Solid tumors start as a local disease, but some are capable of metastasizing to the lymph nodes and distant organs. The hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating cancer progression and metastasis. However, the molecular mechanisms mediating the disseminated cancer cell metastasis remain incompletely understood. Here, we show that C/EBPβ/AEP signaling that is upregulated in breast cancers mediates oxidative stress and lung metastasis, and inactivation of asparagine endopeptidase (AEP, also known as legumain) robustly regulates breast cancer reactive oxygen species (ROS) and metastasis. AEP, a protease activated in acidic conditions, is overexpressed in numerous types of cancer and promotes metastasis. Employing a breast cancer cell line MDA-MD-231, we show that C/EBPβ, an oxidative stress or inflammation-activated transcription factor, and its downstream target AEP mediate ROS production as well as migration and invasion in cancer cells. Deficiency of AEP in the MMTV-PyMT transgenic breast cancer mouse model significantly regulates oxidative stress and suppresses lung metastasis. Administration of an innovative AEP inhibitor substantially mitigates ROS production and cancer metastasis. Hence, our study demonstrates that pharmacologic inhibition of AEP activity might provide a disease-modifying strategy to suppress cancer metastasis.

Metastatic dissemination of cancer cells is the primary cause of death of patients with cancer and the elucidation of the genetic pathways behind this phenomenon is a central focus of cancer science (1). Death and most complications associated with breast cancer are caused by the dissemination of cancer cells to the lymph nodes and distant organs, including bone, lung, liver, and brain (1, 2). Breast cancers can be categorized into various molecular subtypes based on receptor expression, including luminal-A, luminal-B, HER-2-enriched (ErbB2+) and triple-negative breast cancer (TNBC; ref. 3). TNBCs are the most aggressive form of breast cancers that do not express estrogen, progesterone, or HER2 receptors and have high rates of metastasis and poor survival. The etiology of TNBCs is multifactorial. Oxidative stress, which is responsible for protein, lipid and DNA damage, may play an important role in the development and progression of TNBCs (4). The production of reactive oxygen species (ROS) leads to an imbalance between ROS generation and degradation by cellular antioxidant mechanisms, and the deregulation incurs oxidative stress (4). However, the role of oxidative stress in the progression of TNBCs is not fully understood.

CCAAT/enhancer-binding protein β (CEBPβ) is one of seven members of the C/EBP subfamily of bZIP transcription factors. There are at least three isoforms considered to be N-terminally truncated: 38-kDa, 35-kDa and 21-kDa (5, 6). C/EBPβ transcriptional activity is believed to lead to cellular energy metabolism, cell proliferation and differentiation (7, 8). C/EBPβ also plays a role in inflammation (9). Interestingly, enhanced production of C/EBPβ has been detected in breast cancer, ovarian tumors and colorectal tumors (10–13); C/EBPβ-/- mice, on the other hand, are refractory to tumorigenesis (14), confirming the essential function of C/EBPβ in mediating cancer progression. C/EBPβ, a ROS-sensitive transcription factor, has recently been reported to mediate the expression of antioxidative reductases of NQO1 and GSTP1 in glioblastoma, facilitating brain tumor proliferation (15). During tumorigenesis, CEBPβ is often triggered by mutation or gene amplification, causing the mammary epithelium to extend, proliferate, and invade adjacent tissue (16).

Recently, we have reported that CEBPβ acts as a crucial transcription factor for asparaginyl endopeptidase (AEP, also called legumain or lgmn) and regulates the mRNA transcription and protein expression in an age-dependent manner (17). AEP is an endoprotease of lysosomal cysteine and is the only mammalian enzyme that cleaves C to asparagine residues terminally (18). There are higher levels of AEP in sera in patients with breast cancer relative to healthy control subjects. Serum AEP also facilitates tumor invasion and metastasis, while removing AEP activity by inhibitors or antibodies reduces tumor metastasis in mice (19). Hence, elevated serum AEP level is closely related to mammary cancer progression and metastasis, and AEP is a potential target for breast cancer therapy (19). As a result, the aberrant expression of AEP in cancer cells has rendered the enzyme a prodrug therapy target (20). For example, an α(v)β(3) integrin inhibitor is attached to a peptide-containing asparagine to ensure that AEP is selectively triggered in acidic tumors and tumor microenvironments by the prodrug (21). The demonstrated effectiveness of AEP-targeting prodrugs validates the high degree of tumor tissue selectivity that AEP-specific compounds can achieve, thus minimizing possible side effects and toxicity (22). Recently, we reported that a small molecular AEP inhibitor No. 38u, identified from a high-throughput screen, blocks MMP-2 cleavage by AEP and suppresses lung metastasis of breast cancer cells inoculated in mammary fat pads of nude mice (23). In the current research, we explore the C/EBPβ/AEP signaling pathway in breast cancer metastasis using the transgenic breast cancer mouse model. Specifically, we employed MMTV-PyMT transgenic mice to assess the pathologic role of this pathway in breast cancer metastasis to the lung. The blockade of AEP with the newly identified small-molecule inhibitor robustly inhibits AEP protease activities and suppresses breast cancer metastasis in vivo.

Cell lines and cell culture

The human breast cancer cell line MDA-MB-231 was authenticated and purchased from the ATCC (RRID: CVCL_0062). All cell lines used were regularly tested negative for Mycoplasma contamination throughout the duration of this study. In DMEM, 10% FBS (Hyclone), penicillin (100 U/mL), and streptomycin (100 U/mL; ABAM Life Technologies) were applied to the cells in a humidified incubator with 5% CO2 at 37°C (24).

Quantification of cell numbers

MDA-MB-231 cells were seeded (5 × 103) in 96-well plates in a regular growth medium for up to 5 days. At regular intervals, 10 μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL in PBS, Sigma) was added to each well followed by incubation at 37°C for 3 hours. DMSO dissolved the resulting formazan product and the absorbance at 490 nm was measured using a microplate reader (BioTek Instruments Inc.; ref. 25).

Measurement of the formation of reactive species of oxygen

After indicated treatments, cells were collected and analyzed with a Cellular ROS Assay Kit (Red; No. ab186027, Abcam) according to the manufacturer's instructions. After 1-hour incubation at 37°C, fluorescence intensity was measured with a microplate reader at an excitation/emission of 520/605 nm (BioTek Instruments Inc.).

Lactate dehydrogenase release

Cytotoxicity was measured using lactate dehydrogenase (LDH) assay kits (Promega Corporation). Briefly, after collecting supernatants from cells treated with the indicated agents, 50 μL of every well was applied to the LDH detection reagent. Fifty milliliters of Stop Solution was applied to each well after 1 hour of incubation at room temperature. Finally, a microplate reader registered the absorbance at 490 nm (BioTek Instruments Inc.).

Protein carbonyl assay

After collecting the cell lysates, protein carbonyl levels were detected using a Protein Carbonyl Assay Kit (No. ab126287, Abcam) and measuring the measured OD at 375 nm in a microplate reader (BioTek Instruments Inc.).

AEP activity assay

MDA-MB-231 cell lysates or recombinant proteins are incubated in a 200-μL assay buffer (20 mmol/L citric acid, 60 mmol/L Na2HPO4, 1 mmol/L EDTA, 0.1% CHAPS, 1 mmol/L DTT, pH 6.0) containing 20 μmol/L AEP substrate Z-Ala-Ala-Asn-AMC) following the specified treatments (Bachem). By measuring every 10 minutes at 360 nm and 460 nm in a fluorescence plate reader at 37°C in kinetic mode for a total of 1 hour, the AMC released by substrate cleavage was quantified.

Migration and invasion assays

A total of 1 × 104 cells containing 8.0-μm Clear PET Membrane for migration assays were seeded into the top well of a transwell chamber (No. 353097, Corning). The top well was applied to the serum-free mix, and the bottom well was added to a medium containing 10% FBS. The filters were stained with crystal violet after the indicated treatments and incubation at 37°C for 48 hours with 5% CO2. Using a light microscope, five random fields were counted per chamber. The top surface of the polycarbonate membranes was coated with 50-μL Matrigel for invasion assays (No. 354234, Corning), and studied in the same way as the migration assay.

Western blot analysis

Cells were collected after the specified therapies and total proteins were extracted. In our previous research, Western blot assays were carried out as mentioned. The SDS-PAGE gels were filled with equivalent quantities of protein and then moved to a polyvinylidene difluoride membrane. Main antibodies were used for the following targets: C/EBPβ (No. 7964, Santa Cruz Biotechnology); AEP (No. 93627, Cell Signaling Technology); SRPK2 (No. sc-136078, Santa Cruz Biotechnology); MMP2 (No. sc-13595, Santa Cruz Biotechnology); MMP9 (No. sc-13520, Santa Cruz Biotechnology); NQO1 (No. 3187, Cell Signaling Technology); GSTP1 (No. 3369, Cell Signaling Technology); and β-actin (No. 3700, Cell Signaling Technology).

Cell transfection and infection

siRNAs and overexpressing plasmids from Santa Cruz Biotechnology were collected. Using Lipofectamine 3000 and P3000 (No. L3000075, Invitrogen) under the manufacturer's instructions, cells were transfected with 20-nmol/L siRNA or 2-μg plasmids.

Gelatin zymography assay

The samples were dissolved in the sample buffer and electrophoresed in 8% polyacrylamide SDS gels copolymerized with gelatin (1 mg/mL). Following electrophoresis, gels were washed in 100-mL renaturing solution (25% v/v Triton X-100 in dH2O.) and then incubated with 100-mL developing buffer (0.5 mol/L Tris–HCl, pH 7.8, 2 mol/L NaCl, 0.05 mol/L CaCl2, and 0.2% Brij 35) at 37°C for approximately 24 hours in a closed tray. Gels were then dyed for at least 1 hour in Coomassie-blue solution or until the gel was evenly dark blue. Enzyme-digested regions were detected as white bands against a blue background.

LE28 staining

MDA-MB-231 cells were pretreated with the indicated concentrations of AEP inhibitors for 48 hours, followed by the addition of 1 μmol/L LE28 for 5 hours and imaging Cy5 fluorescence with a fluorescent microscope (ref. 26; Lionheart, Biotek).

In vivo mouse model experiments

Animals were maintained and managed in compliance with procedures accepted at Emory University by the Institutional Animal Care and Use Committee (IACUC). The 8-week-old MMTV-PyMT female mice were obtained from The Jackson Laboratory. The LE28 probe via tail vein was injected at a dose of 20 nmol in 20% DMSO/PBS. Six hours later, mice were imaged for Cy5 fluorescence using the IVIS 100 system. Primary tumors and lungs were then removed and imaged ex vivo using the IVIS 100 system. Lungs were then washed and fixed with Bouin's solution for 24 hours. The number of tumor nodules on the whole surface of the lungs was counted using a dissecting microscope. The body weight of each mouse was assessed every 7 days. MMTV-PyMT mice were treated with 10 mg/kg of CP6 or control vehicle by intraperitoneal (i.p.) injection every day for 3 months (n = 8 mice per group). Total blood from three mice per group was collected in EDTA tubes before harvest to perform hematology and blood chemistry testing at the Quality Assurance & Diagnostic Lab, Division of Animal Resources, Emory University, Atlanta, GA.

Hematoxylin and eosin staining and IHC

MMTV-PyMT mice primary tumors and metastatic organs were fixed overnight in 10% of formalin and embedded in paraffin. To detect any histologic changes in primary tumors, lung metastasis, and other organs, sections were stained with H&E. The paraffin-embedded samples have been stained using IHC antibodies Ki67 (No. 550609, BD Biosciences), 4-HNE (No. 46545, Abcam), EGFR(No. sc-373746, Santa Cruz Biotechnology) and RSU1 (No. 11207-1-AP, ProteinTech) using a previously recorded technique. The microscope was used to take images (Olympus, Japan).

Bioinformatic analysis

TCGA data portal (http://cancergenome.nih.gov/dataportal/data/about), UALCAN (http://ualcan.path.uab.edu/index.html) (27), GEPIA (http://gepia.cancer-pku.cn/index.html), Kaplan–Meier Plotter (http://gliovis.bioinfo.cnio.es) and Gene Set Enrichment Analysis (GESA; ref. 28) were used for bioinformatics data analysis.

Statistical analysis

Data visualization and analysis were performed with GraphPad Prism 6 (GraphPad Software Inc.). The analysis was performed using either Student t test or one-way ANOVA. Significant difference among groups was assessed as *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Ethics approval and consent to participate

This study has been conducted in accordance with ethical standards and according to the Declaration of Helsinki and the national and international guidelines, and has been approved by Emory University.

CEBPβ/AEP signaling is overexpressed in TNBCs and can serve as a prognostic biomarker

We evaluated a dataset accessible through The Cancer Genome Atlas (TCGA) with expression and clinical patient data to examine the pathologic functions of CEBPβ in TNBCs. CEBPβ was shown to be highly overexpressed in TNBC patient samples as compared with normal tissues (Fig. 1A). However, there was no significant difference in C/EBPβ expression levels between males and females, and its expression was not age-dependent either (Fig. 1B and C). Low tumor CEBPβ expression was associated with a significantly longer overall survival rate (Fig. 1D). The association between tumor expression of CEBPβ and clinical evidence indicated that CEBPβ could function in patients with TNBC as a prognostic molecular marker. Furthermore, CEBPβ expression levels were significantly increased in samples from patients with high-grade cancers compared with low-grade (Fig. 1E). In addition, a strong correlation between CEBPβ expression and cell migration signaling pathways was demonstrated by Gene Set Enrichment Analysis (GESA; Fig. 1F). A strong positive association between CEBPβ expression and lgmnn was found in parallel with our observations in the TNBC database (Fig. 1G). Therefore, these results indicate that CEBPβ in the TNBC tissues is greatly upregulated, and a lower survival rate is seen in patients with elevated CEBPβ expression.

Figure 1.

C/EBPβ is the prognostic biomarker in patients with TNBC. A, C/EBPβ expression was relative to normal tissues in TCGA (The Cancer Genome Atlas) TNBC samples. The expression of C/EBPβ in the TCGA data set was contrasted between (B) gender and (C) age. D, Overall survival and postprogression survival with stratified C/EBPβ expression in TCGA TNBC patients. E, Studies of the immunohistochemistry of C/EBPβ expression in human TNBC tissues. Scale bar, 100 μm. F, High expression of C/EBPβ correlated with the cell migration signaling pathway, as revealed using the GSE11121 datasets. NES is a normalized enrichment score. G, Correlation between C/EBPβ with lgmn expression. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

C/EBPβ is the prognostic biomarker in patients with TNBC. A, C/EBPβ expression was relative to normal tissues in TCGA (The Cancer Genome Atlas) TNBC samples. The expression of C/EBPβ in the TCGA data set was contrasted between (B) gender and (C) age. D, Overall survival and postprogression survival with stratified C/EBPβ expression in TCGA TNBC patients. E, Studies of the immunohistochemistry of C/EBPβ expression in human TNBC tissues. Scale bar, 100 μm. F, High expression of C/EBPβ correlated with the cell migration signaling pathway, as revealed using the GSE11121 datasets. NES is a normalized enrichment score. G, Correlation between C/EBPβ with lgmn expression. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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AEP is overexpressed in numerous human cancers, and it is a downstream target of C/EBPβ (17). Hence, we also employed a bioinformatic analysis of AEP expression in TNBCs from the TCGA database. As expected, AEP was significantly more abundant in malignant breast tumors than normal tissues, and the high levels of AEP expression coupled with shorter patient survival rates in TNBCs from the TCGA database. No significant sex- or age-dependent expression pattern for AEP was observed. Furthermore, GESA showed a strong correlation between AEP expression and cell migration signaling pathways (Supplementary Fig. S1). Hence, our data demonstrate that the relevance of the CEBPβ/AEP pathway in TNBCs and shorter patient survival rates are inversely correlated.

C/EBPβ/AEP expression mediates oxidative stress in cancer, affecting cell migration and invasion

Metastatic nodules exhibit enriched oxidative stress compared to subcutaneous tumors. To explore whether C/EBPβ/AEP regulates ROS production in breast cancers and mediates the migration and invasion of tumor cells, we transfected C/EBPβ into MDA-MD-231 cells in the presence of control siRNA or that targeting AEP. Overexpression of C/EBPβ significantly increased cell numbers compared with control plasmid, whereas knockdown of AEP had no additional effect (Fig. 2A). Interestingly, an LDH release assay showed that C/EBPβ overexpression decreased cell death, which was not further altered by deletion of AEP (Fig. 2B). Notably, ROS levels and carbonyl expression were decreased upon C/EBPβ overexpression, reflecting reduced oxidative stress and AEP knockdown reversed this effect (Fig. 2C and D). Intriguingly, C/EBPβ overexpression strongly promoted breast cancer cell migration and invasion that were significantly reversed in AEP knocked down cells (Fig. 2E). Immunoblotting confirmed that C/EBPβ was indeed overexpressed in the transfected MDA-MD-231 cell. Its downstream target AEP was subsequently increased and siRNA targeting AEP reduced its expression levels. We also monitored p-C/EBPβ expression and found that suppression AEP will decrease p-C/EBPβ. Moreover, NQO1 and GSTP1 (15), two well-characterized reductases downstream of C/EBPβ, were strongly augmented in C/EBPβ-overexpressed cells. In addition, suppression of AEP expression resulted in a significant abrogation of reductase expression. In alignment with the quantitative ROS levels, the abundance of these reductases was inversely correlated with oxidative stress. SRPK2, a cell cycle-related kinase implicated in human cancers, is cleaved by active AEP at N342 (29). Accordingly, Western blotting showed that SRPK2 was strongly cleaved at N342 in C/EBPβ/AEP elevated cells, suggesting that AEP is activated in C/EBPβ-overexpressed cancer cells (Fig. 2F). Previous studies support that AEP cleaves MMPs and mediates cancer cell migration and invasion (30, 31). Accordingly, we utilized the gelatin zymography to assess whether MMP2 and 9 were affected in breast cancer cells. In consequence, cleaved MMP2 and 9 were greatly elevated in C/EBPβ-overexpressed cells and suppression of AEP expression attenuated their cleavage (Fig. 2G). A fluorogenic substrate assay verified that C/EBPβ overexpression significantly elevated AEP activity and this was reduced upon AEP knockdown (Fig. 2H).

Figure 2.

C/EBPβ/AEP expression mediates oxidative stress in cancer, affecting cell migration and invasion. A, Cell proliferation over 5 days in MDA-MB-231 cells after overexpression C/EBPβ and deletion AEP. The LDH levels (B), the ROS levels (C), and carbonyl expression (D) in MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP. E, Transwell assay to demonstrate the capacity of migration and invasion with overexpression C/EBPβ and deletion AEP. Scale bar, 100 μm. F, Western blot analysis of MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP. G, Gelatin zymography was used to demonstrate the activity of MMP-2 and MMP-9 in the presence of C/EBPβ and AEP. H, AEP enzyme activity of MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP for 60 minutes. Data are means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, n = 3).

Figure 2.

C/EBPβ/AEP expression mediates oxidative stress in cancer, affecting cell migration and invasion. A, Cell proliferation over 5 days in MDA-MB-231 cells after overexpression C/EBPβ and deletion AEP. The LDH levels (B), the ROS levels (C), and carbonyl expression (D) in MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP. E, Transwell assay to demonstrate the capacity of migration and invasion with overexpression C/EBPβ and deletion AEP. Scale bar, 100 μm. F, Western blot analysis of MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP. G, Gelatin zymography was used to demonstrate the activity of MMP-2 and MMP-9 in the presence of C/EBPβ and AEP. H, AEP enzyme activity of MDA-MB-231 cell lines after overexpression C/EBPβ and deletion AEP for 60 minutes. Data are means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, n = 3).

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AEP deficiency results in escalated oxidative stress and decreased breast-to-lung metastasis in the MMTV-PyMT breast cancer model

Mammary gland–specific expression of PyMT under the guidance of transgenic mice MMTV promoter/enhancer (MMTV-PyMT) results in the widespread transformation of the mammary epithelium, development of multifocal mammary adenocarcinomas and metastatic lymph node and lung lesions (32). In comparison, in MMTV-PyMT and MMTV-Neu mice, metastatic spreading occurs largely via hematogenous spread to the lungs and lymph nodes, as opposed to the original spread of cancer cells to the local lymph nodes through lymph nodes in human breast cancer. To explore whether AEP contributes to breast cancer metastasis, we crossed MMTV-PyMT transgenic mice with AEP-deficient mice. We monitored the in vivo activity of AEP in lung metastases that developed after breast tumor onset at 90 to 120 days (33). Specifically, we employed an activity-based probe LE28, which binds to AEP in an activity-dependent manner, resulting in the generation of a fluorescent signal (26). As expected, AEP was greatly activated in breast tumors in MMTV-PyMT mice, and this was reduced in MMTV-PyMT/AEP+/− mice and totally eliminated in MMTV-PyMT/AEP−/− mice. Lung tissues from MMTV-PyMT mice also displayed a similar in vivo LE28 fluorescence (Fig. 3A and B). Ex-vivo LE28 signals in dissected primary breast tumors corroborated the in vivo observations (Fig. 3C). Lung metastases were readily visible indemonstrable in MMTV-PyMT mice; however, they were barely detectable in MMTV-PyMT/AEP KO mice, suggesting that deletion of AEP can diminish breast cancer lung metastasis (Fig. 3D). H&E staining of the lung tissues verified these observations (Fig. 3E). Immunoblotting revealed that C/EBPβ levels were highly increased in lung metastases compared with primary tumors and this was decreased when AEP was deleted in MMTV-PyMT/AEP−/− mice. These data suggest that AEP feeds back to mediate the upstream transcription factor expression. Levels of pro- and active AEP were tightly correlated with C/EBPβ amounts. Interestingly, both MMP2 and MMP9 also oscillated in a similar pattern, as did the downstream SRPK2 N342 fragmentation. Notably, C/EBPβ downstream targets NQO1 and GSTP1 were also attenuated, fitting with C/EBPβ transcription factor levels (Fig. 3F). IHC staining for Ki67, a marker for cell proliferation, revealed that cell proliferation in lung metastatic tumors was enhanced as compared with primary tumors in the breast, and this was strongly reduced when AEP was deleted in MMTV-PyMT/AEP KO mice (Fig. 3G, top).

Figure 3.

AEP deficiency results in escalated oxidative stress and decreased breast-to-lung metastasis in the MMTV-PyMT breast cancer model. A, MMTV-PyMT transgenic mice bearing tumors were injected with LE28. After 6 hours, ex vivo imaging of lungs (B) and tumors (C) extracted from mice and imaged on an IVIS 100 machine. D, The representative mouse lungs of animals in different groups. E, H&E staining of lung slices in various groups. Scale bar, 500 μm. F, Western blot analysis of primary tumor and lung metastasis lysates from the animals. G, IHC staining of Ki67, 4-HNE, EGFR and RSU1 in the primary tumor and lung metastasis slides from different groups of mice. Scale bar, 100 μm. Data are means ± SEM (*, P < 0.05, one-way ANOVA, n = 3).

Figure 3.

AEP deficiency results in escalated oxidative stress and decreased breast-to-lung metastasis in the MMTV-PyMT breast cancer model. A, MMTV-PyMT transgenic mice bearing tumors were injected with LE28. After 6 hours, ex vivo imaging of lungs (B) and tumors (C) extracted from mice and imaged on an IVIS 100 machine. D, The representative mouse lungs of animals in different groups. E, H&E staining of lung slices in various groups. Scale bar, 500 μm. F, Western blot analysis of primary tumor and lung metastasis lysates from the animals. G, IHC staining of Ki67, 4-HNE, EGFR and RSU1 in the primary tumor and lung metastasis slides from different groups of mice. Scale bar, 100 μm. Data are means ± SEM (*, P < 0.05, one-way ANOVA, n = 3).

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In breast cancer, EGFR overexpression is associated with large tumor size, low differentiation and poor clinical outcomes (34). While overexpression of EGFR is observed in all breast cancer subtypes, overexpression of EGFR is more commonly observed in TNBC and inflammatory breast cancer (IBC), which are extremely aggressive (35, 36). IHC staining for EGFR shows similar patterns as Ki67 staining in both primary and metastatic tumors (Fig. 3G, left third panel). In metastasis, extracellular matrix (ECM)-related adhesion proteins are essential. Cell–ECM adhesions are localized to Ras suppressor-1 (RSU-1), a Ras-transformation suppressor. IHC staining with RUS1 demonstrated a similar pattern as to Ki67 (Fig. 3G, bottom). Our finding is consistent with the previous report that RSU-1 is upregulated in metastatic breast cancer samples and possesses the metastasis-promoting properties (37). Hence, in breast-to-lung metastases, EGFR, Ki67 and RUS1 activities are significantly augmented versus the primary cancers, and they are abrogated in MMTV-PyMT/AEP KO mice. In contrast, the deletion of AEP escalated 4-HNE, a surrogate marker for oxidative stress, in alignment with the attenuated reductases NQO1 and GSTP1 (Fig. 3G, left second panel). The quantification is shown in Fig. 3G, right.

Optimized AEP inhibitor CP6 (No. 11A) potently suppresses AEP activity

Accumulating evidence shows that AEP is highly expressed in breast cancers and promotes bone and lung metastasis (19, 23, 38). We have previously reported that a small-molecule AEP inhibitor blocked breast cancer metastasis of subcutaneous MDA-MD-231 cells xenografted in nude mice (23), suggesting that AEP is an effective target for breast cancer therapy. We have characterized a large number of derivatives based on the pharmacophore of the previously reported lead compound (No. 11) and its cocrystal structure with active AEP (39). Accordingly, we analyzed the inhibitory activity of these derivatives using active AEP proteins. Titration assays revealed that some of the derivatives possessed sub-nanomolar IC50 values against recombinant AEP. Notably, thiol-containing CP1 displayed an IC50 of 4.34 nmol/L. Because the thiol group is labile and easily oxidized in vivo, we replaced it with a methyl group in CP2, which exhibited a comparable IC50 of 6.05 nmol/L; however, increasing the volume of substituted group from methyl to ethyl decreased the IC50 in CP3 to 39.6 nmol/L. Because the 1,4-diamino-benzene structure is labile for Michael addition in the liver by GSH conjugation, resulting in liver toxicity, we introduced a hydroxyl-methyl group in CP6 (also called No. 11A). Remarkably, it possessed IC50 of 5.13 nmol/L without reactive metabolite activity (Fig. 4A and B).

Figure 4.

Optimized AEP inhibitor CP6 (No. 11A) potently suppresses AEP activity. A, The structures of AEP inhibitor derivatives. B, The IC50 curves of the AEP inhibitor derivatives against its recombinant proteins. C, Microscopy images of MDA-MB-231 cells exposed to LE28 (red) for 5 hours after different concentrations of CP2 and CP6 treatment. Scale bar, 100 μm.

Figure 4.

Optimized AEP inhibitor CP6 (No. 11A) potently suppresses AEP activity. A, The structures of AEP inhibitor derivatives. B, The IC50 curves of the AEP inhibitor derivatives against its recombinant proteins. C, Microscopy images of MDA-MB-231 cells exposed to LE28 (red) for 5 hours after different concentrations of CP2 and CP6 treatment. Scale bar, 100 μm.

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To further characterize whether these compounds effectively inhibit AEP in intact cells, we employed C/EBPβ overexpressed MDA-MD-231 cancer cells. Titration assay with cell lysates showed that CP6 (No. 11A) exhibited the most robust in vitro cellular IC50 values of 297 nmol/L, and IC50 values for the rest of the compounds are summarized in Supplementary Fig. S2A. Alternatively, we also monitored the cellular AEP inhibition curve with LE28 and found that both CP2 and CP6 dose-dependently suppressed LE28 fluorescence intensities with IC50 values of 351 and 272 nmol/L, respectively (Fig. 4C). Because AEP belongs to the cysteine protease superfamily, to ensure that these derivatives specifically target AEP, but not other cysteine proteases like caspases, we monitored their IC50 against active caspase-3. Except for CP2, other AEP inhibitors did not exhibit inhibition of caspase-3 at concentrations up to 200 μmol/L (Supplementary Fig. S2B), suggesting that these derivatives are specific AEP inhibitors. Compound CP6 (No. 11A) is therefore a highly selective and potent AEP inhibitor in vitro and intact cancer cells. To further characterize the anticancer effects of AEP inhibitors, we evaluated their cytotoxicity in MDA-MB-231 cells in vitro. Cells were incubated with various concentrations (0–10 μmol/L) of AEP inhibitors for 48 hours. Only CP5 decreased cell numbers at 10 μmol/L, while other AEP inhibitors showed little cytotoxicity (Supplementary Fig. S2C). Hence, AEP inhibitors exhibit an anticancer effect independent of their cytotoxicity.

AEP inhibitor regulates ROS and metastasis of C/EBPβ-overexpressed breast cancer cells

Because lead compound CP6 (No. 11A) possesses druggable characteristics and is able to strongly inhibit AEP, its potential as an anticancer drug was subsequently assessed in breast cancer cells. We employed a highly metastatic human breast cancer cell line, MDA-MB-231, which was transfected with C/EBPβ. Although AEP inhibitor CP6 (No. 11A) did not affect the cell numbers or cell death rates in C/EBPβ-transfected MDA-MD-231 cells, it dose-dependently increased ROS production and carbonyl expression levels (Fig. 5AD). Moreover, CP6 (No. 11A) inhibited cancer cell invasion and migration in a concentration-dependent manner (Fig. 5E). Immunoblotting demonstrated that active AEP and p-C/EBPβ levels were gradually attenuated, as the doses of CP6 (No. 11A) progressively. Consequently, SRPK2 N342 cleavage activities were tightly coupled with active AEP levels. Interestingly, both MMP2 and MMP9 were diminished as CP6 (No. 11A) concentrations were escalated (Fig. 5F). In alignment with these effects, gelatin zymography revealed that cleaved MMP2 and MMP9 were dose-dependently repressed (Fig. 5G). AEP enzymatic assay confirmed that the cellular AEP was strongly blocked by the inhibitor in a concentration-dependent manner (Fig. 5H). Thus, CP6 (No. 11A) potently inhibits AEP in C/EBPβ stably transfected MDA-MD-231 cells, increases the oxidative stress and suppresses its migration and invasion.

Figure 5.

AEP inhibitor regulates ROS and metastasis of C/EBPβ-overexpressed breast cancer cells. A, The cell proliferation over 5 days in MDA-MB-231 cells after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L and 5 μmol/L CP6 treatment. The LDH levels (B), the ROS levels (C), and carbonyl expression (D) in MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment for 48 hours. E, Transwell assay to demonstrate the capacity of the indicated cells to migrate and invasion with overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment. Scale bar, 100 μm. F, Western blot analysis of MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L and 5 μmol/L CP6 treatment. G, Gelatin zymography was used to demonstrate the activity of MMP-2 and MMP-9 in the presence of C/EBPβ and AEP inhibitors. H, AEP enzyme activity of MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment for 60 minutes. Data are means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, n = 3).

Figure 5.

AEP inhibitor regulates ROS and metastasis of C/EBPβ-overexpressed breast cancer cells. A, The cell proliferation over 5 days in MDA-MB-231 cells after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L and 5 μmol/L CP6 treatment. The LDH levels (B), the ROS levels (C), and carbonyl expression (D) in MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment for 48 hours. E, Transwell assay to demonstrate the capacity of the indicated cells to migrate and invasion with overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment. Scale bar, 100 μm. F, Western blot analysis of MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L and 5 μmol/L CP6 treatment. G, Gelatin zymography was used to demonstrate the activity of MMP-2 and MMP-9 in the presence of C/EBPβ and AEP inhibitors. H, AEP enzyme activity of MDA-MB-231 cell lines after overexpression C/EBPβ and 0.5 μmol/L, 1 μmol/L, and 5 μmol/L CP6 treatment for 60 minutes. Data are means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, n = 3).

Close modal

AEP inhibitor strongly blocks breast-to-lung metastasis in mice

Because AEP deficiency in MMTV-PyMT mice strongly blunted breast cancer lung metastasis, we next examined the therapeutic efficacy AEP specific inhibitor CP6 (No. 11A) against breast cancer metastasis in MMTV-PyMT breast cancer transgenic mice. CP6 (No. 11A) was administered to the mice via i.p. injection at a dose of 10 mg/kg. The lungs were tested for the presence of metastatic nodules following 90 days of opioid therapy. Lung metastases were significantly decreased upon CP6 (No. 11A) treatment as compared with vehicle control (Fig. 6A). H&E staining on the lung tissues revealed that metastatic cancers in the lung were much smaller after CP6 (No. 11A) treatment versus the vehicle group (Fig. 6B). Consistent with the observation that C/EBPβ and its transcriptional target AEP were escalated in the lung metastatic cancers, we also found C/EBPβ and AEP augmentation in metastatic tissues in the vehicle group, which were prominently suppressed by CP6 (No. 11A), which also showed similar results in IHC staining (Supplementary Fig. S3C). Consequently, AEP downstream targets SRPK2 N342 truncation were almost eliminated by CP6 (No. 11A), whereas MMP9 and MMP2 were reduced by CP6 (No. 11A) in the lung metastatic tissues. As compared with elevated reductases in the metastatic tissues treated by vehicle, CP6 (No. 11A) strongly attenuated both NQO1 and GSTP1 levels (Fig. 6C). Correspondingly, IHC staining with 4-HNE showed that the oxidative stress levels were substantially increased after CP6 (No. 11A) treatment, whereas Ki67, EGFR and metastatic marker RUS1 levels were significantly lessened as compared with vehicle groups in both the primary tumor and metastatic tumors (Fig. 6D).

Figure 6.

AEP inhibitor strongly blocks breast to lung metastasis in mice. A, The representative mouse lungs of animals treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. B, H&E staining of lung slices in various groups. Scale bar, 500 μm. C, Western blot analysis of primary tumor and lung metastasis lysates from treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. D, IHC staining of Ki67, 4-HNE, EGFR, and RSU1 in the primary tumor and lung metastasis slides from treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. Scale bar, 100 μm. Data are means ± SEM (*, P < 0.05, one-way ANOVA, n = 3). E, The changes in mice serum safety assay after the i.p. injection of CP6 (10 mg/kg) or control vehicle groups.

Figure 6.

AEP inhibitor strongly blocks breast to lung metastasis in mice. A, The representative mouse lungs of animals treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. B, H&E staining of lung slices in various groups. Scale bar, 500 μm. C, Western blot analysis of primary tumor and lung metastasis lysates from treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. D, IHC staining of Ki67, 4-HNE, EGFR, and RSU1 in the primary tumor and lung metastasis slides from treated with i.p. injection of CP6 (10 mg/kg) or control vehicle groups. Scale bar, 100 μm. Data are means ± SEM (*, P < 0.05, one-way ANOVA, n = 3). E, The changes in mice serum safety assay after the i.p. injection of CP6 (10 mg/kg) or control vehicle groups.

Close modal

It important to note that chronic CP6 (No. 11A) treatment did not affect the body weight of the MMTV-PyMT mice, indicating that the compound is not toxic (Supplementary Fig. S3A). H&E staining with various tissues including heart, liver, spleen and kidney demonstrated no detectable toxicity (Supplementary Fig. S3B), fitting with the above body weight measures. Nevertheless, AST (aspartate aminotransferase), a marker for the liver inflammation and damage, indicated that CP6 (No. 11A) chronic treatment might elicit the liver side effect, although ALT (alanine aminotransferase), ALP (alkaline phosphatase), GGT (gamma-glutamyl transferase), Phos (phosphate), other liver damage markers, and BUN (blood urea nitrogen), a marker for the kidney damage remained similar between vehicle and CP6 (No. 11A) treatment (Fig. 6E). However, a few days after CP6 (No. 11A) treatment, AST returned to normal levels, suggesting that the liver inflammation is reversible and the side effect from CP6 (No. 11A) is tolerable. Moreover, CBC (complete blood chemistry) analysis showed that CP6 (No. 11A) chronic treatment alleviated the aberrant blood cell indexes in MMTV-PyMT mice (Supplementary Fig. S3D), supporting that this compound might be a promising pharmacological agent for blunting breast cancer metastasis.

In this study, we show that C/EBPβ/AEP pathway is upregulated in human breast cancers and is inversely correlated with poor patient survival rates. Interestingly, we find that overexpression of C/EBPβ in breast cancer cell line MDA-MD-231 significantly reduces ROS levels and escalates migration and invasion activities. Knockdown of AEP in these cells substantially reverses these events. Because AEP is a downstream target of C/EBPβ, accordingly, overexpression of C/EBPβ elevates AEP levels, leading to its proteolytic activation. In consequence, the substrates MMP2 and 9 were cleaved by active AEP, and depletion of AEP greatly attenuates their cleavage. We made similar observations in C/EBPβ-overexpressed MDA-MD-231 breast cancer cells using a small-molecule AEP inhibitor.

In genetically modified mice, specific promoters, including ErbB2/Neu, polyoma middle T antigen (PyMT), simian virus 40 (SV40) T antigen, etc., have been added to drive the expression of several recognized oncogenes directly in the mammary epithelium to induce or modulate breast carcinogenesis in mice. Mammary gland-specific expression of PyMT under the guidance of the transgenic mouse MMTV promoter/enhancer (MMTV-PyMT) results in the widespread transformation of the mammary epithelium and in the production of multifocal mammary adenocarcinomas and metastatic lesions in the lymph nodes and lungs (32). In the MMTV-PyMT breast cancer mouse model, knockout of AEP feeds back and mitigates C/EBPβ levels in both primary tumors and metastatic tumors. Noticeably, the abundance of both MMP2 and 9 was substantially diminished in the absence of AEP. Previous studies indicate that C/EBPβ may mediate both MMP2 and 9 mRNA transcription via binding to their promoters (40, 41). Because NQO1 and GSTP1 are both downstream targets of C/EBPβ, these anti-oxidative enzymes are decreased. Strikingly, the breast to lung metastasis is prominently blocked when AEP is knocked out from MMTV-PyMT breast cancers. In alignment with the genetic deletion of AEP in the breast cancer transgenic animals, administration of an AEP-specific inhibitor CP6 (No. 11A) also demonstrates robust therapeutic efficacy in antagonizing the lung metastasis by selectively inhibiting AEP.

More recently, we have stated that C/EBPβ functions in glioblastoma as a transcription factor for both NQO1 and GSTP1. Manipulation of C/EBPβ levels firmly dictates the expression of NQO1 and GSTP1, controlling ROS and proliferation of GBM cells (15). Therefore, overexpression of C/EBPβ in MDA-MD-231 breast cancer cells significantly reduces ROS levels (Figs. 2 and 5). Remarkably, inactivation of AEP with its specific siRNA or small inhibitor CP6 (No. 11A) escalates ROS and reduces C/EBPβ overexpression–promoted migration and invasion in MDA-MD-231 cells. Moreover, genetic deletion of AEP from MMTV-PyMT mice or blockade of AEP with its small-molecule inhibitor in these animals diminishes C/EBPβ, which suppresses the expression of both NQO1 and GSTP1 in tumors, leading to augmentation of 4-HNE, a biomarker for ROS, in both primary and lung tissues (Figs. 3 and 6). ROS is developed within eukaryotic cells by both enzymatic and nonenzymatic processes and plays an important role in cellular physiology and pathophysiology. Cancer cells are typically subject to higher ROS levels, and by inducing prolonged replication, death avoidance, angiogenesis, invasiveness, and metastasis, further promote the malignant phenotype (4). The supply of ROS results, by enzymatic and nonenzymatic antioxidants, from the equilibrium between its production and its disposal. The imbalance of this mechanism, which has long been described as oxidative stress, contributes to a pro-oxidant environment. In addition, the interaction between Nrf2 and NAD(P)H dehydrogenase quinone 1 (NQO1), an enzyme involved in cancer cell defense against cytotoxic agents, was significant (42). C/EBPβ is in comparison with these reductases, the downstream target gene of Nrf2 (43). In comparison, EGFR overexpression is observed in at least 50% of TNBC cases, which is a greater rate of expression than that seen in other subtypes of breast cancer (44). Interestingly, inactivation of AEP significantly reduces EGFR levels in both primary and metastatic tumors, inversely correlated to the oxidative stress in these tissues (Figs. 3 and 6).

Cancer cells produce high ROS levels that might be involved in neoplastic transformation. Because of the high ROS steady-state concentration in cancer cells, conceivably, a further increase in ROS concentration would overwhelm antioxidant capability and lead to oxidative damage–driven death. The strategy for cancer treatment is called pro-oxidant therapy, postulating that the additional ROS exacerbates the preferential killing of cancer cells sparing nontumoral cells because the latter normally have lower oxidant levels and would be able to cope with the rise without undergoing oxidative damage and death (45). In this context, pro-oxidant therapy triggers a harmful oxidative environment to mitigate tumorigenesis by taking advantage of elevated ROS levels in tumor cells. Our current data support that blunting AEP with its small-molecule inhibitor suppresses the expression of reductases via mitigating the upstream transcription factor C/EBPβ, resulting in ROS escalation in both the primary and metastatic tumors, significantly attenuating tumor growth and metastasis (Fig. 6). This finding is consistent with the pro-oxidant therapy concept. It is important to note that chronic administration of this compound is safe. H&E examination of various major tissues from MMTV-PyMT mice demonstrated no detectable toxicity (Supplementary Fig. S3B). In addition, this drug treatment also improves various parameters in CBC (complete blood chemistry). Although a sensitive marker for liver damage, AST (aspartate aminotransferase) increases after chronic AEP inhibitor treatment, the ALT (alanine aminotransferase) level remains unaltered, and the AST amount declines and recovers after drug treatment stops, supporting that the induced liver inflammation and damage by the drug are minor and reversible (Fig. 6E). This is corroborated by the pathologic examination of liver H&E staining.

Primary tumor masses are often vulnerable to spawning founder cells that migrate out, infiltrate neighboring tissues, and then fly to distant locations where they may succeed in forming new colonies during the growth of most forms of human cancer, including breast carcinoma. Although only a small amount of AEP is found in normal tissues, the enzyme is overexpressed on the surface of cells and in solid tumor cytoplasmic vesicles (30). Increased invasive and aggressive behavior of many cancers, including breast, prostate, colorectal and gastric carcinomas, is consistent with endoprotease production of AEP (30, 46–48). MMP-2 leads to human breast cancer migration and colonization of MDA-MB-231 and MDA-MB-435 cells (49). Therefore, in an attempt to classify other possible drug target candidates, additional review of MMPs has centered on their control. Previous studies indicate that through proteolytic elimination of an N-terminal propeptide, AEP activates MMP-2 (30, 31). Previous experiments have shown that AEP is situated at the apex of invading cells, producing a lamellipodia and invadopodia integrin complex. AEP's binding to these integrins greatly increases AEP's ability to activate pro-MMP2 and cathepsin L proteolytically. The elimination of AEP from breast cancer greatly reduces lung metastasis and enhances tumor oxidative stress (Fig. 3). Our evidence supports redox homeostasis as a possible target for the treatment of breast cancer. AEP inhibitors may therefore be more promising cancer therapeutics than the metalloproteinase inhibitors described above. As predicted, our data show that both MMP2 and 9 protein levels and their proteolytic cleavage are substantially suppressed by the AEP inhibitor, resulting in suppression of lung metastasis in breast cancer. Peptide AEP inhibitors have previously been shown to decrease bone and lung metastasis of breast cancer (19, 38). However, owing to their reduced stability and bioavailability, peptide-based compounds appear to be undesirable drug candidates.

In summary, we have tried to find small-molecule TNBC therapy inhibitors that directly target AEP and theoretically abolish its function. Our data support that C/EBPβ/AEP signaling regulates the oxidative stress in breast cancer metastasis and blocking AEP with its specific small-molecule inhibitor No. 11A presents a promising cancer therapeutic strategy.

K. Ye is a shareholder of Wuhan Yuanzheng Pharmaceuticals, Inc. and Shanghai Braegen Pharmaceuticals Inc. No disclosures were reported by the other authors.

K. Lei: Conceptualization, resources, data curation. S. Kang: Data curation. E. Ahn: Formal analysis. C. Chen: Resources, data curation. J. Liao: Data curation. X. Liu: Supervision. H. Li: Methodology. L.E. Edgington-Mitchell: Resources. L. Jin: Supervision. K. Ye: Conceptualization, writing–original draft.

This work is funded with Federal funds from the NCI, NIH NIH grant RO1 CA186918 (to K. Ye). K. Lei was supported by a grant from the China Postdoctoral Science Foundation (2018M632168). This work is also sponsored by the Program of Shanghai Academic Research Leader 20XD1403400 (to L. Jin).

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

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Supplementary data