 0.1% NP-40, and incubated on ice for 15 minutes. Nuclei were separated from cytosol by centrifugation at 12,000 rpm at 4°C for 15 minutes. The cytosolic supernatants were removed and the precipitated pellets were suspended in 10 mM Tris (pH 8.0) containing 100 mM NaCl and stored on ice for 30 minutes. After agitation for 30 minutes at 4°C, the lysate was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected.

Electrophoretic mobility shift assay (EMSA)
The secquence of biotin-labelled sense NF-κB probe for EMSA is 5′-GAACCTCAGAGAACCCCGAAGAGCC-3′. The cold probe is the same NF-κB sequence without biotin label. The sequence of biotin-labelled mutated sense NF-κB probe is 5′-GAA CCTCA AGAGGTTTTG AAGAGCC-3′ (mutated sequence underlined): 1 ng of the probe was incubated together with 10 to 20 μg of cell extracts or 5 to 10 μg of nuclear extracts for 30 minutes at 25°C in a final volume of 20 μl. The binding reaction was subsequently separated on a 5.5-7% polyacrylamide gel in 1x Tris-Borate-EDTA (TBE) buffer (90 mM Tris, 90 mM boric acid).

Statistical analysis
The statistical significance of differential findings between the experimental and control groups was determined by Student's t-test as implemented by Microsoft Excel 2010 (*P <0.05, **P <0.01 and ***P <0.001).

Results
Alternative UEV1 transcript levels in breast cancer cell lines and samples Two major human UEV1 transcripts (UEV1A and UEV1B) were previously reported [1]. It was determined that only Uev1A, but not Uev1B, is able to physically interact with Ubc13 and promote K63-linked polyubiquitination [10]. It turns out that Uev1B is excluded from the nucleus and involved in endosomal trafficking [40]. Interestingly, database analyses also indicate another splicing variant that would encode a 147-amino acid Uev1 core domain, which we name Uev1C ( Figure 1A), the cellular function of which is currently unknown.
Western blot analysis of endogenous Uev1s using a Uev1-specific monoclonal antibody LN2B [39] could only detect Uev1C in several breast cancer cell lines including MDA-MB-231 and MCF7, and MCF10A, an immortalized normal mammary epithelial cell line ( Figure 1B). qRT-PCT analysis revealed that the relative transcript level of UEV1C is approximately 100-fold higher than that of UEV1A in the above cell lines ( Figure 1C), consistent with observations in other cell lines including HeLa and U2OS (data not shown).
We next examined relative transcript levels of UEV1A and UEV1C in breast cancer lines using MCF10A as a reference. Interestingly, the UEV1A transcript level is elevated in all breast cancer cell lines examined ( Figure 1D), with no significant upregulation of UEV1C ( Figure 1E) or MMS2 (Additional file 2: Figure S1A) in these lines. It has been previously reported that the UEV1A level may be elevated when normal cells undergo immortalization [2]. To further assess relative UEV1A expression in normal versus breast tumor tissues, we measured the UEV1A transcript level in five normal human breast samples and 43 breast cancer samples from TissueScan microarrays. Compared with the five normal human breast samples, 33/43 or 77% of breast cancer samples display UEV1A expression above the highest UEV1A level in normal samples, or at least 1.7-fold higher than the average level among the five normal samples (Additional file 2: Figure  S1B), suggesting that Uev1A may play a role in promoting breast tumorigenesis.

Overexpression of UEV1A promotes breast cancer cell invasion in vitro and metastasis in a xenograft model
To ask whether an elevated UEV1A level is indeed sufficient to promote breast cancer, UEV1A, UEV1C or MMS2 genes were cloned into a pcDNA4.0/TO/HA(+) vector and then transfected into MDA-MB-231-TR cells to construct stable cell lines, and the level of ecotopic gene expression after 10 μg/ml doxycycline (Dox) treatment was monitored by qRT-PCR (Additional file 2: Figure S2A-C) and western blot against the HA tag (Figure 2A), Uev1 (LN2B, Additional file 2: Figure S2D) or Uev1 plus Mms2 (LN3, Additional file 2: Figure S2E).
The cell growth and cell cycle progression of stable MDA-MB-231 transfectants were first measured, with no obvious alterations among each group (Additional file 2: Figure S3). The effects of ecotopic genes on breast cancer cell migration and invasion were then measured. The wound-healing experimental data show that the overexpression of UEV1A doubles the mobility compared with the same cells without ecotopic UEV1A expression or vector-transfected MDA-MB-231-TR cells with Dox treatment, while overexpression of UEV1C or MMS2 does not affect cell mobility compared with uninduced cells ( Figure 2B, C and Additional file 2: Figure S4). In a transwell assay, the invasiveness of UEV1A transfectants after induction was approximately 2.3-fold higher than the  control, UEV1C or MMS2 transfectants, whereas there was no significant difference among control, UEV1C and MMS2 transfectants regardless of Dox treatment ( Figure 2D and E). These results suggest that UEV1A regulates breast cancer cell migration and invasion in vitro.
As an increased ability of cancer cells to migrate and invade in vitro is a faithful indicator of cell metastasis, to further confirm the correlation between UEV1A expression and breast cancer metastasis, we assessed the effects of UEV1A on metastasis using an in vivo xenograft mouse model. Stably-transfected MDA-MB-231-TR cells were injected into the lateral flanks of 4-to 5-week-old BALB/c female nude mice and Dox (625 mg/kg) was added in feed as soon as the cells were injected. Tumor growth and metastasis were then monitored. All mice (10/10) injected with the UEV1A-expressing cells had massively enlarged lymph nodes containing invasive breast cancer cells ( Figure 2F). In contrast, there were no tumor metastasis foci in lymph nodes of mice injected with vector control or UEV1C-expressing cells, although some lymph nodes were enlarged (data not shown). Furthermore, overexpression of UEV1A but not UEV1C accelerated tumor growth compared to vector-transfected cells ( Figure 2G). In the tail-vein injection groups, compared with UEV1C or vector control, overexpression of UEV1A significantly promoted lung metastasis colony fomation ( Figure 2H, and I). These observations collectively demonstrate that elevated expression of UEV1A alone is sufficient to promote tumor growth and metastasis.

Depletion of Uev1 prevents breast cancer cell invasion in vitro and metastasis in nude mice
Compared with MCF-10A cells, there was a 2.7-fold increase in the UEV1A expression in MDA-MB-231 cells ( Figure 1D). To ask whether this moderate overexpression of UEV1A contributes to breast cancer metastasis, the endogenous UEV1A expression in MDA-MB-231 cells was suppressed using an shRNA (shUEV1) delivered by lentiviral particles. It was found that two independent shUEV1 constructs, shUEV1-1 and shUEV1-2, reduced UEV1A expression to 40% and 55% of control shRNA-treated cells, respectively ( Figure 3A). As expected, the cellular UEV1C mRNA and protein levels were also reduced but MMS2 remained unaffected (Additional file 2: Figure S5A-C). Moreover, partial depletion of Uev1 reduced cell migration ( Figure 3B and Additional file 2: Figure S5D) and invasion ( Figure 3C and Additional file 2: Figure S5E). The above findings were further extended by using a xenograft lung metastasis model, in which depletion of Uev1 limited tumor growth to the extent that no tumor was found in nude mice injected with MDA-MB-231 cells in which the Uev1 level was reduced by shUEV1-2 ( Figure 3D and Additional file 2: Figure S5F). Furthermore, depletion of Uev1 in MDA-MB-231 cells significantly reduced the number of lung nodules formed in mice and completely abolished metastasis in lung, even with moderate depletion of Uev1 ( Figure 3E, shUEV1-1, middle panel). These results clearly indicate that the elevated UEV1A expression in MDA-MB-231 cells plays a critical role in breast tumorigenesis and metastasis.
Overexpression of UEV1A activates NF-κB in MDA-MB-231 cells in a Ubc13-dependent manner To understand the mechanism by which Uev1A promotes metastasis in breast cancer cells, we took into account that Uev1A has been reported to activate NF-κB in HepG2 [19], and that NF-κB regulates the expression of a large number of genes critical for tumorigenesis, inflammation and metastasis [41]. As a hallmark of NF-κB activation is its translocation from the cytoplasm to the nucleus, we transfected MDA-MB-231-TR cells with a variety of constructs, induced the target gene expression by adding Dox, fractionated cells and then measured the subcellular distribution of the p65 subunit of NF-κB. As seen in Figure 4A, only overexpression of UEV1A, but not UEV1C or MMS2, was able to increase the phosphorylation of the NF-κB inhibitor IκBα (presumably in the cytoplasm) and enrich p65 in the nucleus. Consistently, depletion of Uev1 by shRNA reduced IκBα phosphorylation and p65 nuclear translocation ( Figure 4B).
It has been previously reported that Uev1A is a cofactor of Ubc13 in the NF-κB signaling pathway [10,17]. To ask whether the above Uev1A function is indeed dependent on Ubc13, we created a Uev1A-F38E mutation as the corresponding Mms2-F12E mutation ( Figure 1A) absolutely abolishes its interaction with Ubc13 and its ability to promote Ubc13-mediated K63 polyubiquitination [9]. Indeed we confirmed that the Uev1A-F38E substitution does not affect its expression (Additional file 2: Figure S2F) but abolishes its interaction with Ubc13 in vivo ( Figure 4C). As expected, overexpression of UEV1A-F38E failed to activate NF-κB, as judged by a lack of IκBα phosphorylation and p65 nucleaar translocation ( Figure 4D).

MMP1 and MMP9 are tightly regulated by UEV1
As NF-κB is a transcriptional factor that regulates the expression of a large number of genes including those involved in metastasis, we measured the transcription of many established or putative NF-κB target genes thought to be involved in metastasis such as COX2 [42], VEGF [43] and MMP family genes, among which MMP1 and MMP9 transcripts were elevated by 4.6-and 3.9-fold, respectively, in UEV1A-overexpression cells, but not in UEV1C or MMS2 overexpression cells ( Figure 5A,B), with corresponding elevation at proteins levels ( Figure 5C). The observed increase is completely dependent on Ubc13, as overexpression of UEV1A-F38E failed to induce MMP1 or MMP9 ( Figure 5B). Similarly, depletion of UEV1 in MDA-MB-231 cells significantly reduced MMP1 and MMP9 transcript ( Figure 5D) and protein ( Figure 5E) levels, and more efficient depletion of UEV1 (shUEV1-1 versus shUEV1-2) resulted in stronger repression of MMP1 and MMP9 expression ( Figure 5D and E), indicating that MMP1 and MMP9 are tightly regulated by Uev1A-Ubc13.

MMP1 is a downstream effector for Uev1A-induced metastasis
To ask whether MMP1 and/or MMP9 are critical effectors for Uev1A-induced metastasis, we depleted MMP1 or MMP9 by siRNA in MDA-MB-231 cells ( Figure 6A). The above treatment did not affect the UEV1A expression ( Figure 6A), but the depletion of MMP1 significantly decreased the invasiveness of MDA-MB-231 cells as determined by a transwell assay, while MMP9 depletion had much less effect ( Figure 6B and Additional file 2: Figure S6A). Similarly, MMP1 depletion in UEV1Aoverexpressed MDA-MB-231 cells (Additional file 2: Figure S6B) also decreased invasiveness (Additional file 2: Figure S6C and D). To ask whether overexpression of MMP1 is indeed sufficient to dictate MDA-MB-231 cell invasion, we constructed an MMP1-expression plasmid and transfected it to MDA-MB-231 cells, which resulted in a 2.6-fold increase in the MMP1 transcript level (Additional file 2: Figure S6E) and a similar increase at the protein level (Additional file 2: Figure S6F), as well as a 2.3-fold increase in invasion ( Figure 6C and Additional file 2: Figure S6G). We then restored MMP1 level in UEV1-depleted cells to that of control cells ( Figure 6D and E), which was sufficient to rescue the invasiveness in both UEV1-depleted MDA-MB-231 cell lines ( Figure 6F). As MMP1 is an important cancer cell metastasis factor [44][45][46], the above findings allow us to conclude that UEV1A regulates metastasis through tightly controlling MMP1 expression.

UevA-Ubc13 control MMP1 expression by regulating NF-κB
To ask whether Uev1A regulates MMP1 through NF-κB, we cloned the 1.8-kb human MMP1 promoter sequence ( Figure 7A) into pGL4.2 and co-transfected it with plasmids expressing UEV1A, UEV1A-F38E or an empty vector into MDA-MB-231 cells. A luciferase assay showed that UEV1A expression activates the MMP1 promoter and that this activation relies on its interaction with Ubc13, as the Uev1A-F38E substitution completely abolished the activation ( Figure 7B). The predicted NF-κB binding site (−1133 to approximately −1125) in the MMP1 promoter was then mutated ( Figure 7A) and the mutated reporter was used to co-transfect with plasmids expressing UEV1A, UEV1C, MMS2 or the empty vector into MDA-MB-231 cells. Overexpression of only UEV1A, but not UEV1C or MMS2, Nuclear or whole-cell extracts were prepared, equal amounts of protein were separated by SDS-PAGE gel, followed by western blotting analysis using an anti-p65 antibody to measure NF-кB nuclear enrichment and an anti-P(S32)-inhibitor of NF-κBα (IκBα) antibody to assess the degree of IκBα phosphorylation as an indication of its degradation and release of NF-κB into the nucleus. (B) NF-κB activation in Uev1-depleted cells. Experimental conditions are as described in Figure 4A. (C) The F38 residue of Uev1A was required for its interaction with Ubc13. MDA-MB-231 cell extracts expressing UEV1A or UEV1A-F38E were immunoprecipitated with an anti-HA monoclonal antibody, followed by western blotting with an anti-Ubc13 monoclonal antibody and an anti-HA monoclonal antibody. (D) Uev1A-F38E failed to activate NF-κB. Experimental conditions were as described in Figure 4A.
activated the wild-type P MMP1 -Luc reporter and this activation absolutely required the intact NF-κB target site ( Figure 7C). To gain direct evidence that the predicted NF-κB binding site indeed interacts with NF-κB, an EMSA was performed using nuclear lysates from TNFα-treated and untreated cells ( Figure 7D). A biotin-labeled synthetic MMP1 promoter probe containing the putative NF-κBbinding sequence was able to interact with the nuclear lysate and this interaction was enhanced when cells were pretreated with TNF-α, which induced nuclear translocation of p65 ( Figure 7E, lanes 4 and 5). This interaction was abolished when the NF-κB binding sequence was mutated (lane 6) or out-competed by adding excess unlabeled probe (lane 7), indicating that the interaction is sequence-specific. We conclude from the above observations that NF-κB directly binds to the MMP1 promoter at the predicted binding site.

Discussion
It has been reported previously that human cells contain two UEV genes, UEV1 and MMS2, which share >90% amino acid sequence-identity in their core domains [4]. Although both Uev1A and Mms2 proteins serve as cofactors for Ubc13-mediated K63-linked polyubiquitination, their biological functions are apparently distinct and only the Uev1A-Ubc13 complex is involved in NF-κB signaling [10]. It has been puzzling us that Uev1A and Mms2 have different molecular weights and migrate differently; however, a monoclonal antibody capable of recognizing both purified Uev1A and Mms2 only detects a single band in western blot analysis, and siRNA depletion of either Mms2 or Uev1 only partially reduced the intensity of this band. A careful examination in this study reveals that in addition to the previously reported two UEV1 splicing variants UEV1A and UEV1B [1], cultured The mutated sequence in the P MMP1 -NF-κBm construct is also shown. (B) The P MMP1 -Luc reporter was co-transfected to MDA-MB-231 cells with constructs that overexpressed wild-type UEV1A or UEV1A-F38E. The data were normalized to the activity of cells transfected with the empty vector (pGL4.2). (C) Only UEV1A, but not UEV1C or hMMS2, was able to activate the MMP1 promoter, and this ability was dependent on its interaction with Ubc13. The experimental conditions were as described in Figure 7B. (D) TNFα activates NF-κB as measured by the nuclear localization of the p65 subunit of NF-κB. HeLa cells were treated with or without TNFα for 30 minutes, nuclear proteins were purified and the relative p65 level in the nuclear fraction was determined by western blot and the nuclear protein Lamin B was used as a loading control. (E) Physical interaction between NF-κB and the putative NF-κB target sequence in the MMP1 promoter as determined by electrophoretic mobility shift assay. Lane 1, biotinlabeled NF-κB target sequence probe (NF-κB*). Lane 2, biotin-labeled mutated NF-κB binding site probe (NF-κBm*). Lane 3, Unlabeled double-strand DNA containing the P MMP1 NF-κB target sequence (NF-κB). Lanes 4 to 7 contain either wild-type or mutated P MMP1 NF-κB target sequence as indicated in the upper panel, plus TNFα-treated or untreated cell extract. Lane 7 also contains excessive unlabeled DNA of P MMP1 NF-κB target sequence.
human cells contain a novel UEV1 splicing variant, UEV1C, lacking the N-terminal 30 amino acid unique region of Uev1A. The resulting 147-residue protein would comigrate with Mms2 during electrophoresis. It turns out that the UEV1C transcript is much more abundant than UEV1A, and a Uev1-specific monoclonal antibody can detect cellular Uev1C but not Uev1A, unless the latter is experimentally overexpressed.
The current study investigates roles of UEV1A, UEV1C and MMS2 in tumorigenesis using a breast cancer model. With comparable levels of ectopic expression, it was found that only UEV1A, but not UEV1C or MMS2, is able to promote cell migration and invasion. Similarly, overexpression of UEV1A, but not UEV1C promotes tumor growth and metastasis in a xenograft mouse model. The above results are highly reliable, as the target gene expression is under tight regulation of a Tet-on promoter, and the phenotypes were only observed under Dox-induced conditions. In a reverse experiment, depletion of Uev1 in cultured breast cancer cells significantly reduces cell migration and invasion, as well as tumor growth and metastasis in a dose-dependent manner, indicating that the cellular Uev1 (presumbly Uev1A) level plays a critical role in breast tumorigenesis and metastasis.
To understand the molecular mechanism by which Uev1A promotes tumorigenesis, we demonstrated that overexpression of UEV1A, but not UEV1C or MMS2, is able to promote IκBα phorsphorylation and NF-κB translocation into the nucleus, and that this effect absolutely relies on its physical interaction with Ubc13. It is conceivable that as previously reported, the Ubc13-Uev1A heteromider serves as an E2 to assemble K63-linked poly-Ub chains along with cognate really interesting new gene (RING)finger E3s like TRAF2 and/or TRAF6 [17,34], which recruit K63 polyUb-binding proteins like NEMO [18] and TAB2/3 [47,48] to phorsphorylate and subsequently degrade IκBα, leading to NF-κB activation.
NF-κB activation promotes the transcription of many downstream genes in the signaling cascade [41]. However, the moderate level of NF-κB activation by UEV1A overexpression does not appear to induce all NF-κB targeting genes. To understand how overexpression of UEV1A leads to tumorigenesis and particularly metastasis in breast cancer cells, we surveyed NF-κB and metastasis-related genes and focused on two candidate genes, MMP1 and MMP9, both of which are highly induced upon UEV1A overexpression. Experimental results as presented in this report indicate that both genes are tightly regulatd by cellular Uev1 levels; however, depletion of MMP9 was not as effective as that of MMP1 on cell migration and invasion. As ecotopic expression of MMP1 to restore the wild-type level in Uev1-depleted cells also restored wild-type level of invasiveness, it is plausible to conclude that MMP1 is the critical downstream effector of UEV1A-induced breast cancer metastasis, although this study does not rule out the contributions of MMP9 and possibly other genes. The signal transduction cascade of Uev1A → NF-κB → MMP1 → metastasis is further confirmed by showing that Uev1A-induced MMP1 expression is dependent on both Ubc13 and the predicted NF-κB binding site located in the MMP1 promoter. Although this report only presents data from one human breast cancer line, we have obtained comparable results with a different breast cancer cell line MCF7 (data not shown), indicating that the tumorigenic and metastatic effects of Uev1A is a general phenomenon in breast cancers.
While the experimental evidence as shown in this report clearly indicates that UEV1A can function as a protooncogene, the clinical relevance of this finding awaits future investigation. Nevertheless, our limited TissueScan microarray data indicate a low (less than 2-fold) variation in UEV1A transcript levels among five normal human breast samples, compared with an increase of up to 20fold in some breast cancer samples. As NF-κB activation is commonly observed in breast cancers [24,25,49] and UEV1 upregulation is also frequently observed in breast cancer samples [12][13][14] and in cultured tumor cell lines [4], and is found to be correlated to tumorigenic indicators [2,3], it is conceivable that a certain percentage of breast cancer samples with NF-κB activation is due to elevated UEV1A expression.
This study demonstrated that the N-terminal region of Uev1A is the molecular determinant of its cellular function(s) in the NF-κB signaling pathway. Although the exact cellular function of Uev1C remains a mystery, our previous studies [10] have shown that truncated Uev1A missing the N-terminal 30 amino acids behaves like Mms2 in terms of subcellular localization and promotion of K63-linked di-Ub versus poly-Ub chains in vitro, suggesting that Uev1C may play an Mms2-related role.
Given the importance of Uev1 in signaling and tumorigenesis, small-molecule inhibitors against Uev1 have been isolated [20,50] based on their interference with the Ubc13-Uev1A interaction, and one appears to be able to inhibit proliferation and survival of diffuse large B-cell lymphoma cells. It is unclear whether these inhibitors also interfere with the Ubc13-Mms2 interaction, as critical residues responaible for the heterdimer formation are conserved between Uev1 and Mms2 [9]. Furthermore, this study provides evidence that a desired inhibitor should target the N-terminal region of Uev1A instead of the Ubc13-Uev1 interface. Hence, this report provides an experimental and theoretical cornerstone for future diagnosis and therapy by targeting Uev1A for the cure of breast cancer.

Conclusions
Among three Uev gene products, only Uev1A promotes breast tumor metastasis, which is primarily through activating the NF-κB target gene MMP1. Hence, UEV1A is considered a proto-oncogene and a therapeutic target for breast cancers.

Additional files
Additional file 1: Clinical data for the TissueFocus™ breast cancer tissue microarray purchased from Origene.
Additional file 2: Figure S1. Ubiquitin conjugating enzyme variant (UEV)1A is overexpressed in breast cancer cell lines and tumor samples. Figure S2. UEV expression levels in MDA-MB-231-TR inducible cells. Figure S3. UEV overexpression does not affect cell cycle progression or proliferation in MDA-MB-231 cells. Figure S4. Representative images of wound-healing assays without doxycycline (Dox) treatment. Figure S5. Uev1 depletion reduces cell invasion in vitro and tumor growth in a xenograft model. Figure S6. Matrix metalloproteinase (MMP)1 is tightly regulated by UEV1.