RNF12 is regulated by AKT phosphorylation and promotes TGF-β driven breast cancer metastasis

Transforming growth factor-β (TGF-β) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-β signaling by binding to the inhibitory SMAD7 and inducing its proteasomal degradation. How RNF12 activity is regulated and its exact role in cancer is incompletely understood. Here we report that RNF12 was overexpressed in invasive breast cancers and its high expression correlated with poor prognosis. RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in zebrafish and murine xenograft models. RNF12 levels were positively associated with the phosphorylated AKT/protein kinase B (PKB) levels, and both displayed significant higher levels in the basal-like subtype compared with the levels in luminal-like subtype of breast cancer cells. Mechanistically, AKT-mediated phosphorylation induced the nuclear localization of RNF12, maintained its stability, and accelerated the degradation of SMAD7 mediated by RNF12. Furthermore, we demonstrated that RNF12 and AKT cooperated functionally in breast cancer cell migration. Notably, RNF12 expression strongly correlated with both phosphorylated AKT and phosphorylated SMAD2 levels in breast cancer tissues. Thus, our results uncovered RNF12 as an important determinant in the crosstalk between the TGF-β and AKT signaling pathways during breast cancer progression.


INTRODUCTION
Transforming growth factor-β, (TGF-β), plays a biphasic role in breast cancer progression by acting as a tumor suppressor during the early stages of cancer progression, and a tumor promoter during the late stages [1][2][3]. Although TGF-β elicits cytostatic responses in normal and pre-malignant breast cells, such responses are abrogated in malignant breast cells, wherein TGF-β stimulates the migration, invasion, and metastasis of malignant cells. TGF-β induces cellular responses by activating heteromeric complexes of TGF-β type I (TβRI) and type II (TβRII) receptors, respectively [4]. Upon TβRI phosphorylation by TβRII kinase, activated TβRI induces the phosphorylation of receptor-regulated (R)-SMAD2 and SMAD3, which form heteromeric complexes with the common mediator, SMAD4. These heteromeric SMAD complexes mediate transcriptional responses in collaboration with other DNA-binding transcription factors [5]. In breast cancer, mutations that inactivate TGF-β receptors and SMADs are not commonly found. Instead, TGF-β/ SMAD-induced growth inhibitory effects are diminished or redirected via the activation of oncogenes [6]. One such pathway is the phosphatidylinositol kinase (PI3K)/AKT pathway, which is strongly activated by growth factors such as the insulin growth factor (IGF), and mediates cellular survival [6,7]. An intricate interplay occurs between TGF-β/SMAD and PI3K/AKT pathways [8]. For example, AKT can sequester R-SMADs in the cytoplasm, thereby inhibiting TGFβ-induced apoptosis [9,10] and both pathways also enforce each other to promote the invasion of breast cancer cells [7,11].
Inhibitory SMAD7 is an important negative regulator of the TGFβ/SMAD pathway. It is rapidly induced by TGF-β, to participate in a negative feedback loop [12]. SMAD7 antagonizes TGF-β family signaling by recruiting SMURF E3 ubiquitin ligases to activated receptors and targeting them for degradation [13,14]. Recently, OTUD1 was identified as a deubiquitinase of SMAD7. OTUD1 acts as a tumor suppressor in breast cancer by mitigating TGFβ-induced pro-oncogenic responses by increasing SMAD7 levels [15]. ARKADIA (RNF111) [16] and RNF12 (RLIM) [11] have been identified as E3 ubiquitin ligases acting on SMAD7, which mediate ubiquitination and proteasomal degradation. These are negative regulators of SMAD7, and promote TGF-β/SMAD signaling [17]. ARKADIA is involved in the early stages of breast cancer metastasis [18]. RNF12/RLIM was originally identified as a protein that binds to LIM homeodomain (HD) proteins and targets a cofactor of LIM protein (CLIM) for degradation [19,20]. CLIM and RNF12 have been identified as opposing regulators of estrogen receptor (ER)dependent transcriptional activity in breast cancer [21]. RNF12 mitigates the transcriptional activity of LIM-HDs but promotes transcriptional activation of ER target genes, and may thereby contribute to the stimulation of breast cancer cell proliferation [21]. RNF12 increases the turnover of the telomeric protein, TTAGGG repeat binding factor 1 (TRF1). Depletion of RNF12 in HT1080 fibrosarcoma cells increases TRF1 levels and induces telomere shortening [22], thereby impairing cell growth. In addition, RNF12 negatively regulates c-Myc/MIZ1 transcriptional activity and inhibits hepatocellular carcinoma cell proliferation [23,24]. Moreover, RNF12 is an X-encoded, dose-dependent inducer of X chromosome inactivation (XCI) in mouse embryonic stem cells (ESCs) [25,26]. Subcellular distribution and function of RNF12 are regulated via Ser214 phosphorylation by Ser/Arg (SR)rich splicing factor (SRSF) protein kinase (SRPK) [27,28]. Together, the hitherto described data suggest a complex role for RNF12 in the regulation of multiple signaling pathways, where its role in cancer progression may be context-dependent. In this study, we show that RNF12 enhances the TGF-β signaling pathway and plays a role in breast cancer cell migration, invasion, and metastasis. Our findings may provide some insights for understanding the underlying mechanism for breast cancer metastasis.

RNF12 is critical for metastatic and invasive traits in breast cancer
To investigate the role of RNF12 in cancer progression, we analyzed the RNA expression levels of RNF12 in different cancer tissue samples in Oncomine. The RNA expression level of RNF12 was high in breast cancer compared with other types of cancer (Fig. 1A). Next, we determined the expression of RNF12 in a tissue microarray that included clinical samples from 16 different common cancers. RNF12 protein levels were nominally higher in breast, bladder, colon, thyroid cancer than in adjacent normal tissues (Fig. 1B), but the reverse was observed for kidney, lung, liver, brain, prostate, skin, pancreas and stomach cancer ( Supplementary Fig. S1). Interestingly, further in-depth analysis of the 45 breast samples included on the array indicated that the expression level of RNF12 protein was high in invasive breast cancer, moderate in non-invasive breast cancer, and low in adjacent normal breast tissue (Fig. 1C). These results suggested that RNF12 may play a specific role in the invasiveness of breast cancer. To obtain further clinical evidence for this hypothesis, we examined RNF12 expression in another larger panel, which included 175 breast cancer tissue samples (ORIGO) [29]. We found that RNF12 expression was higher in lymph node-positive breast cancer samples than in lymph node-negative breast cancer samples (Fig. 1D). Moreover, Kaplan-Meier analysis of 1302 breast cancer patients from Kaplan-Meier plotter dataset (http://kmplot.com/analysis) showed that RNF12 amplification was associated with a poor clinical outcome in lymph node-positive breast cancer but not in the lymph node-negative form, suggesting that high RNF12 levels are associated with worse prognosis in lymph nodepositive cancers. (Fig. 1E). Based on these results, we hypothesized that RNF12 drives breast cancer progression mainly by promoting invasive and metastatic traits.

RNF12 regulates breast tumor progression in vivo
To study the function of RNF12 in breast cancer progression, with particular reference to enhanced breast cancer migration and invasion, we silenced endogenous RNF12 expression in the highly aggressive basal MDA-MB-231 breast cancer cell line. We found depletion of RNF12 in MDA-MB-231 cells resulted in a significant decrease in cell invasiveness in the Transwell migration assay ( Fig. 2A). Next, we tested the role of RNF12 in malignant MDA-MB-231 breast cancer cells in vivo using a previously published zebrafish embryo xenograft breast cancer model [8]. Zebrafish embryos were injected into the Duct of Cuvier (Doc) with mCherry-labeled MDA-MB-231 cells. Circulating cancer cells extravasate into the tail fin, which is enriched with collagen ( Fig. 2B). Extravasation is a key step during the metastatic process. Embryos transplanted with RNF12-depleted MDA-MB-231 cells exhibited significantly less extravasation ability than embryos injected with control cells (Fig. 2B). Furthermore, we tested the role of RNF12 in an experimental breast cancer metastasis model using immunodeficient mice. RNF12-depleted MDA-MB-231 cells and control MDA-MB-231 cells were injected into the tail vein of female nude mice, respectively. We observed significant inhibition of MDA-MB-231 cells colonization in the lung when RNF12 expression was suppressed (Fig. 2C, Supplementary Fig. 2). We also constructed a stable MCF-7 cell line with ectopic expression of RNF12 ( Supplementary Fig. 3A). We found that overexpression of RNF12 in MCF-7 cells can enhance the migration and invasion of MCF-7 cells (Supplementary Fig. 3B-C). Together, these results indicated that RNF12 promotes breast cancer cell migration, invasion, and metastasis in vitro and in vivo.

AKT phosphorylates and stabilizes RNF12 protein
The above studies showed that RNF12 plays an important role in regulating TGF-β/SMAD signaling and breast cancer progression. To study the mechanism underlying the regulation of RNF12 activity, we first analyzed the RNF12 protein sequence. The primary amino acid sequence revealed that RNF12 contains two AKT consensus RxRxxS(T) phosphorylation motifs at Ser215 and Ser347, which are conserved in RNF12 orthologues (Fig. 3A). Next, we analyzed RNF12 protein levels in different breast cancer cell lines that we collected in previous study [37]. RNF12 was expressed at higher levels in the basal-like subtype than in the luminal-like subtype breast cancer cell lines (Fig. 3B). Interestingly, the expression levels of activated AKT (phosphorylated on Ser473; pAKT) in basal-like breast cancer cell lines were also significantly higher than that in luminal-like cell lines (Fig. 3B). Thereafter, we searched for a possible link between RNF12 levels and pAKT levels and observed a positive correlation (Fig. 3B).
Mechanistically, both exogenous and endogenous RNF12 interacted with the AKT protein (Fig. 3C). To examine whether RNF12 acts as a substrate for AKT, we used a phospho-specific antibody that recognizes the optimal AKT phosphorylation consensus motif. The AKT substrate antibody showed that IGF-1 induced phosphorylation of RNF12 wild-type (WT) protein in HEK 293 T cells, but no specific signal was detected with this antibody in the RNF12 double mutant proteins [2SA: RNF12 Ser215Ala (S215A) and Ser347Ala/ Thr349Ala (S347A/T349A)] (Fig. 3D). Both IGF-1 and TGF-β induced Y. Huang et al. phosphorylation of endogenous RNF12 was detected by the AKT substrate antibody (Fig. 3E). Notably, the protein level of RNF12 was significantly increased in cells treated with IGF-1, whereas this response was abrogated in cells treated with MK2206, a selective inhibitor of AKT [38] (Fig. 3E). Consistent with these findings, other results obtained from cycloheximide (CHX) pulse/chase experiments demonstrated that the half-life of RNF12 mutant S215A was shorter than that of RNF12 WT (Fig. 3F), while the stability of RNF12 S347A/T349A was comparable to that of RNF12 WT protein (Fig. 3F). In addition, we found the half-life of endogenous RNF12 protein was shorter when AKT activity was inhibited (Fig. 3G). These results suggested that AKT signaling stabilizes RNF12 protein mainly through the Ser215 site.
AKT signaling is critical for the nuclear localization and function of RNF12 It was reported that AKT played an important role in regulating the nuclear accumulation of its substrates [39]. Therefore, we tested the influence of RNF12 subcellular localization by AKT signaling. Consistent with previous study [28], we found that phosphorylation of Ser215 in RNF12 is needed for its nuclear localization in MDA-MB-231 cells. The RNF12 S215A mutant protein is mainly localized in the cytoplasm. Mutation of serine 347 to alanine (S347A) exerted little effect on RNF12 localization, which, similar to RNF12 WT, remained mainly in the nucleus (Fig. 4A). Moreover, when the subcellular distribution of endogenous RNF12 in MDA-MB-231 cells was analyzed in the absence or presence of AKT activation, it was found that AKT signaling was essential for nuclear localization of RNF12. This result was consistent with those of ectopic expression experiments. Challenging MDA-MB-231 cells with IGF-1 promoted the nuclear localization of RNF12, whereas the selective AKT kinase inhibitor, MK2206 [38], nearly abolished the nuclear localization of RNF12 in cultured cells (Fig. 4B). Next, we explored the effect of AKTinduced changes in the subcellular distribution of RNF12 and its effect on its substrate SMAD7. Inhibitory SMAD7 acts as a substrate for RNF12, which is consistent with a previous report [40], which indicated that when RNF12 and SMAD7 were coexpressed, RNF12 decreased SMAD7 expression levels (Fig. 4C). Under basal growth conditions, SMAD7 is predominantly localized in the nucleus [40]. Notably, we found that inhibition of AKT signaling stabilizes the RNF12 substrate SMAD7 protein. These results were also consistent with the relevant western blotting experiments in vitro (Fig. 4D). Taken together, these findings suggest that nuclear-localized AKT inhibits inhibitory SMAD7 levels and thereby potentiates TGF-β signaling.
AKT-mediated phosphorylation of RNF12 regulates its function in TGF-β signaling RNF12 is not a stable protein and undergoes ubiquitination [41]. RNF12 S215A was degraded faster than RNF12 WT (Fig. 3F). Consistent with this finding, RNF12 S215A demonstrated an increased level of conjugation with polyubiquitin chains in the presence of the proteasome inhibitor, MG132, when compared with RNF12 WT or S347A/T349A (Fig. 5A). These results suggested that S215 was the main site from which AKT signaling regulated RNF12 stability. Impaired protein half-life of RNF12 S215A was likely due to elevated ubiquitylation and proteasomal degradation.
In addition, we examined the effect of AKT on RNF12-mediated SMAD7 protein degradation. We found that knockdown RNF12 in MDA-MB-231 cells increased the protein levels of endogenous SMAD7 (Supplementary Fig. 5A) and RNF12 S215A attenuated the degradation of SMAD7 protein ( Supplementary Fig. 5B). Strikingly, the level of SMAD7 ubiquitination mediated by RNF12 was increased when stimulated using IGF-1 (which triggers AKT activation) but decreased when treated with the selective AKT kinase inhibitor, MK2206 (Fig. 5B). Furthermore, both RNF12 S215A and S347A/T349A mutations inhibited SMAD7 ubiquitination (Fig.  5C). The observation that S215A, and not S347A, affects RNF12 protein stability, indicated that the effect of AKT signaling on RNF12-mediated SMAD7 ubiquitination was not solely due to the regulation of RNF12 stability. Thus, we also detected an association between RNF12 phosphorylation site mutations and SMAD7 (Fig. 5D). We found that both S215A and S347A mitigated its association with SMAD7, with RNF12 S215A showing a more striking effect (Fig. 5D). This may be caused by the predominantly cytoplasmic localization of RNF12 S215A and the nuclear localization of SMAD7 in the absence of TGF-β stimulation [40]. We also found the interaction between endogenous RNF12 and SMAD7 proteins was also significantly attenuated by the AKT inhibitor MK2206 (Supplementary Fig. 5C). These results suggested that AKT-mediated RNF12 phosphorylation is required for RNF12 to relocate to the nucleus and interact with SMAD7.
Next, we analyzed the cooperation between AKT and TGF-β in regulating RNF12 and biological responses. TGF-β-induced migration of MDA-MB-231 cells was inhibited by RNF12 knockdown (Fig. 6A). This response was also inhibited by the selective AKT kinase inhibitor, MK2206 (Fig. 6A), which result was in line with previous reports that support a role for AKT signaling in TGFβ-induced migration and epithelial-mesenchymal transition (EMT) of breast cancer cells [8]. To further investigate the role of RNF12 in IGF-1/AKT signaling, we examined the effect of RNF12 depletion on IGF-1 induced migration of MDA-MB-231 cells. We found that depletion of RNF12 reduced IGF-1 induced cell migration (Fig. 6A,  B), suggesting that RNF12 plays a positive role in AKT-induced MDA-MB-231 migration. Immunoblotting showed that AKT and TGF-β signaling cooperated with each other to induce the expression of EMT-associated markers such as SLUG and SNAIL, which were decreased in RNF12-depleted cells (Fig. 6C). In addition, RNF12 depletion inhibited TGF-β or insulin-induced morphological phenotypes in MDA-MB 231 cells as measured by filamentous (F-) actin staining using phalloidin (Fig. 6D). This result suggests the possibility of the interplay between AKT signaling and RNF12 in the TGF-β pathway. To further support this, we reexamined the 175 clinical samples obtained from breast cancer patients. Notably, the expression of RNF12 was positively correlated with pAKT and pSMAD2 expression levels (Fig. 6E). All the above results indicate that AKT is important for the phosphorylation and nucleus localization of RNF12 in breast cancer cells, which further upregulate TGF-β signaling and promote breast cancer metastasis (Fig. 6F).

DISCUSSION
Aberrant AKT activation and the TGF-β pathway appear to cooperate in promoting breast cancer progression. However, the underlying mechanisms of the crosstalk between AKT and TGF-β pathways remain unclear. We found that the E3 ubiquitin ligase, RNF12, plays a pivotal role in the interplay between these two pathways. Our results suggest a model in which AKT activation by IGF induces RNF12 phosphorylation, which triggers its nuclear accumulation and increases its stability. Fig. 1 RNF12 expression in breast and other types of cancer. A Oncomine box plots of RNF12 mRNA levels in different types of cancer from public datasets (www.oncomine.org). B Statistics of RNF12 protein levels expressed in 499 specimens of different cancer tissues compared with the matched adjacent normal tissues. The two-tailed Student's t-test was used to determine the statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). C Representative images of RNF12 immunohistochemistry result in invasive breast cancer tissues and adjacent normal tissues. The intensity of RNF12 staining is shown in the right-hand panel. Scale bar = 300 μm. D Representative images of RNF12 immunohistochemistry results in lymph node-positive or lymph node-negative breast cancer tissues. The intensity of RNF12 staining in the 175 specimens of breast cancer (ORIGO) is shown in the right-hand panel. E RNF12 correlates with poor prognoses in lymph node-positive breast cancer patients. Kaplan-Meier curves (http://kmplot.com/analysis) show that metastasis-free survival of individuals was negatively correlated with RNF12 expression in lymph node-positive patients (bottom panel) but showed no correlation with RNF12 in lymph nodenegative patients (middle panel).
The phosphorylated RNF12 becomes more effective in inducing the ubiquitination and degradation of SMAD7, therefore, enforces TGF-β-induced invasion and metastasis responses in breast cancer cells (Fig. 6F).
We found that RNF12 is highly expressed in invasive breast cancer cells and its high level was associated with poor prognoses of breast cancer patients. We further observed that RNF12 expression was particularly high in lymph node-positive breast cancer cells, which demonstrate a high probability for breast cancer metastasis. Consistent with these findings, RNF12 was expressed at higher levels in more aggressive basal breast cancer cell lines than in luminal ones. In addition, RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in in vitro and in vivo models. RNF12 is a potent activator of TGF-β signaling [11], which play important role in triggering the invasive and metastatic traits of mesenchymal triple-negative breast cancer cell lines. Consistent with this notion and high RNF12 expression patterns seen in invasive breast cancer, we observed that RNF12 is needed for efficient induction of TGF-β of mesenchymal markers and TGF-β target genes in the malignant phenotypes of MDA-MB 231 cells. These findings hinted at a mechanistic basis for the role of RNF12 in breast cancer cell invasion and metastasis.
Moreover, a strong positive correlation was found between RNF12 and activated pAKT and pSMAD2 in clinical breast cancer samples. This prompted us to consider RNF12 as a possible substrate for AKT. Two potential substrates were identified: Ser215 and S347. We observed that AKT binds and phosphorylates (human) RNF12 at Ser215 and Ser347. Consistent with previous results [27,28], Ser215 was found to be important for nuclear accumulation. Moreover, it promoted the stability of RNF12 and its ability to ubiquitinate SMAD7. The latter result is in line with a recent report in which SRSFPK was shown to phosphorylate RNF12 at (mouse) Ser214, (and other serine residues) which ensured its delivery to nuclear substrates, such as SMAD7, increasing its E3 ubiquitin ligase activity thereby [27]. Our finding that AKT-induced phosphorylation increased the stability of RNF12, while simultaneously increasing ubiquitination activity was somewhat unexpected. RNF12 auto-ubiquitinates and targets itself for proteasomal degradation. Further studies are required to investigate the link between ubiquitination types to explain these findings. Our results demonstrated that S347A RNF12 phosphorylation at S347 is not involved in the regulation of subcellular distribution of RNF12, and only exerted a minor effect on RNF12 function.
Our findings indicated that RNF12 and AKT cooperated to promote breast cancer cell invasion and metastasis, in part by promoting prooncogenic responses to TGF-β. As TGF-β displays both tumorsuppressing and tumor-promoting activities, directly inhibiting TGF-β, via TGF-β receptor kinase inhibitors or antibodies that block ligandreceptor interactions, to treat cancer remains challenging [42]. Therefore, developing new drugs on RNF12 may provide potential avenue to treat breast cancer patients with high RNF12 expression. Furthermore, the combination therapy by targeting AKT, RNF12 and TGF-β in aggressive breast cancer can be studied in future research.

Patient selection
A high-density multiple organ tumor tissue microarray (TMA) with adjacent normal tissue was purchased from US Biomax (MC5002). The breast cancer TMA analyses were performed on TMAs that were constructed from 175 tumor samples from the ORIGO cohort [43]. Patients included in this cohort were diagnosed with a primary breast tumor and treated in the Leiden University Medical Center (LUMC) between 1997 and 2003. Informed consent was obtained from all patients.

Cell lines
The human kidney epithelial HEK293T cell line and the cervical cancer HeLa cell line, as well as breast cancer MDA-MB-231 cell lines, were originally obtained from the American Type Culture Collection (ATCC). All cell lines were cultured in DMEM supplemented with L-glutamine, 10% fetal bovine serum (FBS), and 1:100 penicillin/streptomycin (Gibco, Invitrogen, Blijswijk, Netherlands). All cell lines were regularly tested to ensure the absence of mycoplasma. In addition, the cell lines were authenticated and verified as free of contamination from other cell lines and microbes.

Lentiviral transduction and generation of stable cell lines
Lentiviruses were produced by transfecting HEK293T cells with shRNAtargeting plasmids and the helper plasmids, pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV. Cell supernatants were harvested 48 h after transfection and were used either to infect cells or stored at −80°C. To obtain stable cell lines, cells were infected at the low confluence (20%) for 24 h with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 5 ng/mL of polybrene (Sigma). Forty-eight hours following infection, cells were placed under puromycin selection for 1 week and then passaged before use. Puromycin (2 µg/mL) was used to maintain the MDA-MB-231 cell line. Lentiviral shRNAs were obtained from Sigma (MISSION® shRNA). Five shRNAs were identified and tested, and the two most effective shRNAs were used in the experiments. We used TRCN0000004139 (#1) and TRCN0000004142 (#2) to knock down RNF12. The MCF-7 stable cell line with ectopic expression of RNF12 was constructed by pLV-GFP-RNF12 lentivirus (Genechem Co.,Ltd., Shanghai).

Zebrafish xenograft invasion assay
The transgenic zebrafish line, Tg (fli1: GFP), was raised, staged, and maintained according to standard procedures and in compliance with the stipulations of the local Institutional Committee for Animal Welfare of the LUMC. MDA-MB-231 cells were injected into zebrafish embryos, as previously described [8]. Briefly,~400 mCherry MDA-MB-231 cells were injected into the duct of Cuvier (DoC). After implantation, zebrafish embryos were checked for correct injection, misinjected/non-viable embryos were discarded, and remaining embryos were maintained thereafter at 33°C. Cells that had extravasated from blood vessels and were present in the avascular tail fin area were analyzed for extravasation [8]. Two independent experiments with at least 30 embryos per group were performed.

Nude mice and experimental lung metastasis assay
Nude mice were purchased from the Animal Husbandry Centre of the Beijing Institute of Cell Biology, Academia Sinica, Beijing, China. All experiments were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Peking Union Medical College, Chinese Academy of Medical Science. For lung metastasis formation, 1 × 10 6 MDA-MB-231 cells stably expressing firefly luciferase were injected into the lateral tail vein of nude mice (n = 8 per group) at a volume of 0.1 mL [7]. Bioluminescence images were collected after 21-30 days using a Lumina II imaging system (PerkinElmer), which provides data in a color scale depicting the photon flux (photons per second) emitted from xenografted mice.

Ubiquitination assay
Cells were washed with phosphate buffered saline (PBS) and lysed in two pellet volumes of RIPA buffer (20 mM NAP, pH 7.4, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, and 1% SDS) supplemented with protease inhibitors and 10 mM N-ethylmaleimide. Lysates were sonicated, boiled at 95°C for 5 min, diluted in RIPA buffer containing 0.1% SDS, and centrifuged Fig. 3 AKT phosphorylates and stabilizes RNF12 protein. A Sequence alignment of the AKT phosphorylation site within RNF12 orthologues of different species and known AKT substrates, p27, p21, and SKP2 B RNF12 protein was expressed at higher levels in basal-like breast cancer cell lines. Immunoblot analysis of RNF12 and pAKT expression in different breast cancer cell lines; GAPDH was used as the loading control. Scatterplot showing the positive correlation between pAKT and RNF12 expression in breast cancer cell lines (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; Pearson's coefficient tests were performed to assess significance). C Co-immunoprecipitation of Flag-RNF12 and HA-AKT transfection in HEK293T cells as indicated (left panel). Endogenous interaction between RNF12 and AKT was detected in MDA-MB-231 cells (right panel). Cell lysates were subjected to immunoprecipitation with RNF12 antibody, followed by immunoblotting with an AKT antibody. D AKT phosphorylates RNF12 at Ser215 and 347 in vitro. Flag-RNF12 mutants were transfected into 293T cells, and phosphorylation was detected using a phospho-AKT substrate antibody that recognizes either the RXRXXpS(pT) or the R-KXR-KXXpS(pT) motif.

Transwell and wound-healing assay
For the Transwell assay, cells were plated in medium with 1% FBS in the upper chamber of a Transwell plate (Corning), while a medium containing 10% FBS was placed in the lower well. After incubation for the indicated time, cells were fixed with methanol for 15 min, stained with crystal violet dye for 15 min, washed, and photographed. Experiments were performed at least thrice. For the wound-healing assay, a confluent cell monolayer in a 12-well plate was wounded by directly scraping the cells with a yellow 20 μL pipette tip and treating with growth factors or inhibitors for the indicated time. Results were analyzed by measuring the migrating wound area and comparing it with the initial wound area. Three independent biological experiments were performed.

Immunohistochemical staining and evaluation
Primary antibodies against RNF12 (1:100; Abcam 22813), pAKT-Ser473 (1:100; Cell Signaling D7E), and pSMAD2-Ser465/467 (1:100; Millipore AB3849) were used for the immunohistochemical staining of formalin-fixed paraffin-embedded TMAs, according to previously described staining protocols [29]. Quantification of staining was expressed as an H score. The H score was determined by the formula 3 × the percentage of strongly stained cells + 2 × the percentage of moderately stained cells + 1 × the percentage of weakly stained cells, yielding a range of 0-300. Scoring was performed blindly by two independent investigators and taken the median.

Microscopy and analysis
Fixed cells were imaged using PBS-Tween. Fluorescent image acquisition was performed using a Leica MZ16FA stereo microscope or a Leica SP5 STED confocal microscope. Confocal stacks were processed for maximum intensity projection using the Leica software or Adobe Photoshop CS4 software. Images were adjusted for brightness and contrast using Adobe Photoshop CS4. Overlays were created using Adobe Photoshop CS4.

Statistical analysis
Statistical analyses were performed using Prism 7 software (GraphPad La Jolla, USA). Results are represented by the mean ± SD. Two-way analysis of variance (ANOVA) followed by the two-tailed Student's t-test was used. Statistical significance was set at p < 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6 AKT signaling increases TGF-β/RNF12 signaling and related metastasis. A Control and RNF12 stably depleted MDA-MB-231 cells were plated for the cell wound-healing assay. Sub-confluent MDA-MB-231 cells were wounded using a 20 µL pipette tip and treated with or without the indicated ligands and reagents for 16 h. The wound-healing area at 16 h after wounding was normalized to that at the start time. B Statistical analyses were performed with Student's t-test (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001). For the bar charts, data are plotted as the mean ± SD of at least three independent experiments. C Immunoblotting analysis of mesenchymal marker expression treated with IGF-1, TGF-β, and MK2206 as indicated. D RNF12 depletion inhibits AKT-and TGF-β-induced EMT in MDA-MB 231 cells. Cells were treated with TGFβ for 24 h. F-actin was stained with phalloidin conjugated with tetramethylrhodamine isothiocyanate (TRITC). E Representative images of RNF12 and matched pAKT and pSMAD2 immunohistochemistry results are shown. Graphs in the right panel show the correlation between RNF12 expression levels and those of pSMAD2 in breast cancer TMAs. Spearman's test was used, and the correlation coefficients and the twotailed significance values (p < 0.001) are shown. Scale bar = 300 μm. F Proposed model for the crosstalk between AKT signaling and RNF12 in breast cancer metastasis.