AKR1B1 promotes basal-like breast cancer progression by a positive feedback loop that activates the EMT program

Breast cancer is a heterogeneous disease with four major subtypes according to gene expression profiling: luminal A, luminal B, HER2, and basal like (Vargo-Gogola and Rosen, 2007). Compared with the other subtypes, basal-like breast cancer (BLBC) is associated with an aggressive clinical history, early recurrence, distant metastasis, and shorter survival (Kreike et al., 2007; Rakha et al., 2008). BLBC usually occurs in younger and premenopausal women and has a tendency toward early metastatic spread to the brain and lungs, sites known to be associated with a poor prognosis (Blick et al., 2008; Sarrió et al., 2008; Storci et al., 2008; Bergamaschi et al., 2009; Hennessy et al., 2009). A lack of effective targeted therapies for BLBC often results in a rapidly fatal clinical outcome. An important prerequisite for targeted therapies is to understand the molecular mechanisms underlying the metastatic process associated with BLBC. Notably, BLBC possesses the activated epithelial–mesenchymal transition (EMT) program, which is required for tumor progression and metastasis (Mani et al., 2008; Gupta et al., 2009; Polyak and Weinberg, 2009; Valastyan and Weinberg, 2011; Dong et al., 2013b).

EMT is a phenotypic conversion that facilitates embryonic development, wound healing, and cancer metastasis (Polyak and Weinberg, 2009; Valastyan and Weinberg, 2011; Dong et al., 2012; Ye and Weinberg, 2015). During EMT, epithelial cells lose their epithelial properties and acquire a motile mesenchymal phenotype (Polyak and Weinberg, 2009; Ye and Weinberg, 2015). Importantly, EMT causes the acquisition of cancer stem cell (CSC)–like properties (Mani et al., 2008; Gupta et al., 2009; Valastyan and Weinberg, 2011; Liu et al., 2015). As tumor cells that have undergone EMT often faced a harsh environment of foreign tissue during the metastatic process, they must rewire metabolic and oncogenic pathways to meet the demands of survival and metastasis. Characterization of the causal relationship between EMT and metabolic alteration will help to develop novel interventions for targeting metastatic breast cancer.

Metabolic reprograming has been accepted as a hallmark of cancer (Hanahan and Weinberg, 2011). Lipid metabolism, which represents an important source of bioactive molecules, is implicated in tumor progression (Tammali et al., 2011; Carracedo et al., 2013). Arachidonic acid (AA) is a polyunsaturated fatty acid that is metabolized into prostaglandins (PGs), hydroperoxyeicosatetraenoic acids, and epoxyeicosatrienoic acids through a series of enzyme-catalyzed reactions (Brash, 2001). AA-related enzymes and their metabolites are involved in pathophysiological processes of many diseases, including a variety of cancers (Brash, 2001; Tammali et al., 2011). Aldo-keto reductase 1 member B1 (AKR1B1), which catalyzes the reduction of PGH2 to PGF2α, is a major nicotinamide adenine dinucleotide phosphate reduced (NADPH)–dependent PGF synthase during AA metabolism. PGF2α, a major metabolite of AKR1B1, modulates cell adhesion, migration, and invasion via PGF2α receptor–mediated signaling in endometrial cancer (Sales et al., 2008), suggesting that AKR1B1 may be involved in tumor progression by modulating lipid metabolism. While investigating Twist2-mediated induction of EMT in BLBC, we unexpectedly identified that AKR1B1 expression is elevated by Twist2. AKR1B1 expression provides tumorigenic and metastatic advantage in BLBC through a positive regulatory feedback loop that activates the EMT program and enhances stem cell–like properties. Our study not only reveals a critical mechanism of how AKR1B1 contributes to EMT and metastasis, but also provides a potential target for the treatment of BLBC.

We recently showed that loss of fructose-1,6-biphosphatase (FBP1) by Snail-mediated repression provided metabolic advantages in BLBC (Dong et al., 2013b). To systematically identify other metabolic genes required for BLBC, we analyzed gene expression profiles of various subtypes of breast cancer in two publicly available cDNA microarray datasets (GEO accession no. GSE25066 and dataset NKI295), which contain 508 and 295 breast cancer patients, respectively (van de Vijver et al., 2002; Hatzis et al., 2011). Several known genes previously shown to have important roles in BLBC were observed to have significant differences between BLBC and other subtypes, such as lactate dehydrogenase B (LDHB) and FBP1. Notably, AKR1B1 expression was significantly higher in BLBC than in other subtypes, whereas expressions of other PGH2 reductases such as AKR1C3 and FAM213B did not have obvious changes in different subtypes (Fig. 1 A and Fig. S1 A). To confirm this finding, we collected fresh frozen breast tumor tissues from 9 cases of luminal and 21 cases of triple-negative breast cancer (triple-negative in the expression of ERα, PR, and HER2/neu; sometimes used as a surrogate term for basal like). Consistently, the expressions of AKR1B1 and Twist2 were elevated in triple-negative breast cancer, and their expressions were significantly down-regulated in the luminal subtype of breast cancers (Fig. 1 B and Fig. S1 B). To further extend these observations, we also analyzed AKR1B1 expression in another four gene expression datasets (GEO accession nos. GSE12777, GSE10890, and GSE16732; and ArrayExpress accession no. E-TABM-157), which contain 51, 52, 41, and 51 breast cancer cell lines, respectively (Neve et al., 2006; Hoeflich et al., 2009; Riaz et al., 2009). Similar to the observations in the GSE25066 and NKI295 datasets and in breast cancer tissues, AKR1B1 overexpression correlated with basal subtype of breast cancer cell lines (Fig. 1 C). Then, we confirmed these findings by either semiquantitative RT-PCR or quantitative real-time PCR in a representative panel of breast cancer cell lines that contained five luminal and five BLBC cell lines. Consistently, AKR1B1 mRNA expression was remarkably elevated in five BLBC cell lines (Fig. 1, D and E). We further examined the protein levels of AKR1B1 and EMT markers in these cell lines. BLBC cell lines lost the expression of epithelial molecule (E-cadherin) and contained high levels of mesenchymal markers (vimentin, N-cadherin, and Twist2). Importantly, AKR1B1 protein level also was high in BLBC cell lines, whereas it was absent in luminal cell lines (Fig. 1 F). Together, our data indicate that AKR1B1 overexpression is correlated with BLBC, underscoring the importance of characterizing potential function and mechanism of AKR1B1 for this subtype of breast cancer.

When investigating the correlation between AKR1B1 and BLBC, AKR1B1 and Twist2 displayed similar expression patterns in breast cancer cell lines and tissues (Fig. 1, B and F). When AKR1B1 expression was plotted against Twist2 expression in two microarray datasets (GSE10890 and GSE12777) that contain 52 and 51 breast cancer cell lines, respectively (Hoeflich et al., 2009), AKR1B1 expression positively correlated with Twist2 expression in both datasets (Fig. 2 A). A similar result was obtained in a dataset from The Cancer Genome Atlas (TCGA; 1,215-patient samples; Fig. S2 A). The positive correlation between AKR1B1 and Twist2 in breast cancer prompted us to investigate their mutually causal relationships. To this end, we established stable clones with empty vector or Twist2 expression in T47D cells. As anticipated, Twist2 expression substantially down-regulated E-cadherin expression and induced a morphological change indicative of EMT in T47D cells (Fig. 2 B). Intriguingly, Twist2 but not Twist1, Snail, or Slug expression remarkably promoted AKR1B1 expression in T47D cells (Fig. 2, B and C; and Fig. S2 B). Similar results were obtained in MCF7 cells with exogenous Twist2 expression (Fig. 2 C). We also generated stable transfectants with empty vector or knockdown of Twist2 expression in two BLBC cell lines (MDA-MB231 and SUM159). Strikingly, knockdown of Twist2 expression restored E-cadherin expression and down-regulated AKR1B1 expression (Fig. 2 C). These data suggest that Twist2 as a transcriptional factor may induce AKR1B1 expression via transcriptional regulation.

Given the immediate induction of AKR1B1 by Twist2 and their mutual correlation in breast cancer cell lines and tissues, we sought to determine whether AKR1B1 expression is regulated directly by Twist2. A survey of a DNA sequence in the AKR1B1 locus revealed that the proximal AKR1B1 promoter contains seven potential Twist2-binding E-boxes (CANNTG) from –1374 bp to the transcription start site (TSS; Fig. S2 C). To determine whether these E-boxes are crucial for Twist2-mediated gene transcription, we cloned the human AKR1B1 promoter and generated several deletion mutants of promoter–luciferase constructs based on the location of these E-boxes, including wtA (−1,374 bp), wtA1 (−1,020 bp), and wtA2 (−583 bp; Fig. S2 C). By expressing the wtA in T47D, MCF7, MDA-MB231, and SUM159 cells, Twist2 significantly promoted AKR1B1 promoter luciferase activity (Fig. 2 E). The wtA1 without the region between −1,374 and −1,021 bp still maintained high reporter activity induced by Twist2 (wtA vs. wtA1), whereas the wtA2 (−583 bp) lost the activity to respond to Twist2 expression (Fig. S2 C). Thus, the region between −1,020 and −584 bp might be important for Twist2-mediated AKR1B1 activation. To pinpoint the exact binding E-boxes, we introduced point mutations into E1 and/or E2. Mutation in E1 (mutA1) did not significantly reduce, whereas mutation in either E2 (mutA2) or both E-boxes (mutA3) almost completely abolished, Twist2-mediated activation of the AKR1B1 promoter luciferase (Fig. 2 F), suggesting that Twist2 activates the AKR1B1 promoter in an E-box–dependent fashion and that the E-box at −997 bp is required for Twist2-induced transcriptional activation. We also performed chromatin immunoprecipitation (ChIP) assay after wtA1 as well as its E-box mutants (mutA1, mutA2, and mutA3) were coexpressed with empty vector or Twist2 in HEK293 cells. As expected, mutation in either E2 (mutA2) or both E-boxes (mutA3) dramatically attenuated the binding of Twist2 to the AKR1B1 promoter, indicating the E-box at −997 bp is a critical Twist2-binding site (Fig. S2 D). Furthermore, we revealed that Twist2 directly bound to the AKR1B1 promoter in MDA-MB231 and SUM159 cells by ChIP assay (Fig. 2 G). These data indicate that AKR1B1 is a direct target of Twist2.

To study the molecular function and mechanism of AKR1B1, we established stable transfectants with empty vector or knockdown of AKR1B1 expression in MDA-MB231 and SUM159 cells and clones with empty vector or AKR1B1 expression in T47D and MCF7 cells (Fig. 3, A and C). Because AKR1B1 is a direct transcriptional target of Twist2 that plays critical roles in EMT and tumor metastasis, we first examined whether AKR1B1 was associated with EMT in these cells. Indeed, knockdown of AKR1B1 expression significantly caused an increase in E-cadherin expression, whereas ectopic AKR1B1 expression reversed the up-regulation of E-cadherin in MDA-MB231 and SUM159 cells with stable knockdown of AKR1B expression (Fig. 3 A and Fig. S3 A). Epalrestat, a specific inhibitor of AKR1B1, also restored E-cadherin expression in MDA-MB231 and SUM159 cells (Fig. 3 B). Conversely, in AKR1B1-expressing cells, E-cadherin expression was greatly down-regulated (Fig. 3 C). To further establish the functional relationship of AKR1B1 and EMT, we measured the E-cadherin promoter luciferase activity. We observed that knockdown of AKR1B1 expression or epalrestat reversed the suppression of the E-cadherin promoter in MDA-MB231 and SUM159 cells (Fig. 3, D and E; and Fig. S3 B), whereas AKR1B1 expression significantly decreased E-cadherin promoter luciferase activity in T47D and MCF7 cells (Fig. 3 F). Because suppression of E-cadherin can promote cell migration and invasion in the presence or absence of EMT (Onder et al., 2008) and because AKR1B1 is associated with suppression of E-cadherin, we hypothesized that AKR1B1 is critical for breast cancer cell migration and invasion. As expected, knockdown of AKR1B1 expression or epalrestat dramatically repressed the migration and invasion of MDA-MB231 and SUM159 cells in vitro (Fig. 3, G–J; and Fig. S3, C and D). Together, these observations indicate an important role for AKR1B1 in induction of EMT and acquisition of migratory and invasive ability in breast cancer cells.

To understand the metabolic consequence of AKR1B1 expression, we first measured the production of PGF2a, a major metabolite of AKR1B1 (Fig. 4 A). We noticed that knockdown of AKR1B1 expression or epalrestat significantly reduced PGF2a production in MDA-MB231 and SUM159 cells (Fig. 4 B and Fig. S4 A), whereas exogenous AKR1B1 expression increased PGF2a production in T47D and MCF7 cells (Fig. 4 C). These data indicate that AKR1B1 is required for increased PGF2a production in breast cancer cells. We speculated that AKR1B1 might promote breast cancer cell migration and invasion via the PGF2α-mediated pathway. As anticipated, PGF2a markedly induced the migration and invasion of MDA-MB231 and SUM159 cells in vitro (Fig. 4, D and E). AKR1B1, a monomeric NADPH-dependent cytosolic enzyme, leads to a decrease in the ratio of NADPH/NADP⁺ (Fig. 4 A). A decrease in the NADPH/NADP⁺ ratio could cause oxidative stress (Kinoshita et al., 1981; Srivastava et al., 2005; Gloire et al., 2006). This is consistent with our findings that knockdown of AKR1B1 expression caused a significant decrease of reactive oxygen species (ROS) levels in MDA-MB231 cells, whereas ectopic AKR1B1 expression induced an obvious increase of ROS in T47D cells (Fig. S4 B). Because NF-κB is a redox-sensitive transcription factor that has been proposed to be the sensor for ROS (Li and Karin, 1999; D’Angio and Finkelstein, 2000; Gloire et al., 2006), AKR1B1-mediated increase of ROS may involve NF-κB signaling. Additionally, growing evidence also suggests that AKR1B1 can activate NF-κB signaling via the PGF2a-mediated pathway (Ramana et al., 2004; Tammali et al., 2006). Indeed, knockdown of AKR1B1 expression caused a dramatic decrease of RelA level, whereas AKR1B1 expression led to a marked increase of RelA expression in both the nucleus and cytoplasm (Fig. 4 F and Fig. S4 C). To further confirm the functional relationship between AKR1B1 and NF-κB, we measured NF-κB reporter luciferase. Strikingly, knockdown of AKR1B1 expression decreased the activity of NF-κB reporter in MDA-MB231 and SUM159 cells (Fig. 4 G), whereas AKR1B1 expression enhanced NF-κB promoter luciferase activity in T47D and MCF7 cells (Fig. 4 H).

The NF-κB pathway has been implicated in the regulation of EMT (Huber et al., 2004; Zhou et al., 2004). To investigate the role of AKR1B1-mediated NF-κB signaling in EMT induction, we determined the expressions of RelA and various transcriptional factors of EMT by Western blotting. We observed that knockdown of AKR1B1 expression significantly decreased RelA and Twist2 expression but not Twist1, Snail, and Slug expression in MDA-MB231 and SUM159 cells, whereas AKR1B1 expression significantly increase RelA and Twist2 expression in T47D and MCF7 cells (Fig. 5 A). It is well known that TNF and IL-1β contribute to NF-κB activation. Indeed, RelA expression was induced by TNF or IL-1β in MDA-MB231 and SUM159 cells. Importantly, Twist2 expression also was dramatically up-regulated upon TNF or IL-1β stimulation in these cells (Fig. 5 B). These data suggest that Twist2 expression is positively correlated with RelA expression. To further investigate the relationship between RelA and Twist2, we established stable transfectants with empty vector or knockdown of RelA expression as well as stable clones with empty vector or RelA expression in MDA-MB231 and SUM159 cells. Strikingly, knockdown of RelA expression caused a decrease, whereas RelA expression resulted in an increase, in Twist2 expression (Fig. 5 C), indicating a critical role of RelA in regulating Twist2 expression.

To demonstrate whether Twist2 expression is regulated directly by RelA, four putative NF-κB–binding motifs were noticed in the proximal Twist2 promoter sequence from −222 bp to TSS (Fig. 5 D). We generated an AKR1B1 promoter–luciferase construct (wtT = TSS to −369 bp). By expressing the wtT in T47D, MDA-MB231, and HEK293 cells, a two- to fourfold increase in Twist2 promoter activity was observed in cells undergoing RelA overexpression (Fig. S4 D). To further define the elements inside the human Twist2 promoter that are involved in this activation, several point mutants were generated in the NF-κB–binding motifs (mutT1, mutT2, and mutT3; Fig. 5 D). Either the mutT1 or the mutT2, especially the latter, became less sensitive to, whereas the mutT3 with mutation of each NF-κB–binding motif almost completely lost, RelA-mediated activation of Twist2 promoter luciferase (Fig. 5 E), suggesting that all four of these consecutive NF-κB–binding motifs are important for RelA-mediated Twist2 activation. To further examine whether RelA directly binds to the Twist2 promoter, a ChIP assay was performed using MDA-MB231 and SUM159 cells. We detected a dramatic enrichment of RelA in the Twist2 promoter (Fig. 5 F). These data indicate that RelA up-regulates Twist2 expression by directly binding to the Twist2 promoter.

Many solid tumors contain a small population of highly tumorigenic CSCs, which contribute significantly to tumor initiation and metastasis. Emerging evidence has shown that the EMT program generates cells with stem cell–like properties (Polyak and Weinberg, 2009; Thiery et al., 2009; Nauseef and Henry, 2011). As AKR1B1 is highly expressed and involved in the EMT process in BLBC, we speculated that AKR1B1 expression might confer stem cell–like properties to BLBC cells. To test this notion, we examined tumorsphere formation of these cells. Strikingly, knockdown of AKR1B1 expression or epalrestat greatly suppressed tumorsphere formation in MDA-MB231 and SUM159 cells, whereas AKR1B1 expression in T47D cells enhanced tumorsphere formation (Fig. 6, A–C). As breast CSCs are enriched in cells with a CD44^high/CD24^low phenotype (Fillmore and Kuperwasser, 2007; Blick et al., 2010), we then examined the potential effect of AKR1B1 on cell population with CD44^high/CD24^low markers using flow cytometry analysis. Similar to the observation in tumorsphere formation, knockdown of AKR1B1 expression or epalrestat significantly reduced the percentage of CD44^high/CD24^low population in MDA-MB231 and SUM159 cells, whereas AKR1B1 expression induced a remarkable increase of CD44^high/CD24^low population in T47D cells (Fig. 6, D–H).

CSCs are highly tumorigenic and metastatic (Rosen and Jordan, 2009; Liu and Wicha, 2010; Badve and Nakshatri, 2012). First, we investigated AKR1B1’s role in cell growth. Knockdown of AKR1B1 expression or epalrestat did not cause an apparent effect on the growth of MDA-MB231 and SUM159 cells (Fig. S5, A and B). Then, we examined the effect of AKR1B1 expression on the in vitro tumorigenicity using soft agar assay. Knockdown of AKR1B1 expression or epalrestat caused a dramatic decrease of colonies in MDA-MB231 and SUM159 cells, whereas AKR1B1 expression led to a marked increase of colony formation in T47D cells (Fig. 7, A–C; and Fig. S3 E). We also tested the in vivo tumorigenicity in tumor xenograft experiments. Compared with control cells, MDA-MB231 cells with knockdown of AKR1B1 expression resulted in significantly reduced tumor growth (Fig. 7 D), whereas T47D cells with AKR1B1 expression caused dramatically enhanced tumor growth in vivo (Fig. 7 F). In addition, we examined the expression of AKR1B1, RelA, Twist2, and E-cadherin by Western blotting in tumor samples from a mouse model. Consistent with the analysis of cell lines, knockdown of AKR1B1 expression significantly caused an increase in E-cadherin expression and a decrease in RelA and Twist2 expression (Fig. S5 C), indicating that AKR1B1 regulates the EMT program by the same mechanism in vitro and in vivo. In line with these findings, mice given epalrestat exhibited substantially decreased tumor growth compared with control mice (Fig. S5 D). Then, we evaluated the effect of epalrestat treatment after tumor formation. Epalrestat was administered intragastrically 10 d after injection of MDA-MB-231 cells. Consistently, mice given epalrestat also exhibited significantly decreased tumor growth compared with control mice (Fig. 7 E). Next, we investigated whether inhibition of AKR1B1 expression affected tumor metastasis in a xenograft metastasis model in which MDA-MB231 cells were used to generate pulmonary metastases. Strikingly, knockdown of AKR1B1 expression or epalrestat greatly suppressed lung metastasis in vivo (Fig. 8, A and B). Collectively, these data suggest that AKR1B1 is critical for tumorigenicity and metastasis of BLBC cells, and pharmacologic inhibition of AKR1B1 can prevent tumorigenicity and metastasis of BLBC cells both in vitro and in vivo.

Having identified the critical function of AKR1B1 in breast cancer, we then sought to elucidate its clinical relevance by examining its correlation with patient survival in the NKI295 and GSE25066 datasets (van de Vijver et al., 2002; Hatzis et al., 2011). Patients were divided into two groups according to AKR1B1 expression, with high AKR1B1 expression having shorter survival in these patient cohorts (Fig. 8 C). Next, we evaluated whether AKR1B1 expression was associated with the metastatic sites in a dataset (GEO accession no. GSE12276) that contains 204 breast cancer patients (Bos et al., 2009). Consistent with the metastatic tendency of BLBC, primary tumors with high AKR1B1 expression preferentially metastasized to the brain and lungs (Fig. S5 E). These clinical validations strongly support the critical role of AKR1B1 in BLBC.

AKR1B1 facilitates the tumorigenic and metastatic capacity of BLBC cells through a positive regulatory feedback loop that activates the EMT program. Our study provides several mechanistic and therapeutic insights into the critical roles of AKR1B1 in EMT and BLBC.

The EMT program is a key event promoting carcinoma cell dissociation, invasion, and metastasis (Polyak and Weinberg, 2009; Ye and Weinberg, 2015). Loss of E-cadherin, which represents a hallmark of EMT, is often correlated with high tumor stage and metastasis (Thiery, 2009; Dong et al., 2013a). Several transcription factors, such as Snail, Twist1/2, and Zeb1/2, have been implicated in the transcriptional repression of E-cadherin and the induction of EMT (Yang et al., 2004; Kalluri and Weinberg, 2009; Thiery et al., 2009). Twist1 and Twist2 belong to highly conserved basic helix-loop-helix transcription factors that play pivotal roles in EMT and cancer metastasis (Šošić et al., 2003; Ansieau et al., 2010; Fang et al., 2011). Given the similarities of these two genes in sequence and function, a major unsolved question is whether Twist2 possesses the unique molecular targets and cellular functions. The findings that a frameshift mutation in the Twist2 gene causes Setleis syndrome and mutations in the Twist1 gene leads to Saethre-Chotzen syndrome exhibit different functions of two genes in bone and skin development (Chen and Behringer, 1995). In addition, the transcriptional expression pattern of Twist2 differs from that of Twist1 in various tumors (Ansieau et al., 2010). These observations suggest that both Twist2 and Twist1 have overlapping but distinct functions. Here, our finding revealed that Twist2 functions as a transcriptional activator to directly induce the transcription of AKR1B1 in contrast to Twist1, indicating that Twist2 has unique functions. Gene deletion experiments have shown that Twist2-knockout mice display disturbance of proinflammatory cytokines causing perinatal death (Šošić et al., 2003). However, the mechanism underlying the links between Twist2 and inflammation remains relatively unknown. Our data suggest that Twist2 may regulate inflammatory changes by direct transcriptional activation of AKR1B1 that has been implicated in the development of various inflammatory diseases and cancers.

It has been documented that AKR1B1 modulates NF-κB signaling via the PGF2a-mediated pathway (Ramana et al., 2004; Tammali et al., 2006). Consistent with this notion, AKR1B1 expression elevated PGF2a synthesis and RelA expression, indicating that AKR1B1 is critical part of a cellular program promoting PGF2a pathway flux, thus triggering NF-κB activation in BLBC. Activation of NF-κB is tightly associated with EMT induction (Huber et al., 2004; Zhou et al., 2004). Interestingly, we observed that only Twist2 expression was significantly up-regulated in cells with AKR1B1 overexpression, suggesting the involvement of AKR1B1 in Twist2 expression and EMT induction via the NF-κB signaling pathway. Indeed, our data identified that RelA is a transcriptional activator responsible for Twist2 expression, thus controlling the EMT program in BLBC.

Together, we demonstrate that AKR1B1 regulates the EMT program through a Twist2-mediated positive feedback regulatory loop. In BLBC cells, AKR1B1 expression is transcriptionally up-regulated by Twist2, leading to NF-κB activation via the PGF2a-mediated pathway. NF-κB, in turn, elevates Twist2 expression, which induces EMT, thus bridging AKR1B1 and the EMT program and fulfilling a positive feedback loop in BLBC (Fig. 8 D).

BLBC is defined by expression of markers characteristic of basal/myoepithelial cells and identified as a subtype of breast cancer that may originate from undifferentiated stem cells (termed basal-like cells in the normal breast mammary gland; Polyak, 2011). Consistently, BLBC possesses more CSC and EMT characteristics than the other subtypes of breast cancer (Blick et al., 2008, 2010; Honeth et al., 2008; Sarrió et al., 2008; Storci et al., 2008; Bergamaschi et al., 2009; Hennessy et al., 2009). Elegant studies have showed that EMT is a cellular dedifferentiation program that confers tumor cells with CSC properties (Polyak and Weinberg, 2009; Thiery et al., 2009; Nauseef and Henry, 2011). In line with this notion, AKR1B1 expression leads to increased CSC properties by a positive feedback loop that activates the EMT program in BLBC, indicating that AKR1B1 is critical in controlling the viability of CSCs. Extensive studies have demonstrated that CSCs are essential for tumor initiation, recurrence, and metastasis (Rosen and Jordan, 2009; Liu and Wicha, 2010; Badve and Nakshatri, 2012). Indeed, our experiments showed that AKR1B1 expression in luminal cells increased tumorigenicity, whereas knockdown of AKR1B1 expression in BLBC cells suppressed tumorigenicity and metastasis in vitro and in vivo. Clinically, high AKR1B1 expression occurs specifically in BLBC and predicts poor relapse-free survival. Furthermore, AKR1B1 overexpression is also highly correlated with metastatic dissemination to the brain and lungs, which is completely in concordance with the metastatic propensity of BLBC. Clearly, our study suggests that AKR1B1 overexpression represents an oncogenic event that is responsible for the aggressive behaviors of BLBC cells.

The treatment of BLBC is an unmet medical need because of the absence of effective targeted therapies and poor response to standard chemotherapy. Therefore, identification of the relevant molecular targets in BLBC may facilitate the development of effective tailored therapies to improve patient outcomes. Our study identified the crucial roles of AKR1B1 in EMT and BLBC and showed a tight correlation between AKR1B1 and Twist2 in breast cancer cells. Twist2, as a transcription factor, is difficult to target therapeutically because of the absence of a clear ligand-binding domain. As a downstream target of Twist2, AKR1B1 might be a potentially valuable target for therapeutics against this metastatic disease. Epalrestat is the only AKR1B1 inhibitor that has been approved in Japan for the targeted treatment of diabetic complications, which is easily absorbed by tissues and has minimum adverse effects (Hotta et al., 2006). Intriguingly, epalrestat significantly attenuated the viability of CSCs within breast cancer cell populations. This finding may be especially important because traditional therapies often leave behind the minority CSCs that regenerate tumors (Gupta et al., 2009). Thus, it is conceivable that epalrestat can suppress tumor progression. Indeed, epalrestat dramatically inhibited cancer cell migration and invasion in vitro and suppressed tumorigenicity and metastasis of BLBC cells in mice models, displaying potent efficacy against BLBC.

As the safety profiles of marketed drugs are generally known, drug repositioning has been regarded as an efficient approach to drug discovery and a way to identify new treatment for other diseases (Mizushima, 2011). Epalrestat is generally safe in clinical trials at oral dosages of 50–200 mg three times a day for the targeted treatment of diabetic complications, and the plasma epalrestat concentration of 12 µM is reached 1 h after a single oral dose of 50 mg (Uchida et al., 1995; Ito et al., 2009). Significantly, a clinically achievable dose of epalrestat exhibits apparent inhibitory effect on BLBC progression using cellular and mice models. Because epalrestat is already on the market without any major toxicity, our study provides proof of principle to suggest that epalrestat has the potential to become a valuable targeted drug in clinical treatment of BLBC.

AKR1B1 shRNA was purchased from MISSION shRNA (Sigma-Aldrich). Human AKR1B1 was amplified from a HeLa cDNA library and subcloned into pLenti6.3/V5. The pBABE-Twist2 expression plasmid was provided by G. Ouyang (Xiamen University, Xiamen, China).

Antibodies against AKR1B1 and β-actin were purchased from Sigma-Aldrich. Antibodies against Vimentin and Twist2 were from Neomarkers and Abnova, respectively. Antibodies for Snail and Slug were from Cell Signaling Technology. Antibody for IKBα and RelA was from Santa Cruz Biotechnology, Inc. N-cadherin and E-cadherin antibodies were from EMD Millipore and BD, respectively.

MDA-MB231, SUM159, and MCF7 cells were grown in DMEM/F12 supplemented with 10% FBS. T47D and ZR75 cells were grown in RPMI 1640 plus 10% FBS. SUM1315 cells were cultured in DMEM/F12 supplemented with 5% FBS, 5 µg/ml insulin, and 10 ng/ml epidermal growth factor. For establishing stable transfectants with AKR1B1 expression or knockdown of AKR1B1 expression, BLBC cells and luminal cells were transfected with pLenti6.3/V5-AKR1B1 and AKR1B1 shRNA, respectively. Stable clones were selected with 300 ng/ml puromycin for 4 wk.

Experiments were performed as described previously (Dong et al., 2013b). For immunofluorescent staining, cells were grown on chamber slides, fixed with 4% paraformaldehyde, and treated with primary antibodies. Secondary antibodies were Texas red–conjugated goat anti–mouse or FITC-conjugated goat anti–rabbit (Molecular Probes).

Total RNA was isolated using an RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Specific quantitative real-time PCR experiments were performed using SYBR Green Power Master Mix following the manufacturer’s protocol (Applied Biosystems).

Luciferase reporter assay was performed as described previously (Dong et al., 2012). Cell extracts were prepared 48 h after transfection, and luciferase activity was determined by the Dual-Luciferase Reporter Assay system (Promega). All experiments were performed three times in triplicate.

ChIP assays were performed as described previously (Lin et al., 2010; Dong et al., 2012). Two pairs of primers were used for ChIP assays: 5′-TGGAGTTCCCAATCTTGCAT-3′ and 5′-GATCACCCTAATTGGCAGGT-3′ for the AKR1B1 promoter and 5′-AAGACCCACACAGGTCGAG-3′ and 5′-GTTCTCTCTCTGGGCGTGA-3′ for the Twist2 promoter. The cells were prepared to perform ChIP assay with the Imprint ChIP kit (Sigma-Aldrich) according to the manufacturer’s instructions as described recently (Dong et al., 2012).

The concentration of PGF2a was detected using commercially available enzyme-linked immunosorbent assay kits (Enzo Life Sciences). NADPH levels were tested by an NADPH assay kit (BioVision). Cellular ROS level was measured by incubating cells with DCFH-DA (Jiancheng Bioengineering Institute) at manufacture-recommended concentration for 1 h before analysis in a spectrophotometer (SynergyMx M5 Microplate Reader; Molecular Devices).

Colony formation assay was performed using double-layer soft agar in 24-well plates with a top layer of 0.35% agar and a bottom layer of 0.7% agar. Cells were seeded into 24-well plates in the desired medium and cultured at 37°C for 14–21 d, and the colonies were stained and counted.

Migration and invasion assays were performed in Boyden chambers as described previously (Dong et al., 2013a). The cancer cells were stained and counted with a microscope. All experiments were performed at least twice in triplicate. Statistical analysis was performed using Student’s t test; a p-value of <0.05 was considered significant.

Mammosphere assays were performed following the protocol previously described (Grimshaw et al., 2008) by plating single-cell suspensions into ultralow-attachment 6-well plates (Corning) in mammosphere culturing conditions and counting after 7–14 d.

For the detection of the CD44^high/CD24^low population, cells were incubated in a volume of 100 µl with 2 µl of each monoclonal antibody: CD44-PE/Cy7 and CD24-PE (eBioscience). Cells were treated on ice for 30 min and washed twice before analysis in the cytometer (Cytomic FC 500MCL; Beckman Coulter).

Animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee at Zhejiang University. To examine the effect of AKR1B1 on tumorigenesis, female SCID mice (6–8 wk old) were injected with 10⁶ exogenous AKR1B1-expressing or knockdown cells on the left flank and vector control cells on the right flank. To evaluate the drug efficacy, 50 mg/kg/d epalrestat was administered intragastrically after injection of MDA-MB-231 cells on the left flank of every mouse, and sterile water was used as the vehicle control. Tumor formation was examined every 2–4 d for a 4-wk period. Tumors’ size and weight were measured. To test the effect of AKR1B1 on tumor metastasis, female SCID mice were injected with MDA-MB-231 cells (10⁶ cells/mouse) with stable empty vector or knockdown of AKR1B1 expression via tail vein (six mice per group). For evaluation of epalrestat, mice were injected with MDA-MB-231 cells (10⁶ cells/mouse) via tail vein (six mice per group) and then received 50 mg/kg/d epalrestat or sterile water intragastrically. After 4 wk, lung metastasis was detected using an imagining system (IVIS-100; Xenogen). Mice were also sacrificed; lung metastatic nodules were detected in paraffin-embedded sections stained with hematoxylin and eosin. Data were analyzed using Student’s t test; a p-value <0.05 was considered significant.

Results are expressed as mean + SD or SEM, as indicated. Comparisons were made using two-tailed Student’s t test or one-way ANOVA. Correlations between AKR1B1 and Twist2 were analyzed using Pearson’s correlation method and Spearman’s rank correlation test. Survival curves were analyzed using the Kaplan-Meier method, and differences were performed with the log-rank test. In all statistical tests, P < 0.05 was considered statistically significant.

Fig. S1 shows AKR1B1 is up-regulated in BLBC. Fig. S2 shows AKR1B1 is a target of Twist2. Fig. S3 shows AKR1B1 is associated with migration, invasion, and colony formation of breast cancer cells. Fig. S4 shows AKR1B1-mediated NF-κB activation induces Twist2 expression. Fig. S5 shows silencing of AKR1B1 inhibits breast tumor growth and metastasis.

Information about TCGA, the investigators, and institutions that constitute TCGA research network can be found at http://cancergenome.nih.gov/.

This work was supported by grants from the National Natural Science Foundation of China (81472455 to C. Dong and 81370730 and 81571512 to Q. Fu), the Key Program of the Natural Science Foundation of Zhejiang Province (LZ17H160002 to C. Dong), the Major Research Development Program of China (2016YFC1303200 to C. Dong), the Key Program of the Natural Science Foundation of Shandong Province (ZR2015JL027 to Q. Fu), Fundamental Research Funds for Central Universities of China (C. Dong), and the Thousand Young Talents Plan of China (C. Dong).

The authors declare no competing financial interests.

Author contributions: X. Wu, X. Li, Q. Fu, and C. Dong initiated the research, designed experiments, and analyzed data. X. Wu carried out the immunostaining, ChIP assay, colony formation assay, migration and invasion assays, enzyme-linked immunosorbent assay, and Western blot experiments. X. Li designed and conducted the tumorigenesis and lung metastasis experiments. Q. Fu performed the CSC-related work. Q. Cao and M. Wang generated most of the DNA constructs and performed the luciferase reporter assay. X. Chen performed the semiquantitative RT-PCR and quantitative real-time PCR. J. Yu and J. Yao performed the bioinformatic analysis. J. Long analyzed AKR1B1 expression in breast tumor tissues by Western blotting. H. Liu established stable clones with knockdown of RelA expression. D. Wang generated the Twist2 plasmids. R. Liao performed the ROS assay. C. Dong supervised the work and wrote the manuscript.