Metastatic dissemination is often initiated by the reactivation of an embryonic development program referred to as epithelial-mesenchymal transition (EMT). The transcription factor SNAIL promotes EMT and elicits associated pathological characteristics such as invasion, metastasis, and stemness. To better understand the posttranslational regulation of SNAIL, we performed a luciferase-based, genome-wide E3 ligase siRNA library screen and identified SCF-FBXO11 as an important E3 that targets SNAIL for ubiquitylation and degradation. Furthermore, we discovered that SNAIL degradation by FBXO11 is dependent on Ser-11 phosphorylation of SNAIL by protein kinase D1 (PKD1). FBXO11 blocks SNAIL-induced EMT, tumor initiation, and metastasis in multiple breast cancer models. These findings establish the PKD1-FBXO11-SNAIL axis as a mechanism of posttranslational regulation of EMT and cancer metastasis.

SNAIL is one of the most well established master transcriptional regulators of EMT. However, a comprehensive understanding of the posttranslational regulations of these factors is lacking. We developed a high-throughput luciferase-based RNAi screening strategy to identify E3 ligase(s) targeting the SNAIL protein for ubiquitylation and degradation. We identified FBXO11 as a crucial posttranslational regulator that controls the stability of the SNAIL protein. We further uncovered that this regulation is dependent on Ser-11 phosphorylation of SNAIL by PKD1. Importantly, functional studies and clinical sample analysis established a crucial role of the PKD1-FBXO11-SNAIL axis in regulating breast cancer EMT and metastasis and indicated a potential therapeutic value of targeting this pathway.

Many transcription factors are labile proteins with short half-lives and are actively degraded through the ubiquitin-proteosome pathway. Interestingly, in many cases, E3 ligases recognize and ubiquitylate transcription factor substrates by interacting with their transcriptional activation/repressor domain. This allows the coupling of the transcriptional activity with the protein degradation process to prevent hyperactivation of important transcription factors (). For instance, Mdm2 binds to the transactivation domain of p53, targeting it for ubiquitylation and degradation (). Likewise, the E3 ligase FBW7 interacts with the KLF5 transactivation domain for its degradation (). Although prior studies have identified two E3 ubiquitin ligases, FBXL14 (Ppa in Xenopus) () and FBXW1 (also called β-TRCP or BTRC) (), responsible for SNAIL ubiquitylation and degradation, neither of them interacts with the SNAIL/Gfi-1 domain (SNAG) transcriptional repression domain of SNAIL. This leads us to suspect that there may be additional critical E3 ligase(s) that target(s) the SNAIL protein for degradation through interaction with the SNAG domain. Identifying such E3 ligase(s) and the signaling events regulating SNAIL ubiquitination will provide possible new windows for therapeutic targeting of SNAIL.

SNAIL protein was among the first transcription factors discovered to repress CDH1 gene (encoding E-cadherin protein) transcription and induce EMT (). Recent studies suggest that SNAIL has a much broader impact on cancer progression. In mammary epithelial cells, overexpression of SNAIL induces EMT, coupled with increased tumor-initiating properties (). In melanoma, SNAIL promotes tumor metastasis by suppressing host immune surveillance (). SNAIL also cooperates with chromatin-modifying enzymes to inhibit fructose-1,6-biphosphatase (FBP1) expression, which results in increased glucose uptake, macromolecule biosynthesis, and maintenance of ATP production under hypoxic conditions (). Given the importance of SNAIL in cancer progression, it is not surprising that many signaling pathways have been implicated in the regulation of SNAIL gene expression, including transforming growth factor β (TGF-β), the NOTCH and WNT pathways, reactive oxygen species, and hypoxic stress (reviewed by). A better understanding of the regulatory mechanisms for SNAIL will provide critical information on how to block EMT and related processes in cancer progression.

The majority of cancer-related deaths can be attributed to the spread of cancer cells to distant vital organs (). Epithelial-mesenchymal transition (EMT), a crucial process in embryonic development that allows epithelial cells to lose apical-basal polarity and cell-cell contacts while gaining mesenchymal phenotypes, is believed to be utilized by cancer cells to gain mobility and invasiveness during metastasis (). A hallmark of EMT is the functional loss of E-cadherin, and additional cellular changes, such as reduced expression of the epithelial markers cytokeratin and ZO-1 and the upregulation of the mesenchymal markers N-cadherin, Vimentin, and Fibronectin, are also observed frequently.

In conclusion, our study reveals a functionally important posttranslational control mechanism of the EMT master regulator SNAIL. Our results suggest a model in which SNAIL protein is phosphorylated by PKD1 kinase at the Ser-11 residue. Phosphorylated SNAIL protein is then recognized and polyubiquitylated by the SCF-FBXO11 E3 ligase complex for proteosomal degradation, thereby limiting its ability to induce EMT, tumor initiation, and metastasis ( Figure 8 G).

We used immunohistochemistry (IHC) staining to examine the SNAIL protein expression level with the invasive property in 136 breast tumor samples collected at Sun Yat-Sen University Cancer Center. Nuclear SNAIL protein expression correlates positively with lymph node invasion ( Figure 8 A; Pearson χ= 4.9, p = 0.026). Based on our finding that PKD1 phosphorylation promotes SNAIL degradation, we next investigated the prognosis value of PKD1. Consistent with its role in promoting SNAIL protein degradation, a higher PKD1 expression level correlates with longer relapse-free survival ( Figure 8 B; hazard ratio = 0.79, p < 0.001) in a large public clinical microarray database of breast tumors from 1,354 patients (). Next, we tested the possible correlation between activated PKD1 (pY95-PKD1 antibody staining) and p-Ser-11 SNAIL protein levels using a tissue microarray containing 100 breast cancer patient samples. Consistent with a previous report (), activated PKD1 expression correlates positively with p-Ser-11 SNAIL protein expression ( Figures 8 C and 8D; χ= 10.0, p = 0.0016). Finally, we tested the correlation between FBXO11 and SNAIL expression in the same breast tumor samples and observed a strong negative correlation between FBXO11 and nuclear SNAIL expression because more than 70% of FBXO11-low samples have a high SNAIL level ( Figures 8 E and 8F; χ= 7.49, p = 0.0062).

(G) Schematic of PKD1-dependent SNAIL protein ubiquitylation and degradation by FBXO11. SNAIL protein is first phosphorylated by PKD1 kinase at Ser-11 before it can be recognized and ubiquitylated by the SCF-FBXO11 E3 ligase complex. Polyubiquitylated SNAIL protein is then degraded through the 26S proteosomal degradation pathway.

(A) Correlation study of the SNAIL expression level with lymph node invasion in 136 breast tumor specimens. SNAIL-lo, SNAIL IHC staining lower than median; SNAIL-hi, SNAIL IHC staining higher than median; LN−, without lymph node invasion; LN+, with lymph node invasion. χ 2 = 4.9, p = 0.026 by χ 2 test.

Previous results have demonstrated that WT SNAIL failed to induce EMT in MCF7 cells, possibly because of its rapid turnover rate and low stable protein level in this cell line (). On the other hand, the SNAIL-6SA mutant, with disruption of the GSK-3β phosphorylation sites (the binding site for FBXW1), was able to induce EMT in MCF7 cells. Because the SNAIL-S11V mutant disrupted the PKD1 phosphorylation site and also stabilized SNAIL protein, we sought to determine whether the S11V mutant is also able to induce EMT in MCF7 cells. Similar to what has been reported before (), although SNAIL failed to induce complete EMT in MCF7 cells, the 6SA mutant induced strong EMT phenotypes in MCF7 cells ( Figure 7 H). The S11V mutant and the double S11V/6SA mutants also induced a significant EMT program in MCF7 cells ( Figure 7 H). Immunofluorescent staining revealed a decrease of E-CADHERIN at the cell membrane and an increase of FIBRONECTIN in 6SA-, S11V-, and S11V/6SA-transduced MCF7 cells but not in control or wild-type SNAIL-transduced MCF7 cells ( Figure 7 H). Furthermore, although SNAIL was able to reduce CDH1 expression and increase CDH2 and FN levels, these changes were minimal compared with 6SA-, S11V-, and S11V/6SA-induced changes. 6SA and S11V seem to have similar effects on repression of CDH1 expression and induction of CDH2/FN expression, whereas combining these two mutants presented the strongest EMT phenotype, based on mRNA profiles ( Figure 7 I). To functionally test the effect of these mutants on tumor invasion ability, we performed a Matrigel invasion assay and revealed that the S11V-SNAIL and 6SA-SNAIL mutants induced a much stronger invasive capability compared with WT SNAIL or a vector control ( Figure 7 J). In aggregate, our results demonstrate that PKD1 is responsible for phosphorylation of Ser-11 on SNAIL protein and that this phosphorylation promotes SNAIL protein ubiquitylation and degradation.

We further investigated the role of endogenous PKD1 in regulating SNAIL protein stability. When we knocked down PKD1 expression by siRNAs ( Figure S7 D), we observed a significant decrease in the Ser-11 phosphorylation level, loss of interaction between SNAIL and FBXO11 ( Figure 7 E), and stabilization of the endogenous SNAIL protein ( Figure 7 F; Figure S7 E). Previous reports demonstrated that RhoA is a physiological activator for PKD1 kinase (). Therefore, we used a constitutively activated (CA) RhoA and a RhoA inhibitor, Exoenzyme C3 transferase protein (C3), to test their effects on SNAIL protein degradation. We first confirmed the PKD1 activation status by probing p-PKD1-744/748 in PKD1 pull-down samples (IP with HA antibody) ( Figure S7 F). SNAIL and FBXO11 interaction was also diminished significantly by the RhoA inhibitor ( Figure S7 G). Consistently, SNAIL was stabilized after RhoA inhibition by C3, whereas SNAIL degradation was accelerated in cells cotransfected with the RhoA-CA mutant ( Figure 7 G; Figure S7 H). Taken together, our results demonstrate that SNAIL is phosphorylated by PKD1 at Ser-11, which promotes the interaction, ubiquitylation, and degradation of SNAIL protein.

To directly test whether Ser-11 is phosphorylated by PKD1 in our system, we cotransfected 293T cells with SNAIL-FLAG, HA-FBXO11, and different wild-type or mutant PKD1 plasmids. In one set of cell lysates, we first performed IP with an anti-FLAG antibody to pull down SNAIL protein and examined the Ser-11 phosphorylation status by an antibody that specifically recognizes p-Ser-11-SNAIL. SNAIL protein presented a very strong phosphorylation band at the correct size, whereas PKD1 inhibitor CID755673 treatment resulted in a loss of this phosphorylation band. PKD1 dominant-negative mutant (PKD1-KW), but not the wild-type PKD1, also almost completely abolished SNAIL protein phosphorylation ( Figure 7 A). In the remaining set of cell lysates, we performed co-IP experiments with HA antibody to pull down FBXO11 protein. The PKD1 inhibitor, as well as the dominant-negative PKD1-KW mutant, disrupted the interaction between SNAIL and FBXO11 protein ( Figure 7 B) and stabilized SNAIL protein in the CHX pulse-chase experiment ( Figure 7 C; Figure S7 A) without changing the localization of SNAIL ( Figure S7 B). Consistent with these results, PKD1 inhibitor reduced polyubiquitylated SNAIL protein bands compared with the control ( Figure 7 D). Importantly, both p-Ser-11-SNAIL and FBXO11 localized in the nucleus, based on immunofluorescent staining ( Figure S7 C), suggesting that PKD1-dependent targeting of SNAIL by FBXO11 may occur in the nucleus.

(I) qRT-PCR was performed for CDH1, CDH2, VIM, and FN in the indicated MCF7 stable cell lines. Data represent mean ± SD. ∗ p < 0.05 by two-tailed Student’s t test.

(G) The 293T-SNAIL stable cell line was transfected with HA-PKD1 together with either vector or GST-tagged, constitutively active RhoA-CA, and a CHX pulse-chase assay was performed for SNAIL. The Western blot images in Figure S7 H were quantified using ImageJ software.

(F) MCF10A cells were transfected with control or two PKD1 siRNAs. Two days later, endogenous SNAIL degradation was determined by CHX pulse-chase assay. The Western blot images in Figure S7 E were quantified using ImageJ software.

(E) MCF10A cells were treated with either 10 nM control or PKD1-targeting siRNA before being subjected to co-IP and Western blot analysis using the indicated antibodies. Cells were treated with MG132 for 6 hr before co-IP experiments.

(D) 293T cells were transfected with the indicated plasmids. Cells were treated with DMSO or the PKD1 kinase inhibitor CID755673 before lysing in denature lysis buffer. Cell lysates were subjected to denature IP with SNAIL antibody. Polyubiquitylated SNAIL protein was visualized by HA antibody blotting against HA-ubiquitin. Cells were treated with MG132 for 6 hr before co-IP experiments.

(C) In 293T cells stably transfected with SNAIL, SNAIL protein turnover rates were determined by CHX pulse-chase assays after cells were transfected with FBXO11 together with the indicated plasmids or the inhibitor CID755673. The Western blot images in Figure S8 A were quantified using Image J software.

(B) 293T cells were transfected or treated similarly as in (A) and then lysed, and a co-IP experiment was performed using anti-HA antibody. Cells were treated with MG132 for 6 hr before co-IP experiments.

(A) Detection of Ser11 phosphorylation of immunoprecipitated SNAIL protein when SNAIL was cotransfected with the indicated plasmids or treated with the PKD1 inhibitor CID755673 in 293T cells. Cells were treated with MG132 for 6 hr before co-IP experiments.

To further test the effects of these mutations on SNAIL protein degradation, we transfected these mutants either alone or together with wild-type FBXO11 or FBXO11-ΔF expression vectors into 293T cells and performed the CHX pulse-chase experiment. In the absence of exogenous FBXO11, SNAIL-S11E degraded much faster than wild-type (WT) SNAIL protein, whereas SNAIL-S11V protein was stabilized ( Figure 6 E and 6F). In the presence of exogenous FBXO11, the stable level of SNAIL protein was significantly lower than that of the S11V-SNAIL mutant ( Figure 6 G). In the CHX pulse-chase experiment, SNAIL protein turnover was accelerated by cotransfection with FBXO11 but not the FBXO11-ΔF mutant lacking the SCF complex formation domain. As expected, SNAIL-S11V was more stable compared with wild-type SNAIL protein, even in the presence of FBXO11 ( Figures 6 H and 6I). Taken together, our results suggest that Ser-11 is the critical amino acid for SNAIL-FBXO11 interaction.

As recent literature suggests, there are at least five kinases that phosphorylate SNAIL protein at distinct amino acids and fine-tunes the functions of SNAIL differentially (). Phosphorylation of the Ser-11 site by protein kinase D1 (PKD1, also known as PKCμ) lies within the domain that we identified to be responsible for SNAIL-FBXO11 interaction. Interestingly, aberrant upregulation of PKD1 has been suggested to block SNAIL-induced EMT and anchorage-independent growth (). Therefore, we tested whether the PKD1 consensus phosphorylation sequence LXRXXS in the SNAG domain is essential for SNAIL-FBXO11 interaction. We generated L6A-SNAIL, R8A-SNAIL, and S11A-SNAIL alanine scanning mutants in the PKD1 consensus sequence and found that none of these mutants could interact with FBXO11 anymore ( Figures S6 A and S6B). Importantly, they also became stabilized compared with wild-type SNAIL protein ( Figure S6 C). We further generated two additional mutants in Ser11, SNAIL-S11E and SNAIL-S11V, to mimic and disrupt the phosphorylation of the SNAIL protein at Ser-11, respectively. The phosphorylation-disrupting S11V mutant completely lost the interaction with FBXO11 protein, whereas the phosphorylation-mimicking mutant S11E still interacted with FBXO11 ( Figure 6 D). Previous literature suggests that GSK-3β-dependent phosphorylation on SNAIL protein is critical for its recognition by FBXW1, another E3 ligase targeting SNAIL protein (). We acquired several SNAIL mutants, including the 6SA mutant, which can no longer be phosphorylated by GSK-3β, and performed a co-IP experiment with FBXO11. Our results demonstrated that these mutants did not disrupt the interaction between SNAIL and FBXO11 protein ( Figure S6 D). Therefore, although both FBXO11 and FBXW1 can destabilize SNAIL through UPS, distinct protein kinases are involved in producing different phosphorylated SNAIL proteins for recognition by these E3s.

FBXO11 belongs to a large group of the F-box protein superfamily. These proteins usually exert their E3 ligase function by forming a four-subunit functional complex with Cullin, Skp, and Rbx proteins (SCF complex), with F-box proteins as a substrate recognition component. Interestingly, F-Box proteins generally recognize the protein substrate when they are phosphorylated (). Therefore, it is plausible that SNAIL protein also needs to be phosphorylated before being recognized by FBXO11. To investigate this, we performed a modified co-IP experiment in which cell lysate from 293T cells transfected with SNAIL and FBXO11 was treated with or without alkaline phosphatase (calf intestinal phosphatase [CIP]) to remove protein phosphorylation before IP. SNAIL was pulled down together with FBXO11 protein, and this interaction was completely disrupted by CIP treatment ( Figure 6 A), suggesting that phosphorylation modification of either or both SNAIL and FBXO11 might be critical for the SNAIL-FBXO11 protein interaction. To identify the potential region within the SNAIL protein that is phosphorylated and responsible for SNAIL-FBXO11 interaction, we generated several SNAIL truncation mutations and performed the co-IP experiment. Interestingly, only two truncation mutants, ΔSNAG and Δ194 (both lacking the first 20 amino acid SNAG domain on the SNAIL protein) ( Figure 6 B), lost the ability to interact with FBXO11 ( Figure 6 C). Immunofluorescence analysis indicated that ΔSNAG remained localized in the nucleus, suggesting that loss of interaction between ΔSNAG and FBXO11 was not due to localization change ( Figure S7 A). Therefore, the SNAG domain of SNAIL protein is critical for its interaction with FBXO11.

(H and I) 293T stable cells expressing corresponding wild-type SNAIL or mutant SNAIL were transfected with the indicated FBXO11 expression plasmids (H). A pulse-chase assay was performed 2 days later. Data were quantified in (I) using ImageJ software.

(G) 293T cells were transfected with the indicated expression plasmids, and cell lysates were collected for immunoblotting two days after transfection.

(E) CHX pulse-chase assay for wild-type SNAIL, SNAIL-S11E, and SNAIL-S11V in the absence of exogenously expressed FBXO11 in 293T cells. Data were quantified using ImageJ software.

(D) Wild-type SNAIL and SNAIL-S11E and SNAIL-S11V single amino acid mutants were cotransfected with HA-FBXO11 into 293T cells, and cell lysates were collected for the co-IP experiment using anti-HA antibody.

(C) Wild-type FLAG-tagged SNAIL and SNAIL truncation mutants were cotransfected with HA-FBXO11 into 293T cells, and cell lysates were collected for the co-IP experiment using anti-FLAG antibody.

(B) Schematic of the SNAIL protein structure and the deletion mutation constructs generated to map out the interaction domain for SNAIL-FBXO11 interaction.

(A) 293T cells were transfected with SNAIL-FLAG and HA-FBXO11 for 2 days. Cell lysates were collected and treated either with or without CIP for 1 hr before subjected to a co-IP experiment.

Phosphorylation of Ser-11 on the SNAG Domain Is Essential for SNAIL to Be Recognized by FBXO11

To complement the findings obtained by using SNAIL and FBXO11-overexpressing cells, we knocked down endogenous Fbxo11 in 4T1 cells by short hairpin RNAs ( Figure 5 A). Western blot analysis revealed a 5-fold upregulation of Snail protein after stable knockdown of Fbxo11 ( Figure 5 B). Fbxo11 KD in 4T1 cells induced EMT-like cellular changes, including reduction of E-cadherin and increase of Vimentin at the mRNA ( Figure 5 A) and protein levels by Western blot analysis and immunofluorescence analyses ( Figures 5 B and 5C). Importantly, Fbxo11 KD significantly increased lung metastasis ( Figures 5 D and 5E), despite no difference in primary tumor growth (data not shown). Western blot analysis of primary tumors confirmed that Snail expression was upregulated and that E-cadherin expression was reduced in Fbxo11 KD tumors ( Figure S5 A), consistent with an in vitro analysis. Fbxo11 KD and Snail upregulation were also maintained in lung metastasis nodules ( Figure S5 B). These results indicate that FBXO11 inhibits metastasis by promoting the ubiquitylation and degradation of the EMT-inducing SNAIL protein.

(D) 10 5 tumor cells from various 4T1 cell lines were injected orthotopically into BALB/c mice, and primary tumors were removed 10 days later. Lung metastasis nodules were counted after sacrificing the mice at 38 days postinjection. ∗∗ p < 0.01 by Mann-Whitney U test.

(A) Real-time PCR analysis of Fbxo11, Cdh1, and Vimentin expression in the parental and control 4T1 cell line and two Fbxo11-KD cell lines. Data represent mean ± SD. ∗∗ p < 0.01 by two-tailed Student’s t test.

To test the functional impact of FBXO11 on SNAIL-induced EMT in breast cancer metastasis, we decided to use the epithelial-like 4T1 and EpRas mouse mammary tumor cell lines, which are sensitive to Snail-induced EMT and have much more robust primary tumor growth capability than HMLEN. As expected, FBXO11 expression accelerated SNAIL degradation in 4T1 cells ( Figure S4 A). SNAIL overexpression in the epithelial-like 4T1 and EpRas cell lines induced an EMT-like phenotype, including the reduction of E-cadherin and upregulation of N-cadherin or Vimentin expression at the both mRNA and protein levels ( Figures 4 A and 4B ; Figures S4 B–S4D). Strikingly, FBXO11 expression blocked SNAIL-induced EMT in both cell lines. Gene expression profiling revealed that the enrichment of EMT gene signatures in SNAIL-overexpressing cells was reversed by FBXO11 overexpression ( Figure 4 C). Likewise, overexpression of FBXO11 suppressed the SNAIL expression signature in 4T1 cells ( Figure 4 D). Consistent with cellular phenotype and gene expression changes, the increased invasive capability of SNAIL-overexpressing 4T1 cells was inhibited by FBXO11 ( Figure S4 E). Importantly, although SNAIL overexpression did not affect primary tumor growth when 10tumor cells were injected orthotopically ( Figure S4 F), its expression significantly increased spontaneous lung metastasis in vivo ( Figures 4 E and 4F). The lung metastasis-promoting effect of SNAIL was reversed after FBXO11 overexpression ( Figures 4 E and 4F), again without any significant alteration in the primary tumor growth rate ( Figures S4 F and S4G).

(D) A heatmap was generated using hierarchical clustering. The 1,400 gene probes used for clustering were those showing >2-fold expression changes in SNAIL-overexpressing cells.

(B) Quantitative RT-PCR (qRT-PCR) analysis of EMT marker mRNAs in the indicated 4T1 cell lines. Results were normalized to the GAPDH mRNA level. Data represent mean ± SEM. ∗ p < 0.05 by Student’s t test.

(A) Phase contrast and IF images for 4T1 cells transduced with the indicated lentiviruses. SNAIL induced a strong EMT program in 4T1 cells with downregulation of E-cadherin and upregulation of N-cadherin expression, whereas FBXO11 blocked SNAIL-induced EMT. Scale bars, 25 μm.

Because cells that undergo EMT often gain migratory and invasive capabilities, we tested the Matrigel invasion ability of these cells. As expected, SNAIL expression significantly increased the invasiveness of HMLEN cells, whereas FBXO11 expression blocked SNAIL-induced invasion ( Figure 3 D). HMLE cells that have undergone EMT have also been reported to acquire breast cancer stem cell characteristics (). Indeed, HMLEN-SNAIL cells showed a significant increase of the CD44/CD24population, whereas FBXO11 decreased this population ( Figures S3 C and S3D). In line with the population shift, HMLEN-SNAIL showed increased colony formation efficiency, whereas FBXO11 suppressed SNAIL-induced colony formation ( Figure 3 E; Figure S3 E). To further test the effect of FBXO11 on tumor initiation, we injected 20,000 tumor cells orthotopically into the mammary fat pads of nude mice. HMLEN cells had minimal tumor forming capability. However, significant formation of primary tumors can be observed 8 weeks after inoculation of SNAIL-overexpressing HMLEN cells. Again, FBXO11 completely blocked SNAIL-induced tumor initiation ( Figures 3 F and 3G). In aggregate, our results demonstrate a prominent effect of FBXO11 in inhibiting SNAIL-induced EMT and tumorigenesis in the HMLEN model, and this effect is likely due to FBXO11-mediated ubiquitylation and degradation of SNAIL protein.

To evaluate the global transcriptomic changes associated with expression of SNAIL and FBXO11, we performed gene expression microarray profiling and gene set enrichment analysis (GSEA). Using four previously published EMT/cancer stem cell-related gene signatures (see Supplemental Information), we found that these signatures were enriched in SNAIL-overexpressing cells and that such an enrichment was lost in SNAIL/FBXO11 cells (see Figure 3 B using theClaudin-low/EMT signature as an example). We then generated a gene expression heatmap using a list of 2,327 genes showing >2-fold differential expression between the SNAIL-overexpressing cells and the control cells. As shown in Figure 3 C, FBXO11 expression largely reversed the gene expression changes induced by SNAIL. Taken together, these results suggest that FBXO11 can block the SNAIL-induced EMT program and associated global gene expression pattern.

SNAIL has been shown to induce EMT and increases the tumor-initiating capability in nontransformed or oncogene-transformed HMLE human mammary epithelial cells (). We tested whether FBXO11 can block SNAIL-induced EMT by transducing Neu-transformed HMLEN cells with either SNAIL alone or together with FBXO11. SNAIL expression induced strong EMT phenotypes in HMLEN cells, with loss of E-CADHERIN and upregulation of VIMENTIN and FIBRONECTIN ( Figure 3 A; Figures S3 A and S3B). When SNAIL and FBXO11 were coexpressed in HMLEN cells, the stable level of SNAIL protein was decreased significantly compared with cells only expressing SNAIL ( Figure S3 A). The reduced level of SNAIL led to the reversal of the EMT phenotype in HMLEN cells, with the gain of E-CADHERIN at the cell-cell junctions and the loss of VIMENTIN and FIBRONECTIN expression ( Figure 3 A; Figures S3 A and S3B).

(F and G) 2 × 10 4 of the indicated HMLEN cells were injected orthotopically into nude mice (n = 8), and primary mammary tumor growth was measured weekly after injection (E). Representative tumor images are shown in (G). Data represent mean ± SD. ∗∗ p < 0.01 by two-tailed Student’s t test.

(E) Quantification of the colony formation assay results for the indicated HMLEN cell lines. Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

(D) Boyden chamber invasion assay of HMLEN cells with SNAIL or SNAIL/FBXO11 overexpression. The data are shown as the mean of collected data from three triplicate wells of three independent experiments. Data are presented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

(C) A heatmap was generated using hierarchical clustering of the microarray data for the indicated HMLEN cell lines. The 2,327 gene probes used for clustering were those showing >2-folds expression changes upon SNAIL overexpression. The experiment was performed in duplicates.

(B) GSEA analysis of the environment of the EMT gene signature in HMLEN cells after overexpression of SNAIL with or without FBXO11.

(A) Phase contrast and immunofluorescent (IF) images of HMLEN cells with stable overexpression of SNAIL or SNAIL together with FBXO11. Scale bar, 100 μm (phase contrast images) and 25 μm (IF images).

We confirmed that two previously reported E3s for SNAIL, FBXW1 and FBXL14, also interact with SNAIL, whereas other closely related F-box family members do not bind to SNAIL ( Figures S2 J–S2L). Interestingly, FBXW1 promoted SNAIL protein degradation, whereas FBXL14 had no effect on SNAIL stability ( Figures S2 M and S2N). Conversely, FBXW1 KD increased SNAIL stability, whereas no effect was observed after FBXL14 KD. Importantly, simultaneous KD of FBXO11 and FBXW1 led to increased SNAIL stabilization compared with either FBXO11 or FBXW1 KD alone ( Figure 2 E, right panel; Figures S2 O and S2P). These results suggest that both FBXO11 and FBXW1 are capable of promoting SNAIL degradation and may function collectively to control SNAIL stability.

To test the effect of reducing endogenous FBXO11 expression on SNAIL protein degradation, we used siRNAs to knock down FBXO11 gene expression in MCF10A cells. The stable level of SNAIL protein was found to be upregulated more than 2-fold after siRNA treatment ( Figure 2 E, left panel, without CHX treatment). Furthermore, SNAIL protein was much more stable after FBXO11 KD in a CHX pulse-chase assay ( Figure 2 E). Stabilization of SNAIL after FBXO11 KD was also observed in the LM2 cell line ( Figures S2 H and S2I). Taken together, our results suggest that SCF-FBXO11 is a bona fide E3 ubiquitin ligase targeting SNAIL protein for ubiquitylation and degradation.

Because SNAIL protein has been reported as a marker for a poor prognosis, with its high expression correlated with worse patient outcome (), we hypothesized that E3 ligase(s) targeting SNAIL should be a marker for a good prognosis. Indeed, using the NKI295 breast cancer data set (), we found that higher FBXO11 gene expression correlates significantly with longer metastasis-free survival ( Figure 2 D), whereas SPSB1, SOCS3, and TRIM5 do not correlate with metastasis-free survival ( Figure S2 G).

To directly test the effect of FBXO11 on endogenous SNAIL protein degradation, we generated FBXO11-overexpressing stable cell lines using SUM1315 and the LM2 subline of MDA-MB-231 (). In both cell lines, we observed a dramatic increase of endogenous SNAIL protein degradation ( Figures S2 C–S2F). Next we confirmed the interaction between endogenous SNAIL and FBXO11 proteins by co-IP experiments using MG132-treated LM2 cell lysate ( Figure 2 B). To further investigate whether FBXO11 functions as a bona fide E3 ligase that ubiquitylates the SNAIL protein, we cotransfected 293T cells with SNAIL-FLAG, FBXO11, and HA-ubiquitin and treated the cells with MG132 for 6 hr to prevent protein degradation before performing an ubiquitylation assay. A significant increase of polyubiquitylated SNAIL protein was observed in FBXO11-transfected cells, whereas an F-box (Skp1, Cullin, F-box [SCF] complex formation domain) deletion mutant of FBXO11 (FBXO11-ΔF) was not able to increase SNAIL ubiquitylation ( Figure 2 C).

As an initial step in functionally testing these four E3 ubiquitin ligase candidates, we cotransfected the SNAIL-Luc gene with individual E3 genes and examined the SNAIL-Luc protein stable level. FBXO11 expression strongly reduced the stable SNAIL-Luc protein level by up to 70% ( Figures S2 A and S2B). In a CHX pulse-chase analysis of SNAIL-Luc, FBXO11 accelerated SNAIL-Luc protein degradation, whereas SOCS3, SPSB1, and TRIM5 had only modest or negligible effects on SNAIL-Luc protein degradation ( Figure 2 A; note that, in this and other similar pulse-chase figures, Western blot images with different exposure times were used so that the basal SNAIL-Luc protein band intensity at time = 0 hr is the same across the experimental group to illustrate the differences in the kinetics of degradation).

(E) MCF10A cells were transfected with indicated siRNAs, followed by the CHX pulse-chase assay. Quantification data is shown in Figure S2 P.

(C) 293T cells were cotransfected with plasmids expressing HA-Ub and SNAIL-FLAG together with either a vector control, FBXO11, or the FBXO11-ΔF plasmid. Cells were treated with MG132 for 6 hr before cell lysates were immunoprecipitated using a denature IP protocol to pull down SNAIL protein, and the polyubiquitylated SNAIL protein was detected by anti-HA antibody.

(B) LM2 cells were treated with 10 μM MG132 for 6 hr before the cell lysate was immunoprecipitated with FBXO11 antibody or an immunoglobulin G (IgG) control and subjected to Western blot analysis.

(A) Four E3 ligase candidates or a control pLEX-vector were cotransfected with the SNAIL-Luc plasmid into 293T cells, and a CHX pulse-chase assay was performed. Western blot data were quantified using ImageJ software.

The E3 siRNA library screening revealed that siRNA-mediated inhibition of multiple E3 ligase genes increased the level of SNAIL-Luc protein. Among the 554 known and predicted human E1, E2, and E3 ligases ( Table S1 ), siRNA knockdown of more than 100 genes increased the luciferase activity by more than 2 fold ( Figure 1 G, top panel). This large initial pool of potential hits may include false positive candidates that do not directly regulate SNAIL protein stability. Therefore, further validation of these candidates is needed. Interestingly, in this screening, knockdown of FBXW1, a previously known E3 ligase for SNAIL (), increased the SNAIL-Luc protein level 2.6-fold, whereas no change was observed after knockdown of FBXL14, another SNAIL E3 identified in the Xenopus model (). The cell lysates from these initial positive hits were then subjected to immunoblotting for SNAIL-Luc fusion protein ( Figures S1 G and S1H), for most of which an upregulation of SNAIL-Luc protein was observed. Among them, 21 E3 candidates, when knocked down individually, increased the SNAIL-Luc protein level at least 6-fold in a luciferase assay or in immunoblot analyses, the stringent criteria we set for identifying potential candidates ( Table S2 ). A second round of siRNA screening for these 21 genes was performed, confirming that 21 siRNAs were able to consistently increase the luciferase activity and protein stable level ( Figure 1 G, bottom panels; Figure S1 I). We cloned the cDNA coding sequences of 13 candidate genes into a lentiviral expression vector with an N-terminal HA epitope ( Figure S1 J). The remaining eight genes were not cloned successfully. To test the interactions between these 13 E3 ubiquitin ligases and SNAIL, we performed a co-IP experiment to test the interactions between these E3 candidates with SNAIL. Only four E3 ligases were able to interact with SNAIL: FBXO11, SOCS3, SPSB1, and TRIM5 ( Figure S1 J). This result was first confirmed by repeating the co-IP experiment for these four E3 ligase proteins using HA antibody ( Figure 1 H) and then by reciprocal IP of the SNAIL-FLAG protein with an anti-FLAG antibody ( Figure 1 I). Therefore, we focused on these four E3 genes in further functional studies.

We followed the procedures outlined in Figure 1 F to identify potential E3 ligase candidates for SNAIL protein. We first knocked down individual human E3 ligase by pooled siRNA (three siRNAs per gene) in SUM-SNAIL-Luc/R-Luc cells and looked for E3 ligase genes whose knockdown (KD) resulted in more than a 2-fold increase in luciferase activity of the SNAIL-Luc fusion protein ( Figure 1 F). The cell lysates from these positive hits were immunoblotted for SNAIL-Luc fusion protein to confirm protein stabilization. Positive E3 ligase hits were then examined by a second round of siRNA screening to confirm the results. We then cloned all positive E3 ligase genes and determined whether they can interact with SNAIL protein by coimmunoprecipitation assay (co-IP) and reciprocal co-IP for further confirmation. Only the proteins that interacted with SNAIL were considered as E3 candidates for SNAIL protein and selected for further functional testing to determine whether they can affect exogenous and endogenous SNAIL protein degradation.

The identification of E3 ligases responsible for the degradation process of certain protein substrates has traditionally relied on serendipitous discoveries or candidate gene approaches. To identify potential E3 ligase(s) responsible for SNAIL degradation in an unbiased manner, we designed a luciferase-based small interfering RNA (siRNA) screening strategy that can be easily adapted in future discovery efforts to identify new E3 ligase-protein substrate pairs. We fused the SNAIL protein coding sequence in-frame with the firefly luciferase gene coding sequence (SNAIL-Luc) to produce the SNAIL-Luciferase fusion reporter protein ( Figure 1 B). This reporter allowed us to monitor the SNAIL protein stable level and its degradation dynamics by monitoring the luciferase activity. The SUM1315 cell line was first transduced with a lentiviral vector containing the SNAIL-Luc fusion gene and subsequently with the Renilla-Luciferase retrovirus to generate a dual luciferase reporter cell line with Renilla luciferase (R-Luc) serving as an internal control. This cell line is denoted as “SUM-SNAIL-Luc/R-Luc” to facilitate description below ( Figure 1 B). The SNAIL-Luc fusion protein is localized in the nucleus ( Figures S1 B and S1C) and has degradation dynamics similar to those of the endogenous SNAIL protein ( Figures 1 C and 1D and Figure S1 D). In contrast, the F-Luc protein alone is very stable ( Figures S1 E and S1F). The luciferase activity of the SNAIL-Luc fusion protein is also readily detectable by standard firefly luciferase reporter assay and correlates with the fusion protein level ( Figure 1 E). Therefore, the firefly luciferase activity in this cell line reliably represents the stable level of the SNAIL-Luc protein, whereas R-Luc activity is used as an internal control for cell number and viability.

To confirm that the SNAIL protein is posttranslationally controlled by the ubiquitin-proteosome system (UPS) in breast cancer, we first blocked protein synthesis using cycloheximide (CHX) and pulse-chased the SNAIL protein in the SUM1315 human breast cancer cell line. Indeed, SNAIL was degraded rapidly and became undetectable within 4 hr of CHX treatment ( Figure 1 A; Figure S1 A available online). Furthermore, treatment of cells with the proteosomal inhibitor MG132 increased the stable SNAIL protein level, confirming that SNAIL is degraded through the UPS ( Figure 1 A; Figure S1 A).

(H and I) 293T cells were transfected according to the panel labels. The co-IP experiment was performed using either an HA antibody to pull down HA-tagged E3 ligase proteins (H) or an anti-FLAG antibody against SNAIL-FLAG protein (I).

(G) Luciferase-based siRNA library screen against human E3 ligases identified multiple E3 candidates that, when knocked down in SUM-SNAIL-Luc/R-Luc cells, increased luciferase activity more than 2-fold (top panel). A second round of siRNA screening (bottom left panel) and immunoblotting (bottom right panel) was performed for confirmation of candidates.

(C and D) CHX pulse-chase experiment demonstrating the degradation of SNAIL-F-Luc fusion protein in SUM-SNAIL-Luc/R-Luc cells (C). Western blot data are quantified in (D). Data are presented as mean ± SEM.

(A) In SUM1315 cells, endogenous SNAIL protein was detected by Western blot analysis after CHX (left panel) or MG132 treatment (right panel) for the indicated hours. Western blot data are quantified in Figure S1 A.

Discussion

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et al. Global identification of modular cullin-RING ligase substrates. Previous results have demonstrated that SNAIL is a labile protein with a very short half-life (). Although E3 ligases often recognize their transcriptional factor substrates by binding to their transcriptional regulatory domains, neither one of the two previously identified E3 ligases for SNAIL, FBXW1/β-TRCP, and FBXL14 interacts with the SNAG transcriptional repression domain of SNAIL (), leading us to suspect that there exist(s) additional E3 ligase(s) targeting the SNAG domain under the control of important kinase signaling pathways. Conventionally, identifying such E3 ligases is a time-consuming and often serendipitous process. Proteomic analyses have been used to identify protein substrates for certain E3 ligases (). However, this method is not suitable to search for E3 ligases that can degrade a specific protein substrate of interest. We developed a luciferase-based screening method to discover candidate E3 ligases for a specific protein substrate (SNAIL). It is worth noting that other candidate SNAIL-targeting E3s identified in our initial screen, including SOCS3, SPSB1, and TRIM5, can interact directly with SNAIL and that the knockdown of these E3s increased the steady-state level of SNAIL. However, overexpression of these three E3s did not accelerate SNAIL degradation. It is possible that these E3s are already expressed at relative high levels in 293T cells and that further overexpression of these three E3 ligases will not further enhance their activity. The other potential explanation is the off-target effect of siRNA knockdown. For these reasons, we only consider an E3 as a true candidate when it has strong effects on SNAIL protein degradation in both knockdown and overexpression experiments. Overall, our study serves as the proof of principle for adopting such a screening and validation strategy to effectively identify other E3 ligase-substrate pairs.

Functionally, FBXO11 blocks SNAIL-induced EMT in HMLEN cells, a commonly used breast cancer EMT model, by promoting SNAIL protein degradation. Although HMLEN-SNAIL cells become more mesenchymal-like with enhanced tumor-initiating properties, FBXO11 coexpression completely reverses these phenotypes. FBXO11 also prevents SNAIL-induced EMT and in vivo metastasis in the 4T1 and EpRas mammary tumor cell lines by ubiquitylating and degrading SNAIL protein. Knockdown of endogenous Fbxo11 also stabilizes SNAIL protein, induces EMT in 4T1 cells, and promotes lung metastasis. In line with its role as a negative regulator of the prometastatic protein SNAIL, higher FBXO11 expression correlates with longer metastasis-free survival in breast cancer patients. In our preliminary analysis, we did not find any correlation of FBXO11 expression with the breast cancer subtype using the publically available NKI295 data set, which has a limited sample size. However, future studies using larger data sets are needed to further analyze whether FBXO11 is correlated negatively with the Claudin-low subtype, in which the gene expression signature of tumor samples resembles that of normal and cancerous stem cells of the mammary gland.

Previous publications identified five kinases that can phosphorylate SNAIL protein. In our search, we found that PKD1-dependent phosphorylation of Serine-11 on the SNAG domain is the rate-limiting step for FBXO11-mediated SNAIL protein ubiquitylation and degradation. Mutation of the consensus PKD1 phosphorylation site on SNAIL, particularly Ser-11, blocks its phosphorylation by PKD1 and disrupts the interaction of SNAIL with FBXO11. Stabilized SNAIL-S11V protein promotes EMT in MCF7 breast cancer cell lines, whereas the wild-type SNAIL fails to do so. The SNAIL-FBXO11 complex is likely formed in the nucleus, where both p-Ser-11-SNAIL and FBXO11 are detected. Although p-Ser-11-SNAIL is most abundant in the nucleus, we cannot rule out the possibility that PKD1-dependent phosphorylation can still occur in the cytoplasm because many transcriptional factors are shuttling constantly between the nucleus and the cytoplasm.