Abstract
Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Mitochondrial function is dysregulated frequently in cancer cells to allow for adaptation to a harsh tumor microenvironment. Targeting mitochondrial biogenesis and bioenergetics is, therefore, an attractive therapeutic strategy. In this study, we performed quantitative proteomic analyses in human parental and metastatic breast cancer cell lines to identify mitochondrial proteins involved in TNBC metastasis. We found that single-strand DNA-binding protein 1 (SSBP1) was downregulated in highly metastatic breast cancer cells. Moreover, SSBP1 downregulation promoted TNBC cell metastasis in vitro and in vivo. Mechanistically, SSBP1 loss decreased mitochondrial DNA copy number, thereby potentiating calcineurin-mediated mitochondrial retrograde signaling that induced c-Rel/p50 nuclear localization, activated TGFβ promoter activity, and TGFβ-driven epithelial-to-mesenchymal transition. Low SSBP1 expression correlated with tumor progression and poor prognosis in patients. Collectively, our findings identified SSBP1 as a novel metastasis suppressor and elucidated the mechanisms by which dysregulated mitochondrial signaling contributes to metastatic potential, providing potential new prognostic indicators for patients with TNBC. Cancer Res; 76(4); 952–64. ©2015 AACR.
Introduction
Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype associated with poor prognosis. TNBC tumors are generally larger in size and of a higher grade, often have lymph node involvement at the time of diagnosis and tend to more frequently affect younger patients (1–6). However, the absence of well-defined molecular targets for this tumor subtype makes TNBC treatment challenging (7–9). As the major energy and metabolite source in cells, mitochondrial function is often dysregulated in cancer, and there is growing interest in understanding how altered mitochondrial function may be targeted to inhibit tumor progression. The modified bioenergetic and biosynthetic states of mitochondria communicate with the nucleus through mitochondrial retrograde signaling to modulate signaling pathways and transcriptional circuits to meet the requirements of cancer cells. Thus, the discovery of novel biomarkers involved in this pathophysiologic process can provide broad new insights into the mechanisms of tumorigenesis and tumor progression.
In our previous study, the highly metastatic cell line MDA-MB-231HM was developed from parental MDA-MB-231 cells by in vivo selection in mice. Accordingly, MDA-MB-231HM cells exhibited increased invasiveness to the lungs compared with parental MDA-MB-231 cells. Another highly bone-metastatic cell line, MDA-MB-231Bo, which was previously isolated via in vivo selection and also had stronger lung metastasis capacity compared with MDA-MB-231 cells (Supplementary Fig. S1A), was obtained from Dr. Toshiyuki Yoneda (The University of Texas, Houston, TX; refs. 10, 11). Because of the similar genetic background of these cells, they provide a unique model for identifying candidate metastasis-associated biomarkers and potential therapeutic targets for TNBC. To discover mitochondrial biomarkers that may be associated with TNBC progression, we thoroughly investigated the mitochondrial proteome profile of the MDA-MB-231 cell line series. Using iTRAQ-labeling technology followed by nanoscale high-performance liquid chromatography (nano-HPLC)-MS/MS, we identified single-strand DNA-binding protein 1 (SSBP1) as a differentially expressed protein associated with the metastatic potential of TNBC.
SSBP1 (also known as mtSSB) exists in eukaryotic mitochondria (12). cDNA analysis revealed that SSBP1 is translated as a 148-amino acid polypeptide, and the first 16 amino acids are proposed to be removed after the import of this protein into the mitochondria, resulting in a mature protein consisting of 132 amino acids (13). SSBP1 is distinct from nuclear SSB and plays an important role in mtDNA replication, recombination, and repair by binding to single-stranded mtDNA. SSBP1 has been shown to stimulate the DNA polymerase activity of pol γ in humans (14, 15) and the unwinding activity of human mtDNA helicase (16). In addition, SSBP1 stimulates strand-displacement DNA synthesis promoted by the combined action of mtDNA helicase (17). Recently, it has been reported that SSBP1 is associated with human cancer. The level of SSBP1 protein correlates with the aggressiveness of human osteosarcoma cells, suggesting a link between mtDNA replication and cancer progression (18). SSBP1 has also been identified as a novel binding partner of the tumor suppressor p53 in mitochondria biophysical measurements (19). In this study, we show for the first time that SSBP1 is a suppressor of TNBC metastasis.
Materials and Methods
iTRAQ-nano-HPLC-MS/MS analyses
The cell lysates of parental MDA-MB-231 and highly metastatic MDA-MB-231 (MDA-MB-231HM and MDA-MB-231Bo) breast cancer cells were quantified using a Bradford assay, labeled with iTRAQ-labeling reagents (Applied Biosystems), and digested with trypsin. The resulting peptides were fractionated with a Waters ultra-performance liquid chromatography device, and the fractions were then separated by nano-HPLC (Eksigent Technologies) on a secondary reverse-phase (RP) analytical column. A Triple TOF 4600 mass spectrometer (MS) was operated in information-dependent data acquisition mode to automatically switch between MS and tandem MS (MS/MS) acquisition. MS/MS spectra were extracted and charge state deconvoluted using an MS Data Converter from AB Sciex.
Cell culture
All breast cancer cell lines, normal breast MCF10A cells and HEK 293T cells were obtained from the Shanghai Cell Bank, Type Culture Collection Committee of Chinese Academy of Science (Shanghai, China) in 2014, and they were authenticated using short tandem repeat profiling by the cell bank. These cell lines were conserved in our laboratory and subjected to routine cell line quality examinations (e.g., morphology, Mycoplasma) by HD Biosciences every 3 months. The cells for experiments were passaged for less than 6 months. The MDA-MB-231Bo cells, obtained from Dr. Toshiyuki Yoneda (The University of Texas, Houston, TX) were cultured in ATCC-formulated Leibovitz L-15 medium supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The remaining cell lines were maintained in complete growth medium as recommended by the manufacturers.
Study population
This study involved 250 breast cancer patients with a diagnosis of pathologically invasive ductal breast cancer with a follow-up period of at least 5 years. The diagnoses were verified by two independent pathologists in the Department of Pathology of the Fudan University Shanghai Cancer Center (FDUSCC). Samples were collected from these patients in the Department of Breast Surgery of FDUSCC from August 2001 to March 2006. This study was approved by the Ethics Committee of FDUSCC, and each participant signed an informed consent document.
Tissue microarrays, IHC staining, and IHC variable evaluation
Tissue microarrays (TMA; Supplementary Table S1) were constructed from archival formalin-fixed, paraffin wax-embedded samples of carcinomas obtained from the 250 breast cancer patients described above. Tissue cylinders with diameters of 2 mm were punched from a previously marked tumor area in each block (donor block) and inserted into a recipient paraffin wax block, resulting in a 10 × 10 array. Tissue samples from all 250 patients were punched twice into the microarray to compare staining patterns in different areas of the same tumor. The brief details of IHC staining, and IHC variable evaluation are described in the Supplementary Methods.
Quantitative real-time PCR
Total RNA was extracted with the TRIzol reagent (Invitrogen) and reverse transcribed using the PrimeScript RT Reagent Kit (Perfect Real-Time; TaKaRa Biotechnology). Subsequently, real-time PCR was performed with SYBR Premix Ex Taq (TaKaRa Bio) using an ABI Prism 7900 instrument (Applied Biosystems). mtDNA content was measured by real-time PCR using specific primers for the mtDNA-encoded cytochrome oxidase I and nuclear DNA-encoded GAPDH.
The primer sequences used in this study are as follows:
GAPDH: F 5′-GGTGGTCTCCTCTGACTTCAACA-3′,
GAPDH: R 5′-GTTGCTGTAGCCAAATTCGTTGT-3′,
SSBP1: F 5′-TGGAGTCGTGTGTTTTGGCT-3′,
SSBP1: R 5′-TAGGCTTTTCCTGAAAACCGAGG-3′,
TGF-β: F 5′-CCCTGGACACCAACTATTGC-3′,
TGF-β: R 5′-CTTCCAGCCGAGGTCCTT-3′,
TβR1: F 5′-CCCTGGACACCAACTATTGC-3′,
TβR1: R 5′-CTTCCAGCCGAGGTCCTT-3′,
SMAD3: F 5′-AGACCCCACCCCCTGGCTACCTG-3′,
SMAD3: R 5′-GGGGACATCGGATTCGGGGA-3′,
SMAD7: F 5′-TGGATGGCGTGTGGGTTTA-3′,
SMAD7: R 5′-TGGCGGACTTGATGAAGATG-3′,
IL-11: F 5′-ACTGCTGCTGCTGAAGACTC-3′,
IL-11: R 5′-CCACCCCTGCTCCTGAAATA-3′,
CXCR4: F 5′-CAGTGGCCGACCTCCTCTT-3′,
CXCR4: R 5′-CAGTTTGCCACGGCATCA-3′,
PTHrP: F 5′-TTCTTCCCAGGTGTCTTGAG-3′,
PTHrP: R 5′-TTTACGGCGACGATTCTTCC-3′,
Snail1: F 5′-CGAGTGGTTCTTCTGCGCTA-3′,
Snail1: R 5′-CTGCTGGAAGGTAAACTCTGGA-3′,
Snail2: F 5′-GGCTGGCCAAACATAAGCAG-3′,
Snail2: R 5′-GCTTCTCCCCCGTGTGAGTT-3′.
Plasmids and shRNA
Human SSBP1 cDNA was subcloned from the MDA-MB-231 breast cancer cell line into the pCDH-CMV-MCS-EF1-Puro lentiviral vector with a Flag tag. The cloned primer sequence is as follows:
Forward: 5′-CCGGAATTCGCCACCATGGACTACAAGGACGATGATGACAAG CTCGATGGAGGAATGTTTCGAAGACCTGTATT-3′
Reverse: 5′-CGCGGATCCCTACTCCTTCTCTTTCGTCT-3′
SSBP1 shRNAs and the negative control were purchased from GeneChem and expressed in the GV248 backbone. The target sequences are as follows:
shRNA-1: 5′-AGTTTGGTTCTTGAAAGAT-3′;
shRNA-2: 5′-TGGCACAGAATATCAGTAT-3′.
Lentivirus packaging and infection
Briefly, 293T cells were cotransfected with lentiviral vectors and the packaging vectors PCDH (or GV248), psPAX2 and pMD2G. Forty-eight hours after transfection, the viral supernatants were collected, filtered, and concentrated by ultracentrifugation. Polybrene (Sigma-Aldrich) was added at a working concentration of 8 μg/mL. The cells were incubated with virus for 12 hours, and then medium containing FBS was added. Twenty-four hours later, the infected cells were subjected to selection with 2 μg/mL puromycin for 1 week.
Western blot analysis
Whole-cell lysates were generated using Pierce T-PER (Tissue Protein Extraction Reagent; Thermo Fisher Scientific Inc.) containing protease inhibitor cocktail tablets (Roche) and phosphatase inhibitors (Roche). In total, 30 μg of the cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Pall). The membranes were blocked in 5% milk or 5% BSA, and then incubated with various primary antibodies followed by the appropriate horseradish peroxidase–conjugated secondary antibodies. Immunoreactive bands were identified using enhanced chemiluminescence, according to the manufacturer's instructions, and quantified by densitometry. The antibodies used in this study are listed in Supplementary Table S2.
Transwell assays
Cells (5 × 105 for the migration assay and 1 × 106 for the invasion assay) were plated in the top chamber of a non-coated membrane or Matrigel-coated Transwell chambers (BD Biosciences) in medium without FBS. Medium supplemented with serum was used as a chemoattractant in the bottom chamber. The cells were incubated for 15 to 20 hours, and cells that did not migrate through the pores were removed with a cotton swab. The cells on the lower surface of the membrane were stained with methanol and 0.1% crystal violet and then counted.
Calcineurin activity
Calcineurin activity was measured using a calcineurin activity assay kit (Millipore) according to the manufacturer's instructions. Briefly, total phosphatase activity in cellular extracts was measured by incubation with a calcineurin-specific RII phosphopeptide substrate in the presence or absence of EGTA. The detection of free phosphate release is based on the malachite green assay in the presence of EGTA, which was subtracted from the total activity to obtain calcineurin-specific phosphatase activity.
Measurement of mitochondrial membrane potential
Tumor cells plated in phenol red–free growth medium were treated with 1 mg/mL JC-1 dye for 30 minutes at 37°C and analyzed by FACS. Red or green JC-1 fluorescence signals were resolved by detection in the FL1 and FL3 channels, respectively. Loss of mitochondrial membrane potential was measured by a reduction in the red/green fluorescence intensity ratio.
Measurement of mitochondrial reactive oxygen species
Mitochondrial-specific superoxide production was measured by labeling live cells with MitoSOX-Red (Invitrogen). Cells were incubated with 5 μmol/L MitoSOX-red for 20 minutes at 37°C in serum-free DMEM, washed twice with Dulbecco PBS, and then immediately analyzed by FACS.
Phosphoprotein profiling with the Phospho Explorer antibody microarray
The Phospho Explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc., contains 1,318 antibodies. Each antibody has two replicates that are printed on a coated glass microscope slide together with multiple positive and negative controls. The antibody array experiment was performed according to an established protocol.
Immunofluorescence
MCF10A-stable cell lines grown on coverslips were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.5% Triton X-100 for 5 minutes at 4°C, and incubated with primary antibodies for 2 hours at 37°C. The slides were then incubated with Alexa 695–conjugated (red; Abcam) or Alexa 594–conjugated (red; Invitrogen) secondary antibodies for 40 minutes at room temperature. Images were captured with a confocal laser microscope (Leica TCS SP5 II). At least 100 cells were analyzed for each group.
Metastasis assays in nude mice
All animal work was performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Fudan University under approved protocols. In total, 5 × 105 cells were washed in PBS and injected intravenously into female BALB/c nude mice (n = 6) to study lung metastasis activity. Noninvasive bioluminescence imaging was performed to quantify the metastasis burden in target organs using an in vivo imaging system.
Luciferase assay
In the luciferase reporter assays, HEK 293T cells were seeded in 96-well plates at a density of 1 × 104 to 2 × 104 cells per well and cultured for 24 hours followed by cotransfection with c-Rel or the expression control vector at 150 μg/well; TGFβ luciferase constructs of different promoter lengths at 150 μg/well; or the promoter control vector pGL3-basic with 7.5 ng/well of the control Renilla luciferase reporter construct pRL-TK (Promega). After 48 hours, luciferase assays were performed as recommended by the Dual-Glo Luciferase Assay System (Promega) detected by a SynergyHT Multi-Mode Microplate Reader (BioTek) at 595 nm. Primers for plasmid construction are listed in Supplementary Table S3.
Chromatin immunoprecipitation
After incubating with 1% formaldehyde at room temperature for 10 minutes, the cells were pelleted and resuspended in 200 mL of lysis buffer (1% SDS; 10 mmol/L EDTA; 50 mmol/L Tris-HCl, pH 8.0). The cell lysates were then sonicated (Sonics & Materials, Inc.) until the DNA was cleaved into fragments of 200 to 500 bp. These extracts were immunoprecipitated using anti–c-Rel or normal mouse IgG (Santa Cruz Biotechnology, Inc.), and chromatin immunoprecipitation (ChIP) experiments were performed using the EZ–Magna G Chromatin Immunoprecipitation Kit (Millipore, Merck KGaA) according to the manufacturer's instructions. Subsequently, PCR for the TGFβ promoter was performed using specific primers, which are listed in Supplementary Table S4.
Statistical analyses
Results are reported as the mean ± SD or the mean ± SEM as indicated in the figure legends. The results were analyzed using the SPSS 16.0 software (SPSS) and PRISM 5.0 (GraphPad Software Inc.). Comparisons of quantitative data between two groups were analyzed with the Student t test (two-tailed; P < 0.05 was considered significant). The χ2 test was used to compare qualitative variables. Cumulative survival time was calculated using the Kaplan–Meier method, which was analyzed using the log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. P values <0.05 were considered significant.
Results
Identification of SSBP1 as a downregulated gene in highly metastatic breast cancer cells
Using the iTRAQ-labeling method with our model system, we analyzed MDA-MB-231 (human breast cancer) cell and its derived highly metastatic sublines MDA-MB-231HM and MDA-MB-231Bo (Fig. 1A). Our analysis spanned two pairs of cell lines allowing for overlapped comparisons of mitochondrial proteins from matched parental and derivative cell lines. To generate mitochondrial proteins based on quantitative abundance data, we considered proteins upregulated or downregulated at least 1.5-fold in highly metastatic variants as compared with their matched, parental cell lines. This analysis yielded quantitative mitochondrial proteins consisting of 50 and 66 proteins for the 231HM/231 and 231Bo/231 comparisons, respectively (part of results showed in Supplementary Table S5). We focused our interesting candidate on one protein SSBP1, which is significantly downregulated in MDA-MB-231HM and MDA-MB-231Bo cells compared with the parental MDA-MB-231 cells (Fig. 1B and Supplementary Fig. S1B–S1E). In addition to these two metastatic breast cancer cell lines, a panel of breast cancer cell lines was also evaluated to observe the general trend of SSBP1 gene expression. In accordance with the iTRAQ results, SSBP1 mRNA expression was generally observed in luminal breast cancer cells (MCF-7, T47D, and ZR-75-30), which are considered weakly metastatic cell lines, but SSBP1 was downregulated in basal-like breast cancer cells (BT549, MDA-MB-468, and MDA-MB-231). In particular, the highly metastatic cell lines (MDA-MB-231HM and MDA-MB-231Bo) had greater decreases in SSBP1 expression levels (Fig. 1C). In addition, Oncomine expression analysis revealed SSBP1 downregulation in various human cancers (Fig. 1D). We also studied the prognostic value of SSBP1 using an online Kaplan–Meier plot for survival analysis. For the entire cohort of patients and patients with basal-subtype tumors, low SSBP1 expression had a negative impact on relapse-free survival (Fig. 1E). However, SSBP1 expression did not correlate with relapse-free survival for patients with ER-positive and Her-2–positive breast tumors (Fig. 1E). Furthermore, patients in The Cancer Genome Atlas (TCGA) cohort with stomach adenocarcinoma and SSBP1 gene alterations had notably shorter lifespans than those with stomach adenocarcinoma without an SSBP1 gene alteration (Supplementary Fig. S2A). Taken together, these data suggest that SSBP1 may play an oncogenic role in multiple cancer types, making it worthy of further investigation.
Loss of SSBP1 promotes breast cancer cell migration and metastasis in vitro and in vivo
To further investigate the role of SSBP1 in tumor migration and metastasis, we used three breast cancer cell line models (MDA-MB-231, MDA-MB-468, and MDA-MB-231Bo) for in vitro Transwell migration and in vivo metastasis studies. First, we used two independent shRNAs targeting SSBP1 in MDA-MB-231 and MDA-MB-468 cells. An empty vector (shCon) served as control. Western blot analysis demonstrated that both shRNA constructs strongly reduced SSBP1 protein levels (Fig. 2A). Furthermore, we generated an SSBP1 stably overexpressing MDA-MB-231Bo cell line (Fig. 2A), and the results demonstrated that SSBP1 knockdown in MDA-MB-231 and MDA-MB-468 cells significantly promoted cell migration and invasion in Transwell assays (Fig. 2B–E). Using a complementary inverse approach, elevated SSBP1 expression suppressed the invasion ability of MDA-MB-231Bo cells in an invasion assay (Fig. 2F and G).
Next, to examine whether SSBP1 affects invasion and metastasis in vivo, we constructed mouse models. We labeled SSBP1 knockdown MDA-MB-231 cells and SSBP1 stably overexpressing MDA-MB-231Bo cells with a retroviral construct expressing a GFP/luciferase fusion protein (20), and tumor cell colonization and outgrowth in the lungs were monitored by noninvasive bioluminescent imaging (BLI) 6 weeks after the intravenous delivery of cells into the nude mice. Our results demonstrated that low SSBP1 expression significantly increased lung metastasis in vivo, and the lung metastasis-promoting effect was reversed after SSBP1 overexpression in MDA-MB-231Bo cells (Fig. 2H and I and Supplementary Fig. S2B). These findings confirm our hypothesis that low SSBP1 expression promotes breast cancer cell metastasis in vitro and in vivo.
Downregulation of SSBP1 induces mitochondria retrograde signaling
As SSBP1 is essential for mtDNA replication (14, 15), we hypothesized that SSBP1 downregulation would reduce the mtDNA content in cells. First, we assessed the relative mtDNA copy number by specific primers. We found that compared with control cells, the mtDNA copy number in MDA-MB-231 shSSBP1#1 and MDA-MB-468 shSSBP1#1 cells was significantly decreased without obvious differences in the amplification of the GAPDH nuclear gene (Fig. 3B). Furthermore, MDA-MB-231Bo cells had a greater decrease in mtDNA copy number compared with the parental MDA-MB-231 cells (Fig. 3A), indicating that the mtDNA copy number is associated with tumor metastasis. Next, to determine whether the reduced mtDNA content potentiates mitochondrial reactive oxygen species (ROS) generation and loss of the mitochondrial membrane potential (ΔΨm), we examined the level of mitochondrial ROS generation and ΔΨm in MDA-MB-231 and MDA-MB-468 cells. As shown in Fig. 3C and D, there was a significant increase in mitochondrial ROS generation and ΔΨm loss in SSBP1-downregulated cells. In addition, it showed that all these downstream changes similarly generated when mtDNA copy number was reduced in MDA-MB-231 cells by treated with ethidium bromide (EB). We used 30 ng/mL of EB, which is the concentration required for partial depletion of mtDNA (Supplementary Fig. S3A). The mtROS levels and p-SMAD3 levels were increased markedly in “mtDNA-reduced” MDA-MB-231 cells (MDA-MB-231 EB; Supplementary Fig. S3B and S3C). Accordingly, the “mtDNA-reduced” cells acquired increased migratory capacity (Supplementary Fig. S3D).
Dysfunctional mitochondria involved in increased ROS production and ΔΨm loss in mammalian cells can trigger changes in mitochondrial Ca2+ signaling, reducing the energetic drive for mitochondria Ca2+ import and increasing the cytosolic Ca2+ concentration, thus activating calcineurin (21). For these reasons, we assessed the role of SSBP1 in calcineurin activation and found that SSBP1 downregulation played a role in increased calcineurin activity in both MDA-MB-231 and MDA-MB-468 cells (Fig. 3E). Conversely, SSBP1 overexpression in MDA-MB-231Bo cells decreased calcineurin activity (Fig. 3E). Taken together, these results indicate that SSBP1 loss activates the calcineurin-dependent mitochondrial retrograde signaling pathway in breast cancer cells.
Knockdown of SSBP1 induces c-Rel/p50 nuclear localization and activates TGFβ promoter activity
Next, to identify the intracellular signaling network underlying the enhanced metastasis due to decreased SSBP1 expression, we surveyed potential signaling pathways using a phospho-antibody microarray technique. Analysis of the array revealed the induction of several key components of the NF-κB and TGFβ/SMAD pathways as a result of SSBP1 depletion, including NF-κB-p105/p50 (phospho-Ser893), NF-κB-p105/p50 (phospho-Ser337), SMAD2 (phospho-Ser467), and SMAD3 (phospho-Ser204; Fig. 4A). As mitochondrial retrograde signaling may serve to reprogram the nucleus, we first confirmed activation of the NF-κB pathway. The expression of NF-κB-p105/p50 (phospho-Ser893) was evaluated in the two SSBP1-downregulated breast cancer cell lines MDA-MB-231 and MDA-MB-468 (Fig. 4B). Activation of the canonical NF-κB pathway leads to nuclear translocation of the c-Rel/p50 dimer, which functions as a transcriptional activator (22). Compared with control shRNA-infected MDA-MB-231 cells, SSBP1 shRNA induced the inactivation of IκBβ (Supplementary Fig. S3E), the nuclear translocation of c-Rel/p50, and reduced cytoplasmic c-Rel. Moreover, compared with mock-infected MDA-MB-231Bo cells, SSBP1-overexpressing MDA-MB-231Bo cells reduced nuclear c-Rel and p50 expression as demonstrated by fractionation assays (Fig. 4C). Furthermore, we used MDA-MB-231 cells treated with CCCP- (the mitochondria-specific ionophore that can induce disrupted ΔΨm) and CnA (the catalytic subunit of calcineurin) overexpressing MDA-MB-231 cells as positive controls. Results showed that addition of CCCP and overexpression of CnA caused reduced cytoplasmic IκBβ levels and increased nuclear c-Rel/p50 levels, which was similar as transfection with SSBP1 shRNA. In addition, more results showed that all these changes could be reversed by FK506, a calcineurin inhibitor (23). FK506 treatment was used as a control to ascertain the role of shSSBP1-induced calcineurin activation in IκBβ reduction and c-Rel/p50 nuclear translocation. The addition of FK506 resulted in restoration of IκBβ and decreased c-Rel/p50 in both cells transfected with SSBP1 shRNA and CnA (Supplementary Fig. S3E and S3F).
Moreover, as a result of SSBP1 downregulation, MDA-MB-231 and MDA-MB-468 cells produced higher amounts of TGFβ protein and mRNA. In contrast, SSBP1 overexpression inhibited TGFβ production in MDA-MB-231Bo cells. Thus, we hypothesized that the c-Rel–p50 complex could activate TGFβ transcription. To investigate this possibility, we performed ChIP assays of SSBP1-downregulated MDA-MB-231 cells followed by PCR of the conserved TGFβ promoter region encompassing the consensus c-Rel–binding motifs. Chromatin immunoprecipitates from the c-Rel antibody were enriched for the TGFβ promoter region compared with those precipitated with an IgG control, confirming that this region contains a c-Rel–binding site. To further identify the role of SSBP1 depletion in regulating TGFβ promoter transcription, a DNA fragment between −1,033 and +316 relative to TGFβ transcription start site (TSS) was cloned into a pGL3-Basic plasmid to yield pGL3–TGFβ recombinant vector delivering TGFβ promoter (Fig. 4D and E). Results showed that sequence constructs P1, P2, and P3 increased the luciferase activity in 293T cells, but sequence P4 remained the luciferase activity similar as the control, indicating that c-Rel was able to increase the transcriptional activity of the TGFβ promoter by binding to the region located between −500 and +316 upstream of the TSS (Fig. 4F). These findings confirm that the loss of SSBP1 induces c-Rel/p50 nuclear localization, leading to increased TGFβ transcription.
SSBP1 regulates TGFβ signaling and TGFβ-induced epithelial-to-mesenchymal transition
Analysis of the antibody array revealed the induction of several key proteins within the TGFβ–SAMD pathway as a result of SSBP1 depletion. We next investigated the effects of SSBP1 modulation on key components of the TGFβ–SMAD pathway and found the elevated production of TGFβ and increased phosphorylation of SMAD3 (S423+S425), a key mediator of TGFβ signaling, in both MDA-MB-231 and MDA-MB-468 SSBP1-deficient cells (Fig. 5A–C). In contrast, SSBP1 overexpression in MDA-MB-231Bo cells markedly reduced TGFβ secretion and downregulated p-SMAD3 protein levels (Fig. 5A–C). Moreover, we examined the expression of other components of the TGFβ pathway, including TGFβ receptor 1 (TβR1) and SMAD7, but no changes were observed (Fig. 5C).
TGFβ stimulates the epithelial-to-mesenchymal transition (EMT), migration, invasion, and metastasis of breast cancer cells. MCF10A cells are a classical in vitro EMT experimental model (24, 25). MCF10A cells with stable SSBP1 depletion have typical EMT characteristics. Western blot and immunofluorescence assays demonstrated that SSBP1 deficiency resulted in lower E-cadherin expression and higher N-cadherin and fibronectin expression. Of note, treatment with GW788388 and MitoTEMPO, an inhibitor of TGFβ/SMAD3 signaling and mitochondrial ROS, respectively (26–28), mitigated the effects of SSBP1 deficiency on the cell invasive ability and EMT (Fig. 5D–G). In addition, it showed that EMT phenotype changes similarly generated when mtDNA copy number was reduced in MCF10A cells by treated with EB. And treatment with MitoTEMPO reversed the effects of EB on EMT in MCF10A-EB cells (Supplementary Fig. S3G). Furthermore, qRT-PCR analysis of MDA-MB-231 and MDA-MB-468 cells after shRNA-mediated SSBP1 knockdown indicated that SSBP1 depletion was required for the efficient induction of metastasis-related TGFβ target genes, such as IL11, PTHrP, Snail1, and Snail2. Together, these results suggest that SSBP1 depletion potentiates TGFβ/SMAD signaling and TGFβ-driven EMT.
Low SSBP1 expression correlates with SMAD3 activation and poor clinical outcome
To determine the clinical relevance of the above findings in advanced human cancers, we first analyzed the Oncomine database and found that SSBP1 is downregulated in various human cancers (Fig. 1D). On the basis of these data, downregulation or loss of SSBP1 may be an early event in tumorigenesis that contributes to metastasis. In addition, using the cBio database (29), patients with stomach adenocarcinoma and SSBP1 gene alterations have shorter lifespans than those with stomach adenocarcinoma without an SSBP1 alteration in the TCGA cohort.
In light of our observations, we sought to determine whether SSBP1 and p-SMAD3 levels correlated with breast cancer patient prognosis. We examined the expression of SSBP1 and p-SMAD3 in TMAs containing 250 breast tumor specimens by immunohistochemical analysis (Fig. 6A). Of note, patients with low SSBP1 expression were significantly associated with histologic grades (grades III and IV; P = 0.01, Pearson χ2 test; Fig. 6B) and lymph node metastasis (P = 0.009; Fig. 6B). Importantly, low SSBP1 expression and high p-SMAD3 expression, alone or combined, correlated with worse disease-free survival (Fig. 6C–E). Moreover, univariate analysis indicated that low SSBP1 expression at diagnosis was associated with a higher risk for disease relapse (Table 1). Additional multivariate COX analysis exhibited a similar trend as the univariate analysis (Table 1). Finally, we tested the correlation between SSBP1 and p-SMAD3 expression in the same breast tumor samples and observed a significant inverse correlation between their expressions (Spearman ρ = −0.282, P < 0.001; Fig. 6F and G). These data suggest the potential use of SSBP1 for prognostic stratification of patients with breast cancer and confirm our hypothesis that SSBP1 loss promotes human breast cancer metastasis by controlling SMAD-dependent TGFβ signaling.
. | Univariate analysis . | Multivariate analysis . | ||
---|---|---|---|---|
. | HR (95% CI) . | P . | HR (95% CI) . | P . |
Age | 0.978 (0.573–1.668) | 0.935 | 0.933 (0.523–1.665) | 0.815 |
Timor size | 1.373 (0.795–2.371) | 0.256 | 0.953 (0.532–1.707) | 0.871 |
Lymph node status | 2.701 (1.563–4.668) | 0.000 | 2.230 (1.247–3.988) | 0.007 |
Histologic grade | 1.769 (1.031–3.035) | 0.038 | 1.339 (0.744–2.410) | 0.330 |
ER status | 0.815 (0.471–1.409) | 0.464 | 1.379 (0.657–2.895) | 0.395 |
PR status | 0.484 (0.228–1.026) | 0.058 | 0.371 (0.142–0.967) | 0.043 |
Her-2 status | 0.915 (0.530–1.582) | 0.752 | 0.709 (0.378–1.329) | 0.283 |
SSBP1 | 0.348 (0.202–0.598) | 0.000 | 0.385 (0.211–0.703) | 0.002 |
. | Univariate analysis . | Multivariate analysis . | ||
---|---|---|---|---|
. | HR (95% CI) . | P . | HR (95% CI) . | P . |
Age | 0.978 (0.573–1.668) | 0.935 | 0.933 (0.523–1.665) | 0.815 |
Timor size | 1.373 (0.795–2.371) | 0.256 | 0.953 (0.532–1.707) | 0.871 |
Lymph node status | 2.701 (1.563–4.668) | 0.000 | 2.230 (1.247–3.988) | 0.007 |
Histologic grade | 1.769 (1.031–3.035) | 0.038 | 1.339 (0.744–2.410) | 0.330 |
ER status | 0.815 (0.471–1.409) | 0.464 | 1.379 (0.657–2.895) | 0.395 |
PR status | 0.484 (0.228–1.026) | 0.058 | 0.371 (0.142–0.967) | 0.043 |
Her-2 status | 0.915 (0.530–1.582) | 0.752 | 0.709 (0.378–1.329) | 0.283 |
SSBP1 | 0.348 (0.202–0.598) | 0.000 | 0.385 (0.211–0.703) | 0.002 |
NOTE: Bold values are statistically significant (P < 0.05).
Abbreviations: ER, estrogen receptor; PR, progesterone receptor; CI, confidence interval.
Discussion
In this report, we demonstrate that SSBP1 downregulation induces a significant decrease in the mtDNA copy number in breast cancer cells, which initiates the calcineurin-mediated mitochondrial retrograde signaling pathway to alter nuclear gene expression. The importance of retrograde signaling has been demonstrated for changes in mitochondrial Ca2+ signaling and mitochondrial ROS production (30). Decreases in mitochondrial membrane electrochemical potential reduce the energetic drive for mitochondria to import Ca2+, disrupting Ca2+ homeostasis in the cytoplasm (21, 31). Numerous studies have supported a role for mtDNA defects in tumorigenesis in a wide range of human cancers (32–36), but the upstream regulators of mtDNA alteration remain largely elusive. Here, our data demonstrate that SSBP1 downregulation regulated mtDNA content, activated mitochondrial retrograde signaling, and drove EMT in breast cancer cells. This finding led to the identification of SSBP1 as a novel mitochondrial biomarker for breast tumor progression.
The TGFβ–SMAD pathway has been shown to play a role in tumor progression and EMT. Tumor cells normally secrete abundant TGFβ, which is the most potent inducer of EMT in epithelial cancers and promotes cell invasion and metastasis (37, 38). TGFβ exerts its effects through the activation of its receptor and phosphorylated major effector SMADs (SMAD2/SMAD3) translocate to the nucleus to activate EMT (39). Although substantial data have clarified the downstream molecular networks that trigger EMT through TGFβ, the regulatory controls of the oncogenic functions of TGFβ have not been well elucidated. More importantly, elucidation of the regulatory controls governing the function of TGFβ is critical for the design of novel therapies targeting the oncogenic arm of the pathway. Our data show that SSBP1 loss results in calcineurin-mediated mitochondrial retrograde signaling, which plays a causal role in inducing c-Rel/p50 nuclear localization and activating TGFβ transcription. These results indicate that SSBP1 may serve as a novel negative regulator of TGFβ–SMAD3-driven EMT.
When activated, EMT is thought to be a driver of invasion and metastasis in different types of epithelial cancers and to potentiate cancer cell resistance to current chemotherapies (40). The results from MCF10A cells with SSBP1 downregulation are consistent with the induction of a mesenchymal transdifferentiation program. Loss of SSBP1 in MCF10A cells results in a partial EMT morphology change, including the upregulation of fibronectin and N-cadherin, well-known markers of early EMT induction. Thus, cumulative results indicate that SSBP1 loss may play an early role in the induction of EMT.
In addition, using Oncomine expression analysis, we showed that SSBP1 undergoes a copy-number reduction in many human cancers, including breast cancer, suggesting that the potential importance of SSBP1 in initiation and progression is not limited to breast cancer. On the basis of data from the TCGA cohort, patients with stomach adenocarcinoma and SSBP1 gene alterations have shorter lifespans than those with stomach adenocarcinoma without an SSBP1 alteration. Moreover, p-SMAD3 levels correlated with SSBP1 levels in our breast cancer TMA, further validating that SSBP1 plays an essential role in the control of TGFβ–SMAD pathways. Furthermore, low SSBP1 expression is closely associated with breast cancer metastasis in mouse cancer models. TMAs of samples from breast cancer patients revealed that low SSBP1 and high p-SMAD3 expression correlated with poor prognosis. Our results suggest that further studies should examine the potential clinical utility of SSBP1 and p-SMAD3 as prognostic biomarkers for aggressive disease.
In summary, using a combination of in vivo and in vitro functional metastasis assays and extensive clinical correlation analysis, we provide compelling evidence that SSBP1 is a tumor suppressor involved in breast cancer and provide a novel paradigm for SSBP1-mediated activation of mitochondrial retrograde signaling, increased TGFβ transcription, and subsequent TGFβ–SMAD3-driven cell invasion and cancer metastasis (Fig. 6H). In contrast, in the presence of SSBP1, TGFβ secretion decreases, and the oncogenic TGFβ–EMT signaling is restrained. In recent years, many studies have demonstrated that various TGFβ antagonists have partial success in clinical trials (41, 42). Our findings that SSBP1 deficiency promotes invasion and metastasis, whereas its overexpression restricts this process provide insights into mechanisms and strategies for the therapeutic intervention of the TGFβ pathway and cancer metastasis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: H.-L. Jiang, H.-F. Sun, W. Jin
Development of methodology: H.-L. Jiang, H.-F. Sun
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.-L. Jiang, H.-F. Sun
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.-L. Jiang, H.-F. Sun, S.-P. Gao, L.-D. Li, S. Huang
Writing, review, and/or revision of the manuscript: H.-L. Jiang, H.-F. Sun
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Hu, S. Liu, J. Wu, Z.-M. Shao
Study supervision: W. Jin
Acknowledgments
The authors thank Dr. Toshiyuki Yoneda (The University of Texas, Houston, TX) for the high-osteolytic metastasis (Bo) MDA-MB-231 Bo cells.
Grant Support
This work was supported by the grants from National Natural Science Foundation of China (31170778 and 81272923) and Program for New Century Excellent Talents in University (NCET-12-0127).
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