Genomic alterations are crucial for the development and progression of human cancers. Copy-number gains found in genes encoding metabolic enzymes may induce triple-negative breast cancer (TNBC) adaptation. However, little is known about how metabolic enzymes regulate TNBC metastasis. Using our previously constructed multiomic profiling of a TNBC cohort, we identified decaprenyl diphosphate synthase subunit 1 (PDSS1) as an essential gene for TNBC metastasis. PDSS1 expression was significantly upregulated in TNBC tissues compared with adjacent normal tissues and was positively associated with poor survival among patients with TNBC. PDSS1 knockdown inhibited TNBC cell migration, invasion, and distant metastasis. Mechanistically, PDSS1, but not a catalytically inactive mutant, positively regulated the cellular level of coenzyme Q10 (CoQ10) and intracellular calcium levels, thereby inducing CAMK2A phosphorylation, which is essential for STAT3 phosphorylation in the cytoplasm. Phosphorylated STAT3 entered the nucleus, promoting oncogenic STAT3 signaling and TNBC metastasis. STAT3 phosphorylation inhibitors (e.g., Stattic) effectively blocked PDSS1-induced cell migration and invasion in vitro and tumor metastasis in vivo. Taken together, our study highlights the importance of targeting the previously uncharacterized PDSS1/CAMK2A/STAT3 oncogenic signaling axis, expanding the repertoire of precision medicine in TNBC.

Significance:

A novel metabolic gene PDSS1 is highly expressed in triple-negative breast cancer tissues and contributes to metastasis, serving as a potential therapeutic target for combating metastatic disease.

Breast cancer is a heterogeneous disease consisting of four major molecular subtypes: luminal A, luminal B, HER2/ERBB2, and basal-like (1). Clinically, the basal-like subtype tends to be negative for expression of the estrogen receptor, progesterone receptor, and HER2, also referred to as triple-negative breast cancer (TNBC); this phenotype accounts for 15%–20% of breast cancer cases (2). In contrast to the successful development of therapies for hormone receptor–positive and/or HER2-positive breast cancers, little progress has been made in identifying positively expressed molecular targets in TNBC that are druggable (3). Because of the highly aggressive nature of TNBC and the absence of effective therapeutics, elucidating the determinants of its aggressiveness and identifying potential therapeutic avenues are high priorities (4).

Metabolic alterations contribute to the reprogramming of metabolic and oncogenic signaling pathways to meet the requirements of cancer cells for their biosynthesis processes, growth, and metastasis of cancer cells (5). Genomic alterations can also result in copy-number gains and losses in genes encoding metabolic enzymes, which may induce vulnerabilities (6, 7). Therefore, we aimed to identify activated and/or overexpressed metabolic genes in TNBC as high-potential targets.

We performed our bioinformatics analysis of TNBC on the cohort from the Fudan University Shanghai Cancer Center (FUSCC, Shanghai, P.R. China; ref. 8) and identified decaprenyl diphosphate synthase subunit 1 (PDSS1), which elongates the prenyl side chain of coenzyme Q10 (CoQ10) in the quinone biosynthesis pathway, as a prometastatic gene. Previous study revealed that loss of PDSS1 expression disrupted the biosynthesis of CoQ10, which regulates various cellular processes in mammalian cells (9, 10). Specifically, it was reported that CoQ10 enhances the migratory phenotype, regulates mitochondrial electronic transport chain activity, acts as an antioxidant, participates in de novo pyrimidine synthesis and increases intracellular calcium levels (11–15). CoQ10 deficiency may cause several diseases, including a type of infantile multisystem disorder (16, 17). Additionally, CoQ10 supplementation tended to induce disease recurrence in patients with postsurgical breast cancer (18).

The role of PDSS1 in human cancers remains uninvestigated. In this study, we found that PDSS1 is frequently overexpressed in TNBC due to copy-number gain. Thus, we aimed to reveal the function of PDSS1, elucidate the molecular mechanism by which PDSS1 contributes to TNBC progression as well as explore the therapeutic potential of PDSS1 in relevant models.

Tissues and datasets

A total of 10 pairs of primary TNBC tumor tissues and adjacent normal breast tissues (usually 2 to 5 cm away from the lesion) were obtained from patients with TNBC who underwent surgery at FUSCC. These patients did not receive any therapy before surgery. Freshly resected samples were immediately stored at −80°C. All procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Ethics Review Board of FUSCC. Written informed consent was obtained from all of the patients. In addition, several public datasets [FUSCC TNBC, The Cancer Genome Atlas (TCGA), GSE1456, GSE25066, Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), GSE65216, and GSE21653] were also analyzed.

Cell lines

An immortalized normal human mammary epithelial cell line (HMEC), several human TNBC cell lines (MDA-MB-436, MDA-MB-231, Hs-578t, BT-549, MDA-MB-453, and BT-20), and a human embryonic kidney cell line (HEK293T) were obtained from ATCC. These cells were cultured following the instructions from ATCC. MCF10A-CA1a (CA1a) cells were kindly provided by Guo-Hong Hu (Shanghai Institutes for Biological Sciences). The CA1a cell line was cultured in DMEM/F12 medium (Gibco) containing 5% horse serum (Gibco), 20 ng/mL epidermal growth factor (Invitrogen), 0.5 μg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma), and 10 μg/mL insulin (Sigma). All cell lines were authenticated by short tandem repeat analyses and tested negative for Mycoplasma contamination. All cell lines were passaged less than 30 times and maintained at 37°C in an atmosphere containing 5% CO2.

Transfection and virus infection

Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) or Neofect transfection reagent (Tengyi Biotech) was used for transient plasmid transfection with the corresponding standard protocol. siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen). The silencing efficiency was assessed 48–72 hours after transfection.

To generate stable cell lines expressing specific shRNAs or cDNAs, HEK293T cells were transfected with the appropriate lentiviral expression vector and packaging plasmids (psPAX2 and pMD2.G) using Neofect DNA transfection reagents. Each virus-containing supernatant was collected 48 hours after transfection, filtered through a 0.45 μmol/L syringe filter and used to infect target cells in the presence of 8 μg/mL polybrene (Santa Cruz Biotechnology) prior to drug-based selection with 1 μg/mL puromycin (InvivoGen) for 1 week. The knockdown or overexpression efficiency was validated with qRT-PCR or immunoblotting.

Plasmid and siRNA construction

The cDNA sequences of PDSS1 and the mutant PDSS1-D308E were cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro (System Biosciences). Flag-CAMK2A was purchased from GeneChem and HA-STAT3 was purchased from Vigene. To generate various truncated HA-STAT3 and Flag-CAMK2A constructs, the corresponding cDNAs were amplified by PCR and cloned into the pCDH-CMV-MCS-EF1-Puro vector. The short hairpin (shRNA) oligos were annealed and cloned into the pLKO.1-Puro vector. The plasmid constructs expressing the shRNAs were designed by Sigma-Aldrich. Specific siRNAs were synthesized by RiboBio. The related sequences are listed in Supplementary Table S1.

RNA isolation and qRT-PCR

RNA was extracted from treated cells using TRIzol reagent (Invitrogen) and reverse transcribed to cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) following the manufacturer's recommended protocols. Primers were designed from information available on the PrimerBank website. qRT-PCR was performed using SYBR Premix Ex Taq (TAKARA) in an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). qRT-PCR data for mRNA expression levels are shown relative to the expression levels of the reference gene. The formula 2(−ΔCt) was used to calculate the relative expression values. The gene-specific primers used are listed in Supplementary Table S2.

Western blotting

For immunoblot analysis, proteins were resolved by SDS-PAGE, after which, the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore) that were incubated with the indicated primary and secondary antibodies. Corresponding antibody-specific signals were detected with enhanced chemiluminescence substrate (Pierce Biotechnology). Image acquisition was performed with a Molecular Imager ChemiDoc XRS+ system with Image Lab Software (Bio-Rad).

Immunoprecipitation

To immunoprecipitate exogenously expressed and endogenous proteins, cell extracts were incubated with primary antibodies or control IgG in a rotating incubator overnight at 4°C and were then incubated with protein A/G magnetic beads (Bimake) for another 2 hours at 4°C. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting.

Antibodies

The following primary antibodies were used: anti-ACTB (Cell Signaling Technology), anti-PDSS1 (Novus), anti-VCL (Cell Signaling Technology), anti-SNAI2 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-phospho (p)-STAT3-705 (Cell Signaling Technology), anti-Flag (Sigma), anti-HA (Cell Signaling Technology), anti-JAK2 (Cell Signaling Technology), anti-p-JAK2 (Cell Signaling Technology), anti-HDAC1 (Cell Signaling Technology), anti-α–tubulin (Cell Signaling Technology), anti-p-CAMK2A (Abcam), and anti-CAMK2A (Proteintech). Horseradish peroxidase–conjugated anti-mouse (1:5,000 dilution) and anti-rabbit secondary antibodies (1:5,000 dilution) were purchased from Jackson ImmunoResearch.

Measurement of intracellular free Ca2+ concentrations

The indicated cells were plated in 4-well chamber slides (Nunc Lab-Tek II Chamber Slide, Thermo Fisher Scientific). After 24 hours, the cells were washed with Hank's Balanced Salt Solution (HBSS; Invitrogen) and preincubated with 2 μmol/L Fluo4-AM (Dojindo) and 1 μg/mL Hoechst 33258 (Beyotime) dissolved in HBSS at 37°C for 30 minutes. After they were washed, the cells were incubated in HBSS at 37°C for 20 minutes. Representative images were acquired with a Leica confocal microscope. In addition, the cells were digested and collected, and the fluorescence intensity was evaluated and quantified by flow cytometry according to the manufacturer's instructions.

Purification of cytoplasmic/nuclear proteins

Nuclear and cytoplasmic proteins were purified using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions.

Cell migration and invasion assays

For the cell migration and invasion assays, cells were suspended in serum-free medium and seeded (4–20 × 104 cells per well) in the top chambers (Corning). The top chambers were coated with Matrigel (Corning) for the invasion assay; the migration assay did not include a Matrigel coating step. The lower chambers contained 600 μL of medium supplemented with 10% FBS or 20% horse serum. After incubation for 4–16 hours, the cells on the underside of the filter membrane were fixed with methanol for 20 minutes. The unmigrated and uninvaded cells were removed using cotton swabs, and the cells that had migrated or invaded through the filter membrane were then stained with 0.1% crystal violet, photographed, and counted using ImageJ (NIH, Bethesda, MD). In addition, to exclude the secondary effect of cell proliferation caused by PDSS1, cells were also treated with 10 μg/mL mitomycin C for 2 hours to inhibit proliferation prior to performing cell migration and invasion assays.

Wound-healing assay

For the wound-healing assay to measure cell migration, the indicated cells were seeded in 6-well plates (0.5–2 × 106 cells per well) and incubated at 37°C. Upon reaching 100% confluence, the cell monolayers were scratched with sterile 200 μL pipette tips and washed with medium to remove any detached cells. Images were acquired at 0 and 12 hours (or 24 hours), and the wound-healing (% coverage area) rate was calculated and analyzed.

In vivo metastasis assays

For metastasis assays, 6 to 8 weeks old female NOD/SCID mice were injected via the tail vein with MDA-MB-231 cells (stably expressing shPDSS1 or shCTRL; 1 × 106 cells per mouse) or MCF10A-CA1a cells (stably expressing pCDH, PDSS1-WT, or PDSS1-D308E; 1 × 106 cells per mouse). 4T1 cells (stably expressing shPDSS1 or shCTRL; 2 × 105 cells per mouse) were injected via the tail vein into 6 to 8 weeks old female BALB/c mice. The number of injected cells was quantified by d-luciferin injection and IVIS Spectrum (Bruker) immediately after the injection as a reference signal. Lung metastasis bioluminescence imaging (BLI) signals were monitored regularly. The mice were euthanized at the indicated time, and lungs were collected by 4% paraformaldehyde, followed by fixation and hematoxylin and eosin (H&E) staining to analyze the metastatic burden. For chemical drug treatment in vivo, Stattic or cisplatin was administered 1 week after injection of cells. Stattic (2 mg/kg) or cisplatin (2 mg/kg) in a volume of 100 μL was administered intraperitoneally twice weekly. All animal procedures were approved by the Animal Care and Use Committee of Fudan University (Shanghai, P.R. China).

IHC staining

Paraffin-embedded tissue sections were deparaffinized at 60°C for 20 minutes, cleared in xylene, and rehydrated in a graded alcohol series. For H&E staining, the slides were stained with Mayer's hematoxylin (Sigma), blued with 0.1% sodium bicarbonate, and counterstained with eosin Y solution (Sigma). IHC staining was performed as described previously (19) with anti-p-STAT3 (1:100) and anti-PDSS1 (1:50) primary antibodies. Images visible under an Olympus BX43 microscope were acquired. Interpretation of the IHC results was performed by two independent pathologists who were blinded to the clinicopathologic information. Slides were evaluated using light microscopy and assigned a standard semiquantitative immunoreactivity score as described previously (19). The percentage of positively stained area (0 = negative, 1 < 10%, 2 = 10%–50%, 3 > 50%) and the staining intensity (0 = no, 1 = weak, 2 = moderate, 3 = strong) were recorded for each sample, and an immunoreactivity score (0–9) was calculated by multiplying the positive staining score by the staining intensity score. Low and high expression were defined according to the median immunoreactivity score.

RNA sequencing

RNA was extracted from MDA-MB-231 cell lines treated with control siRNA or two independent PDSS1-targeted siRNAs using TRIzol reagent (Invitrogen). In brief, RNA sequencing (RNA-seq) library construction was performed using a VAHTS mRNA-seq V2 Library Prep Kit for Illumina (#NR601-01, Vazyme) prior to sequencing on the HiSeq-2500 platform (Illumina). Sequencing reads were aligned to the hg38 genome assembly using HISAT2 software. Transcripts per kilobase of exon model per million mapped reads values for each gene were calculated using cufflinks. A fold change ≥ 1.5 or ≤ 0.67 was set as the threshold for identifying differentially expressed genes (DEG).

Chemical products

The following chemical products were used: Stattic, a potent STAT3 phosphorylation inhibitor (MedChemExpress); napabucasin, a STAT3 phosphorylation inhibitor (MedChemExpress); mitomycin C, a cell-cycle inhibitor (Selleck); KN-93, a CaMKII inhibitor (MedChemExpress); BAPTA-AM, a membrane-permeable Ca2+ chelator (MedChemExpress); cisplatin, a chemotherapeutic drug (MedChemExpress); IL6, a pleiotropic cytokine (PeproTech); and CoQ10, an endogenous human metabolite (MedChemExpress).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7.0 and R software. Survival curves were plotted using the Kaplan–Meier method and compared using log-rank tests. Relapse-free survival (RFS) was assessed from the date of surgery to the date of local relapse or distant relapse. Overall survival was assessed from the date of surgery to the date of death or last follow-up. Patients without events or death were censored at the last follow-up. P < 0.05 indicates a statistically significant difference. The statistical details and methods used are indicated in the figure legends or text as appropriate.

Other methods

Methods used for supplementary figures are provided in the Supplementary Materials and Methods.

PDSS1 copy-number gain and overexpression are common in TNBCs and associated with poor prognosis

To identify dysregulated metabolic genes involved in TNBC, an unbiased bioinformatic analysis of metabolic genes was conducted with data from the FUSCC TNBC cohort (8). A total of 133 previously reported high-priority genes (6) were included in the screening of DEGs in paired tumor and adjacent normal tissues, and 59 of the selected genes were found to be upregulated in TNBC (fold change > 2; Fig. 1A). We further sought to identify candidates from the above gene set whose overexpression could be explained by gene amplification. There are merely three genes (DHTKD1, PDSS1, PFKP) that are not only located on chromosome 10p, a chromosomal region frequently reported to be amplified in TNBC (20–23), but also showed a high correlation (R > 0.3) between gene amplification and expression along with a relatively strong copy-number gain and amplification at the genomic level.

Figure 1.

PDSS1 is upregulated in TNBC samples and its high expression predicts poor outcomes in TNBC patients. A, Flow chart of bioinformatic screening of metabolic genes from the FUSCC TNBC cohort. R, Pearson correlation coefficient. B, Transwell assays to assess migration. Quantification of relative migrated cells is shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. C, Correlation of the PDSS1 mRNA level with its DNA copy number in TNBC tissues from the FUSCC and TCGA datasets. D,PDSS1 mRNA levels in 88 TNBC tissues and matched normal breast tissues. Two-tailed Student t test. E, PDSS1 protein expression was quantified in 10 primary TNBC tissues and matched adjacent normal tissues by Western blot analysis. F,PDSS1 expression in TNBC tissues of different histologic grades (left). PDSS1 expression in TNBC tissues of different subtypes (right). Wilcoxon rank-sum test. G, The mRNA expression profiles of PDSS1 in TNBC and non-TNBC samples from the GSE65216 and GSE21653 datasets. Wilcoxon rank-sum test. H, PDSS1 protein expression according to PAM50 subtype in 45 patients subjected to proteomic analysis. I, Kaplan–Meier survival analysis for TNBC patients from two independent cohorts (FUSCC and TCGA) stratified by PDSS1 mRNA level. ***, P < 0.001; ns, not significant.

Figure 1.

PDSS1 is upregulated in TNBC samples and its high expression predicts poor outcomes in TNBC patients. A, Flow chart of bioinformatic screening of metabolic genes from the FUSCC TNBC cohort. R, Pearson correlation coefficient. B, Transwell assays to assess migration. Quantification of relative migrated cells is shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. C, Correlation of the PDSS1 mRNA level with its DNA copy number in TNBC tissues from the FUSCC and TCGA datasets. D,PDSS1 mRNA levels in 88 TNBC tissues and matched normal breast tissues. Two-tailed Student t test. E, PDSS1 protein expression was quantified in 10 primary TNBC tissues and matched adjacent normal tissues by Western blot analysis. F,PDSS1 expression in TNBC tissues of different histologic grades (left). PDSS1 expression in TNBC tissues of different subtypes (right). Wilcoxon rank-sum test. G, The mRNA expression profiles of PDSS1 in TNBC and non-TNBC samples from the GSE65216 and GSE21653 datasets. Wilcoxon rank-sum test. H, PDSS1 protein expression according to PAM50 subtype in 45 patients subjected to proteomic analysis. I, Kaplan–Meier survival analysis for TNBC patients from two independent cohorts (FUSCC and TCGA) stratified by PDSS1 mRNA level. ***, P < 0.001; ns, not significant.

Close modal

We next examined whether these three genes affect the migratory properties of TNBC cells in vitro, as the ability of TNBC cells to invade surrounding tissues and metastasize to distant organs is an important hallmark of TNBC. Three siRNAs targeting each gene were synthesized and used in combination to inhibit the expression of each gene in MDA-MB-231 cells, a TNBC cell line (Supplementary Fig. S1A). Transwell assays showed that silencing PDSS1 or PFKP, but not DHTKD1, significantly inhibited the migration ability of MDA-MB-231 cells (Fig. 1B; Supplementary Fig. S1B). PFKP was previously reported to be associated with the progression of several cancers (24–26). However, only one study has shown that SNPs in PDSS1 are associated with survival in cutaneous melanoma (27), and to date, no reports have linked the function of PDSS1 with cancer progression or metastasis. Hence, we were particularly interested in exploring the potential prometastatic roles of PDSS1 and focused on this gene in the subsequent work described below.

To study the clinical significance of PDSS1 expression, TNBC samples from the FUSCC and TCGA databases were analyzed. The mRNA levels of PDSS1 in TNBC were highly correlated with the degree of PDSS1 gene amplification; copy-number gain was detected in more than half of the FUSCC TNBC samples (Fig. 1C). The mRNA (Fig. 1D) and protein (Fig. 1E) levels of PDSS1 were higher in TNBC tissues than in paired adjacent normal breast tissues in the FUSCC cohort. Moreover, PDSS1 expression was higher in grade 3 tumors than in grade 2 tumors (Fig. 1F, left). In contrast to other subtypes, the basal-like subtype of TNBC exhibited markedly upregulated PDSS1 expression (Fig. 1F, right). Further analysis of PDSS1 expression among all patients with breast cancer showed that those with TNBC or the basal-like subtype had significantly higher PDSS1 expression levels than those with other molecular subtypes of breast cancer (Fig. 1G; Supplementary Fig. S1C). Consistently, PDSS1 protein expression was also found to be upregulated in the basal-like subtype compared with other subtypes based on proteomics analysis of a public dataset (Fig. 1H; ref. 28). Correlation analysis of PDSS1 mRNA and protein levels showed that they were tightly correlated (r = 0.75) in breast cancers (Supplementary Fig. S1D).

We then evaluated the prognostic significance of PDSS1 expression in patients with TNBC. Kaplan–Meier survival analysis indicated that the high expression of PDSS1 was significantly associated with reduced RFS in the FUSCC TNBC cohort (Fig. 1I, left). Moreover, the prognostic significance of PDSS1 expression was further validated in data from TCGA TNBC cohort (Fig. 1I, right). In particular, multivariate Cox regression analysis showed that PDSS1 overexpression was an independent risk factor for decreased survival times in patients with TNBC (Supplementary Table S3). Together, these findings suggest that PDSS1 contributes to the clinically relevant features of TNBC and plays an important role in TNBC metastasis.

PDSS1 promotes TNBC migration, invasion, and distant metastasis

To examine the effects of the PDSS1 on tumor progression, we first analyzed the PDSS1 protein expression levels in a normal HMEC line and several well-characterized TNBC cell lines (Fig. 2A). We then stably overexpressed PDSS1 in BT-549 and Hs-578t cells with low PDSS1 levels via lentiviral infection, as confirmed by Western blotting (Fig. 2B). We also established two model cell lines with shRNA-mediated PDSS1 knockdown based on the MDA-MB-231 and MBA-MB-436 cell lines (Fig. 2C). CCK-8 assays indicated that ectopic expression of PDSS1 slightly promoted TNBC cell proliferation, whereas knockdown of PDSS1 led to a slight reduction in proliferation (Supplementary Fig. S2A and S2B). The results of the colony formation assays suggested that overexpression or knockdown of PDSS1 mildly enhanced or impaired the clonogenicity of TNBC cells, respectively (Supplementary Fig. S2C and S2D). To confirm these findings, we investigated the effects of the PDSS1 gene on tumor growth in vivo and found that silencing of PDSS1 slightly impaired the growth of TNBC tumors (Supplementary Fig. S2E).

Figure 2.

PDSS1 drives TNBC cell migration, invasion, and distant metastasis. A, Western blot analysis of PDSS1 expression in HMECs and TNBC cell lines. B, Overexpression of PDSS1 in BT-549 and Hs-578t cells was confirmed by Western blot analysis. C, Knockdown of PDSS1 by two independent shRNAs in MDA-MB-231 and MDA-MB-436 cells was confirmed by Western blot analysis. D–G, Wound-healing assays were performed to evaluate the effects of PDSS1 gain and loss of function on the migration of the indicated TNBC cells. Representative images and quantification of wound closure are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. H–K, Transwell assays were performed to evaluate the effects of PDSS1 gain and loss of function on the migration and invasion of the indicated TNBC cells. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. Scale bars, 100 μm. NOD/SCID mice were injected with shCTRL or shPDSS1#1 MDA-MB-231 cells via tail vein. L–N, BLI signals at the indicated time points; representative images of BLI (L), overall survival times (n = 8 mice per group; M), representative lung H&E staining images and quantification of lung metastatic areas (n = 6 mice per group; N) are shown. Data are mean ± SEM. Scale bars, 2 mm. Two-tailed Student t test. ***, P < 0.001.

Figure 2.

PDSS1 drives TNBC cell migration, invasion, and distant metastasis. A, Western blot analysis of PDSS1 expression in HMECs and TNBC cell lines. B, Overexpression of PDSS1 in BT-549 and Hs-578t cells was confirmed by Western blot analysis. C, Knockdown of PDSS1 by two independent shRNAs in MDA-MB-231 and MDA-MB-436 cells was confirmed by Western blot analysis. D–G, Wound-healing assays were performed to evaluate the effects of PDSS1 gain and loss of function on the migration of the indicated TNBC cells. Representative images and quantification of wound closure are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. H–K, Transwell assays were performed to evaluate the effects of PDSS1 gain and loss of function on the migration and invasion of the indicated TNBC cells. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. Scale bars, 100 μm. NOD/SCID mice were injected with shCTRL or shPDSS1#1 MDA-MB-231 cells via tail vein. L–N, BLI signals at the indicated time points; representative images of BLI (L), overall survival times (n = 8 mice per group; M), representative lung H&E staining images and quantification of lung metastatic areas (n = 6 mice per group; N) are shown. Data are mean ± SEM. Scale bars, 2 mm. Two-tailed Student t test. ***, P < 0.001.

Close modal

We next assessed the role of PDSS1 in cell migration and invasion. Wound-healing assays showed that increased PDSS1 expression in BT-549 and Hs-578t cells significantly promoted cell migration (Fig. 2D and E), while reduced PDSS1 expression in MDA-MB-231 and MBA-MB-436 cells significantly suppressed cell migration (Fig. 2F and G). Similar patterns were observed in transwell assays, which demonstrated that the expression of PDSS1 was positively related to the migration and invasion abilities of TNBC cells (Fig. 2HK). The impact of PDSS1 on TNBC cell migration and invasion was also verified, with cellular proliferation inhibited by mitomycin C (Supplementary Fig. S3A–S3D). Given that PDSS1-mediated signaling was found to be associated with cell migration and invasion, we sought to determine whether inhibition of PDSS1 would affect tumor lung metastasis in vivo. Remarkably, PDSS1 knockdown reduced tumor cell colonization in the lung, as evidenced by BLI signals (Fig. 2L), and led to a shorter overall survival compared with that of control mice (Fig. 2M). Our H&E analysis supported this observation, showing a significant decrease in the area of metastatic nodules in the lungs of mice that were injected with PDSS1-knockdown cells (Fig. 2N). Importantly, this impaired metastatic ability caused by PDSS1 silencing was also repeated in immunocompetent mice (Supplementary Fig. S4A–S4D). These in vitro and in vivo gain- and loss-of-function studies implicate that PDSS1 is critical for promoting the metastasis of TNBC cells.

PDSS1 activates the STAT3 signaling pathway in TNBC cells

To gain insights into the molecular mechanism underlying PDSS1-mediated metastasis, we performed transcriptome sequencing and bioinformatics analysis to define the transcriptional program controlled by PDSS1. RNA-seq identified transcripts that were differentially expressed upon PDSS1 knockdown in MDA-MB-231 cells (Supplementary Fig. S5A and S5B; Supplementary Table S4). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that overlapping downregulated genes were significantly enriched in pathways associated with focal adhesion, extracellular matrix–receptor interaction and cancers, which was also reflected in the Gene Ontology (GO) analysis (Supplementary Fig. S5C). Gene set enrichment analysis (GSEA) indicated that downregulated genes in the siPDSS1 groups were correlated with IL6-JAK2-STAT3 signaling (Fig. 3A; Supplementary Fig. S5D, left) and epithelial-to-mesenchymal transition (EMT; Fig. 3B; Supplementary Fig. S5D, right), both of which play critical roles in cancer metastasis. We therefore tested whether PDSS1 regulates downstream genes related to these pathways. Specifically, PDSS1 siRNA-transfected cells exhibited lower mRNA levels of genes associated with both pathways (e.g., VIM, CDH2, SNAI2, SOX2, and CD44) than did control cells (Fig. 3C).

Figure 3.

PDSS1 promotes TNBC migration and invasion by enhancing STAT3 phosphorylation. A and B, Representative GSEA plot showing downregulation of genes involved in the IL6/JAK/STAT3 signaling pathway (A) and the EMT (B) in MDA-MB-231 cells expressing siPDSS1#1. C, Relative mRNA levels of selected genes in MDA-MB-231 cells with transient knockdown of PDSS1. Data are shown as mean ± SD of triplicate experiments. D, The effects of PDSS1 overexpression on p-JAK2 and p-STAT3 levels in BT-549 and Hs-578t cells. E, The effects of PDSS1 knockdown on p-JAK2 and p-STAT3 levels in MDA-MB-231 and MDA-MB-436 cells. F, Cytoplasmic and nuclear p-STAT3 protein levels after PDSS1 knockdown or overexpression. G, Western blot analysis of p-STAT3 and STAT3 levels in MDA-MB-231 and MDA-MB-436 cells stably expressing control or PDSS1 shRNA and treated with or without IL6 (20 ng/mL). H, Transwell assays to assess migration and invasion after induction with IL6 (20 ng/mL). Quantification results of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. I, Confirmation of p-STAT3 and STAT3 levels after treatment with Stattic [an inhibitor of p-STAT3 (Y705), 2 μmol/L] in BT-549 and Hs-578t cells transfected with pCDH or PDSS1. J, Transwell assays to detect the migration and invasion of cells treated with Stattic (2 μmol/L). Quantification results of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. ***, P < 0.001; ns, not significant.

Figure 3.

PDSS1 promotes TNBC migration and invasion by enhancing STAT3 phosphorylation. A and B, Representative GSEA plot showing downregulation of genes involved in the IL6/JAK/STAT3 signaling pathway (A) and the EMT (B) in MDA-MB-231 cells expressing siPDSS1#1. C, Relative mRNA levels of selected genes in MDA-MB-231 cells with transient knockdown of PDSS1. Data are shown as mean ± SD of triplicate experiments. D, The effects of PDSS1 overexpression on p-JAK2 and p-STAT3 levels in BT-549 and Hs-578t cells. E, The effects of PDSS1 knockdown on p-JAK2 and p-STAT3 levels in MDA-MB-231 and MDA-MB-436 cells. F, Cytoplasmic and nuclear p-STAT3 protein levels after PDSS1 knockdown or overexpression. G, Western blot analysis of p-STAT3 and STAT3 levels in MDA-MB-231 and MDA-MB-436 cells stably expressing control or PDSS1 shRNA and treated with or without IL6 (20 ng/mL). H, Transwell assays to assess migration and invasion after induction with IL6 (20 ng/mL). Quantification results of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. I, Confirmation of p-STAT3 and STAT3 levels after treatment with Stattic [an inhibitor of p-STAT3 (Y705), 2 μmol/L] in BT-549 and Hs-578t cells transfected with pCDH or PDSS1. J, Transwell assays to detect the migration and invasion of cells treated with Stattic (2 μmol/L). Quantification results of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. ***, P < 0.001; ns, not significant.

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On the basis of the alterations of IL6-JAK2-STAT3 downstream signaling, we explored the expression of key players in this pathway (29, 30). We first performed qRT-PCR to examine the mRNA expression levels of IL6, JAK2, and STAT3, which remained unchanged after PDSS1 knockdown (Supplementary Fig. S5E). This result was echoed at the protein level, which showed that the levels of STAT3 and JAK2 were not altered upon PDSS1 overexpression (Fig. 3D) or knockdown (Fig. 3E). However, STAT3 phosphorylation was elevated in response to ectopic expression of PDSS1 in both cell lines tested (Fig. 3D), while knockdown of PDSS1 decreased STAT3 phosphorylation (Fig. 3E). In contrast, the level of JAK2 activation was not changed upon PDSS1 overexpression or knockdown. As reported previously, persistent STAT3 activation has frequently been linked to malignant behaviors, including invasion, metastasis, stemness, and chemotherapeutic resistance (30, 31). Transfection of either of two different siRNAs targeting STAT3 (Supplementary Fig. S5F) abolished the migration capacity of MDA-MB-231 cells (Supplementary Fig. S5G). As STAT3 acts as a transcription factor and the nuclear accumulation of p-STAT3 is critical for its activity (30), we analyzed the p-STAT3 levels in the nuclear and cytoplasmic subfractions. Western blot analysis showed that PDSS1 knockdown decreased both the cytoplasmic and nuclear levels of p-STAT3 (Fig. 3F, left), whereas PDSS1 overexpression did the opposite (Fig. 3F, right). Hence, these data suggest that PDSS1-activated STAT3 (p-STAT3) can translocate into the nucleus and function as a transcription factor.

We next evaluated whether ligand-induced STAT3 activation differed between control and PDSS1 knockdown cells. To this end, we assessed the expression level of total and phosphorylated STAT3 in MDA-MB-231 and MDA-MB-436 cells transfected with control-shRNA and PDSS1-shRNA in response to IL6. IL6-induced STAT3 activation was markedly reduced in PDSS1-knockdown cells compared with control cells (Fig. 3G). Moreover, PDSS1 knockdown significantly impaired the IL6-induced enhancement of cell migration and invasion in both MDA-MB-231 and MDA-MB-436 cells (Fig. 3H; Supplementary Fig. S5H). In addition, we examined whether the observed PDSS1-mediated increase in migration and invasion was dependent on STAT3 activation. Preincubation of cells stably expressing PDSS1 with the STAT3 phosphorylation inhibitor Stattic (32) effectively blocked the increase in STAT3 phosphorylation induced by PDSS1 (Fig. 3I). As expected, the enhanced effect of PDSS1 overexpression on cell migration and invasion was inhibited by Stattic treatment (Fig. 3J; Supplementary Fig. S5I). Taken together, these data strongly support the notion that PDSS1 leads to the activation of STAT3 and promotes TNBC migration and invasion.

The enzymatic activity of PDSS1 is required for its prometastatic effect

Because PDSS1 is best known for its metabolic function, we sought to determine whether its ability to affect TNBC cells is dependent on its canonical catalytic activity. Therefore, we generated the catalytically inactive mutant PDSS1-D308E, which exhibits significant loss of its metabolic function (9). First, we examined the effects of this mutant expression on the p-STAT3 levels in three TNBC cell lines and observed that the PDSS1 mutant was unable to phosphorylate STAT3 (Fig. 4A). Unlike that of wild-type PDSS1 (PDSS1-WT), overexpression of PDSS1-D308E did not affect the migration and invasion of TNBC cells (Fig. 4B; Supplementary Fig. S6A and S6B). We also depleted endogenous PDSS1 from MDA-MB-231 cells and then restored the expression of either PDSS1-WT or PDSS1-D308E (Fig. 4C). Overexpression of PDSS1-WT but not the catalytically inactive mutant PDSS1-D308E restored both the migration and invasion abilities of these cells (Fig. 4D; Supplementary Fig. S6C).

Figure 4.

PDSS1 facilitates TNBC migration, invasion, and distant metastasis in a catalytic activity-dependent manner. A, The level of p-STAT3 in Hs-578t, BT-549, and MCF10A-CA1a cells stably expressing pCDH, PDSS1-WT or PDSS1-D308E was determined by Western blot analysis. B, Quantified results of migration and invasion assays using the indicated stable cell lines (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. C, Knockdown and re-overexpression of PDSS1-WT or PDSS1-D308E in MDA-MB-231 cells were confirmed by Western blot analysis. D, Quantification of cell migration and invasion of the indicated MDA-MB-231 cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. E and F, Effects of dimethylformamide (DMF) or CoQ10 (35 μmol/L) treatment on the migration and invasion of Hs-578t, MCF10A-CA1a, and MDA-MB-231 cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. G, MCF10A-CA1a stable cell lines expressing control vector (pCDH), PDSS1-WT, or PDSS1-D308E were injected into NOD/SCID mice via the tail vein (n = 6 mice per group). BLI signals at the indicated timepoints (left) and representative images of BLI (right) are shown. Data are mean ± SEM. Two-tailed Student t test. ***, P < 0.001; ns, not significant.

Figure 4.

PDSS1 facilitates TNBC migration, invasion, and distant metastasis in a catalytic activity-dependent manner. A, The level of p-STAT3 in Hs-578t, BT-549, and MCF10A-CA1a cells stably expressing pCDH, PDSS1-WT or PDSS1-D308E was determined by Western blot analysis. B, Quantified results of migration and invasion assays using the indicated stable cell lines (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. C, Knockdown and re-overexpression of PDSS1-WT or PDSS1-D308E in MDA-MB-231 cells were confirmed by Western blot analysis. D, Quantification of cell migration and invasion of the indicated MDA-MB-231 cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. E and F, Effects of dimethylformamide (DMF) or CoQ10 (35 μmol/L) treatment on the migration and invasion of Hs-578t, MCF10A-CA1a, and MDA-MB-231 cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. G, MCF10A-CA1a stable cell lines expressing control vector (pCDH), PDSS1-WT, or PDSS1-D308E were injected into NOD/SCID mice via the tail vein (n = 6 mice per group). BLI signals at the indicated timepoints (left) and representative images of BLI (right) are shown. Data are mean ± SEM. Two-tailed Student t test. ***, P < 0.001; ns, not significant.

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As reported previously, it is known that PDSS1 is an enzyme involved in the CoQ10 synthesis pathway (9, 10). Thus, we sought to determine the function of CoQ10 with respect to migration and invasion. Supplementation with CoQ10 in culture medium enhanced the phosphorylation of STAT3 (Supplementary Fig. S6D). Moreover, functional validation showed the enhanced migration and invasion abilities of MCF10A-CA1a and Hs-578t cells with CoQ10 (Fig. 4E; Supplementary Fig. S6E and S6F). At the same time, CoQ10 supplementation significantly enhanced the suppressed phosphorylation of STAT3, cell migration and invasion in PDSS1-knockdown MDA-MB-231 cells (Fig. 4F; Supplementary Fig. S6G and S6H).

To confirm the above findings, we further examined the effects of modulating PDSS1 expression on the in vivo tumor metastasis of TNBC xenografts. Consistent with the in vitro data, PDSS1-WT, but not PDSS1-D308E, enhanced the incidence of lung metastasis, as evidenced by BLI signals and H&E staining analysis (Fig. 4G; Supplementary Fig. S6I). Collectively, the results of these in vitro and in vivo gain- and loss-of-function studies demonstrate that the enzymatic activity of PDSS1 is required for its prometastatic effect.

PDSS1 increases intracellular calcium levels and mediates STAT3 phosphorylation

CoQ10 was reported to positively modulate the intracellular calcium concentration (12). Consistent with this report, GSEA showed that genes involved in the calcium signaling pathway were downregulated in PDSS1 knockdown MDA-MB-231 cells compared with control cells (Fig. 5A). To better understand the relationship of PDSS1 with intracellular calcium signaling, we assessed intracellular calcium levels via fluorescence microscopy and then quantified the intracellular calcium concentration by Fluo-4 AM staining followed by flow cytometry. PDSS1 silencing decreased the Fluo-4 AM fluorescence intensity, indicating a decrease in the calcium level, in MDA-MB-231 cells (Fig. 5B and D). Moreover, overexpression of PDSS1-WT, but not the PDSS1-D308E mutant, increased the Fluo-4 AM fluorescence intensity in Hs-578t cells (Fig. 5C and E). To explore the potential mechanisms underlying the regulatory effect of PDSS1 overexpression, TNBC cells were treated with BAPTA-AM, an intracellular calcium chelator. As shown in Fig. 5F and G, treatment with BAPTA-AM reduced the PDSS1-induced increase in STAT3 phosphorylation in both Hs-578t and MCF10A-CA1a cells. Consistent with this finding, treatment with BAPTA-AM decreased the numbers of migrated and invaded PDSS1-overexpressing cells, as reflected by the results of the Transwell assays (Fig. 5H and I). Together, these data demonstrated that silencing PDSS1 inhibited the migration and invasion of TNBC cells by decreasing the intracellular free calcium concentration.

Figure 5.

PDSS1 upregulates STAT3 phosphorylation by increasing the intracellular Ca2+ concentration. A, Representative GSEA plot showing downregulation of the calcium-mediated signaling pathway in MDA-MB-231 cells expressing siPDSS1#1. B and C, Representative recordings of Ca2+ alterations in MDA-MB-231 cells stably expressing control or PDSS1 shRNA (×400; B) or in Hs-578t cells stably expressing empty vector (pCDH), PDSS1-WT or PDSS1-D308E (×400; C). D and E, Flow cytometric analysis of Ca2+ levels in Fluo-4 AM–labeled MDA-MB-231 cells with PDSS1 knockdown (D) or Hs-578t cells with PDSS1 overexpression (E). Average changes in intensity were analyzed in these cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. F and G, Western blot analysis of the effects of BAPTA-AM (20 μmol/L) treatment for 2 hours on PDSS1-induced phosphorylation of STAT3 in Hs-578t (F) and MCF10A-CA1a cells (G). H and I, Transwell assays to detect the migration and invasion of cells treated with DMSO or BAPTA-AM (20 μmol/L). Representative images and quantification results are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. ***, P < 0.001.

Figure 5.

PDSS1 upregulates STAT3 phosphorylation by increasing the intracellular Ca2+ concentration. A, Representative GSEA plot showing downregulation of the calcium-mediated signaling pathway in MDA-MB-231 cells expressing siPDSS1#1. B and C, Representative recordings of Ca2+ alterations in MDA-MB-231 cells stably expressing control or PDSS1 shRNA (×400; B) or in Hs-578t cells stably expressing empty vector (pCDH), PDSS1-WT or PDSS1-D308E (×400; C). D and E, Flow cytometric analysis of Ca2+ levels in Fluo-4 AM–labeled MDA-MB-231 cells with PDSS1 knockdown (D) or Hs-578t cells with PDSS1 overexpression (E). Average changes in intensity were analyzed in these cells (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. F and G, Western blot analysis of the effects of BAPTA-AM (20 μmol/L) treatment for 2 hours on PDSS1-induced phosphorylation of STAT3 in Hs-578t (F) and MCF10A-CA1a cells (G). H and I, Transwell assays to detect the migration and invasion of cells treated with DMSO or BAPTA-AM (20 μmol/L). Representative images and quantification results are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. ***, P < 0.001.

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CAMK2A activation promotes STAT3 phosphorylation

The multifunctional serine/threonine protein kinase calcium/calmodulin-dependent protein kinase II (CaMKII) is a major calcium sensor in cells (33, 34). This protein is composed of four different chains: alpha, beta, gamma, and delta. Activation of CaMKII requires an increase in the intracellular calcium level and binding of Ca2+/calmodulin to its regulatory domain, after which, the activated kinase [phosphorylated at threonine 286 (T286) for the α isoform and Thr287 for the β, γ, and δ isoforms] can phosphorylate many substrates (33, 35). In breast cancers, phosphorylation and activation of CaMKII at T286 mediates the oncogenic signaling of anoctamin-1 (ANO1; ref. 36). To characterize how intracellular calcium signaling participated in PDSS1-mediated STAT3 phosphorylation in TNBC cells, we evaluated the roles of CAMK2A, CAMK2B, CAMK2D, and CAMK2G in STAT3 phosphorylation. Three siRNAs against each gene were synthesized and used independently to inhibit the corresponding gene expression in MDA-MB-231 cells (Supplementary Fig. S7A). Knockdown of CAMK2A alone evidently suppressed STAT3 activation (Fig. 6A). We also examined the expression of CAMK2A, CAMK2B, CAMK2D, and CAMK2G and found that CAMK2A was upregulated in TNBC tumor tissues compared with adjacent normal tissues (Fig. 6B; Supplementary Fig. S7B). Upon treatment with BAPTA-AM, MDA-MB-231, and MDA-MB-436 cells exhibited reduced phosphorylation levels of both STAT3 and CAMK2A compared to those in cells with DMSO treatment (Fig. 6C, left). In addition, the CaMKII inhibitor KN-93 (37) was employed to study the effects of pharmacologic CAMK2A inhibition on STAT3 activity. STAT3 activation was effectively suppressed by KN-93 treatment (Fig. 6C, right). Furthermore, CAMK2A knockdown resulted in a dramatic decrease in p-STAT3 levels, while CAMK2A overexpression led to a significant increase in p-STAT3 levels (Fig. 6D; Supplementary Fig. S7C). At the same time, CAMK2A knockdown or overexpression impaired or enhanced, respectively, the cell migration and invasion abilities of TNBC cells (Supplementary Fig. S7D). Stattic effectively decreased the levels of p-STAT3 and the numbers of migrated/invaded cells, which were induced by CAMK2A expression (Fig. 6E; Supplementary Fig. S7E and S7F).

Figure 6.

CAMK2A promotes TNBC migration and invasion by enhancing STAT3 phosphorylation. A, Western blot analysis was used to assess alterations in STAT3 and p-STAT3 levels after knockdown of the CaMKII isoforms. B, The mRNA levels of CAMK2A in 88 paired samples of tumor and adjacent normal breast tissues from the FUSCC TNBC dataset (two-tailed paired t test). C, p-CAMK2A and p-STAT3 levels in MDA-MB-231 and MDA-MB-436 cells preincubated with the Ca2+ chelator BAPTA-AM (20 μmol/L) or the CaMKII inhibitor KN-93 (10 μmol/L). D, The level of p-STAT3 was analyzed by Western blotting after knockdown or overexpression of CAMK2A in the indicated cell lines. E, Western blot analysis of the effects of Stattic (2 μmol/L) on CAMK2A-induced p-STAT3 in Hs-578t and BT-549 cells. F, The levels of p-STAT3 and STAT3 in Hs-578t and BT-549 cells stably expressing pCDH, CAMK2A-WT, or CAMK2A-T286A were determined by Western blot analysis. G, Quantitative analysis of migration and invasion assays using the indicated stable cell lines (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. H, Cytoplasmic and nuclear proteins of Hs-578t cells stably expressing pCDH, CAMK2A-WT, or CAMK2A-T286A were separated and immunoblotted. I, Coimmunoprecipitation of exogenous CAMK2A and STAT3 in BT-549 cells. J, Assessment of the interaction of endogenous CAMK2A and STAT3. K, Left, schematic diagram showing the structure of CAMK2A and the truncation mutants used. Flag-tagged wild-type CAMK2A or CAMK2A truncation mutants were coexpressed with HA-STAT3 in HEK293T cells. Right, extracts were immunoprecipitated with an anti-Flag antibody. L, Coimmunoprecipitation in PDSS1-knockdown cells using CAMK2A as bait. M, Coimmunoprecipitation in HEK293T cells overexpressing HA-STAT3, Flag-CAMK2A, or Flag-CAMK2A-T286A using the Flag tag as bait. ***, P < 0.001; ns, not significant.

Figure 6.

CAMK2A promotes TNBC migration and invasion by enhancing STAT3 phosphorylation. A, Western blot analysis was used to assess alterations in STAT3 and p-STAT3 levels after knockdown of the CaMKII isoforms. B, The mRNA levels of CAMK2A in 88 paired samples of tumor and adjacent normal breast tissues from the FUSCC TNBC dataset (two-tailed paired t test). C, p-CAMK2A and p-STAT3 levels in MDA-MB-231 and MDA-MB-436 cells preincubated with the Ca2+ chelator BAPTA-AM (20 μmol/L) or the CaMKII inhibitor KN-93 (10 μmol/L). D, The level of p-STAT3 was analyzed by Western blotting after knockdown or overexpression of CAMK2A in the indicated cell lines. E, Western blot analysis of the effects of Stattic (2 μmol/L) on CAMK2A-induced p-STAT3 in Hs-578t and BT-549 cells. F, The levels of p-STAT3 and STAT3 in Hs-578t and BT-549 cells stably expressing pCDH, CAMK2A-WT, or CAMK2A-T286A were determined by Western blot analysis. G, Quantitative analysis of migration and invasion assays using the indicated stable cell lines (mean ± SD; n = 3 biological replicates). Two-tailed Student t test. H, Cytoplasmic and nuclear proteins of Hs-578t cells stably expressing pCDH, CAMK2A-WT, or CAMK2A-T286A were separated and immunoblotted. I, Coimmunoprecipitation of exogenous CAMK2A and STAT3 in BT-549 cells. J, Assessment of the interaction of endogenous CAMK2A and STAT3. K, Left, schematic diagram showing the structure of CAMK2A and the truncation mutants used. Flag-tagged wild-type CAMK2A or CAMK2A truncation mutants were coexpressed with HA-STAT3 in HEK293T cells. Right, extracts were immunoprecipitated with an anti-Flag antibody. L, Coimmunoprecipitation in PDSS1-knockdown cells using CAMK2A as bait. M, Coimmunoprecipitation in HEK293T cells overexpressing HA-STAT3, Flag-CAMK2A, or Flag-CAMK2A-T286A using the Flag tag as bait. ***, P < 0.001; ns, not significant.

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Reports have indicated that when intracellular calcium levels rise, calcium binds to calmodulin, which activates CaMKII and leads to phosphorylation of CaMKII at T286 (31, 33). Thus, to explore whether the phosphorylation of CAMK2A at T286 is critical for its regulation of the level of p-STAT3, we introduced the T286 mutation to generate the kinase-deficient mutant CAMK2A-T286A. As anticipated, none of the phenotypes observed in cells expressing CAMK2A-WT (increased STAT3 phosphorylation and cell migration and invasion compared with those in control cells) were observed in cells expressing CAMK2A-T286A (Fig. 6F and G; Supplementary Fig. S7G). Notably, nuclear and cytoplasmic p-STAT3 accumulation was observed with CAMK2A overexpression (Fig. 6H). Taken together, these results confirm that the regulatory role of CAMK2A in TNBC cells is dependent on its T286 phosphorylation status.

Considering the regulation of STAT3 signaling by CAMK2A, we sought to determine whether CAMK2A and STAT3 interact. The results from the coimmunoprecipitation experiments showed the interaction between exogenously expressed CAMK2A and STAT3, confirming our hypothesis (Fig. 6I; Supplementary Fig. S8A and S8B). The interaction between endogenous CAMK2A and STAT3 was also confirmed in BT-549 and Hs-578t cells (Fig. 6J). To identify the region of each protein responsible for this interaction, various truncation constructs of CAMK2A and STAT3 were generated (Fig. 6K, left; and Supplementary Fig. S8C) and coexpressed in HEK293T cells. The catalytic domain of CAMK2A and the N-terminal domain of STAT3 were identified as the critical regions of each protein necessary for their interaction (Fig. 6K, right; and Supplementary Fig. S8D). To assess the effect of PDSS1 on the CAMK2A–STAT3 interaction, lysates from shCTRL- or shPDSS1-transfected MDA-MB-231 cells were immunoprecipitated with an anti-CAMK2A antibody. As anticipated, knockdown of PDSS1 impaired the binding of CAMK2A to STAT3 (Fig. 6L). In addition, ectopic expression of the kinase-deficient mutant CAMK2A-T286A in HEK293T cells led to reduced pulldown of STAT3 compared with that in cells expressing CAMK2A-WT (Fig. 6M). Together, these data suggest that CAMK2A phosphorylation is required for the CAMK2A–STAT3 interaction and that this interaction is positively regulated by PDSS1.

PDSS1 silencing diminishes the migration and invasion of TNBC cells by attenuating CAMK2A-mediated STAT3 signaling

To further examine whether PDSS1 promotes STAT3 activation by activating CAMK2A, we used Western blotting to determine the levels of p-CAMK2A, p-STAT3, and the STAT3 target gene Slug (38, 39) after PDSS1 knockdown and overexpression. The levels of p-CAMK2A, p-STAT3, and Slug were decreased in PDSS1 knockdown TNBC cells compared with control cells (Fig. 7A). Furthermore, after overexpression of PDSS1, the opposite effects were observed, but these effects were not observed when the PDSS1-D308E mutant was overexpressed (Fig. 7B; Supplementary Fig. S9A). To further assess CAMK2A on the phenotypic effects of PDSS1 overexpression and knockdown, we knocked down CAMK2A with two independent shRNAs in PDSS1-overexpressing cells. The increases in the levels of p-STAT3 and Slug induced by PDSS1 overexpression were suppressed by CAMK2A knockdown (Fig. 7C; Supplementary Fig. S9B). CAMK2A knockdown also weakened the enhanced cell migration and invasion abilities caused by PDSS1 overexpression (Fig. 7D). In addition, the PDSS1 knockdown-induced downregulation of p-STAT3 and Slug was reversed by CAMK2A overexpression (Fig. 7E), and CAMK2A overexpression rescued the suppressive migration and invasion abilities of PDSS1-knockdown cells (Fig. 7F).

Figure 7.

CAMK2A is required for PDSS1-mediated STAT3 phosphorylation. A, Immunoblot and quantitative analysis of p-STAT3, p-CAMK2A, and Slug protein levels in MDA-MB-231 cells with PDSS1 knockdown. Mean ± SD of triplicate experiments. Two-tailed Student t test. B, Immunoblot and quantitative analysis of p-STAT3, p-CAMK2A, and Slug in Hs-578t cells overexpressing PDSS1. Mean ± SD of triplicate experiments. Two-tailed Student t test. C and D, The levels of p-STAT3, p-CAMK2A, and Slug (C), and the migration and invasion abilities (D) were assessed in Hs-578t cell lines stably expressing pCDH or PDSS1, followed with infection of indicated control or CAMK2A shRNA. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. E and F, The levels of p-STAT3, Slug (E), migration and invasion abilities (F) were assessed after overexpressing CAMK2A in PDSS1-knockdown MDA-MB-231 cells. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. G and H, Quantification of migration assays conducted with Hs-578t and MCF10A-CA1a cells transfected with control (pCDH) or PDSS1 plasmid and treated with 2 μmol/L Stattic (G) or 0.5 μmol/L napabucasin (H) as indicated. Two-tailed Student t test. I, Representative IHC images of PDSS1 and p-STAT3 staining in specimens from TNBC patients. Scale bar, 100 μm. J, Pearson correlation analysis between PDSS1 and p-STAT3 levels based on IHC staining. K, Kaplan–Meier survival analysis of patients with TNBC according to PDSS1 staining intensity. L, A schematic model showing how the PDSS1/CAMK2A/STAT3 axis promotes TNBC metastasis. *, P < 0.05; ***, P < 0.001; ns, not significant.

Figure 7.

CAMK2A is required for PDSS1-mediated STAT3 phosphorylation. A, Immunoblot and quantitative analysis of p-STAT3, p-CAMK2A, and Slug protein levels in MDA-MB-231 cells with PDSS1 knockdown. Mean ± SD of triplicate experiments. Two-tailed Student t test. B, Immunoblot and quantitative analysis of p-STAT3, p-CAMK2A, and Slug in Hs-578t cells overexpressing PDSS1. Mean ± SD of triplicate experiments. Two-tailed Student t test. C and D, The levels of p-STAT3, p-CAMK2A, and Slug (C), and the migration and invasion abilities (D) were assessed in Hs-578t cell lines stably expressing pCDH or PDSS1, followed with infection of indicated control or CAMK2A shRNA. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. E and F, The levels of p-STAT3, Slug (E), migration and invasion abilities (F) were assessed after overexpressing CAMK2A in PDSS1-knockdown MDA-MB-231 cells. Representative images and quantification of relative migrated/invaded cells are shown (mean ± SD; n = 3 biological replicates). Scale bars, 100 μm. Two-tailed Student t test. G and H, Quantification of migration assays conducted with Hs-578t and MCF10A-CA1a cells transfected with control (pCDH) or PDSS1 plasmid and treated with 2 μmol/L Stattic (G) or 0.5 μmol/L napabucasin (H) as indicated. Two-tailed Student t test. I, Representative IHC images of PDSS1 and p-STAT3 staining in specimens from TNBC patients. Scale bar, 100 μm. J, Pearson correlation analysis between PDSS1 and p-STAT3 levels based on IHC staining. K, Kaplan–Meier survival analysis of patients with TNBC according to PDSS1 staining intensity. L, A schematic model showing how the PDSS1/CAMK2A/STAT3 axis promotes TNBC metastasis. *, P < 0.05; ***, P < 0.001; ns, not significant.

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Because PDSS1 promotes metastasis via STAT3 effector molecules, we next investigated whether PDSS1 expression would alter the sensitivity of cells to Stattic treatment. The migration ability of TNBC cells was weakened by treatment with Stattic, and the extent to which migration was inhibited was much greater in PDSS1-overexpressing cells than in control cells (Fig. 7G; Supplementary Fig. S10A). Consistently, napabucasin (40, 41), an FDA-approved p-STAT3 inhibitor, also effectively suppressed the migration of PDSS1-overexpressing cells (Fig. 7H; Supplementary Fig. S10B), supporting the conclusion that STAT3 phosphorylation is a biologically relevant downstream effector of PDSS1. Notably, neither drug treatment triggered apparent apoptosis, as detected by flow cytometry analysis (Supplementary Fig. S10C and S10D).

To investigate the clinical correlation between PDSS1 and p-STAT3, we performed IHC staining analysis and detected PDSS1 and p-STAT3 in TNBC specimens (Fig. 7I). We observed a significant positive correlation between PDSS1 expression and p-STAT3 levels (Fig. 7J). Moreover, patients with higher PDSS1 expression had a poorer prognosis than those with lower PDSS1 expression levels (Fig. 7K). Taken together, our data support the role of the PDSS1/CAMK2A/STAT3 axis in promoting TNBC metastasis and suggest that PDSS1 is a biomarker and potential therapeutic target for TNBC.

Stattic in combination with cisplatin decreased the metastasis of TNBC cells

Cisplatin has been used to treat numerous human cancers, including breast cancer. Its mechanism of action has been linked to its ability to cross-link with purine bases in DNA and its inducing effect on reactive oxygen species (ROS) production (42–45). Recently, a clinical trial associated the use of CoQ10 with an increased risk of recurrence in patients with breast cancer (18). Because CoQ10 is an antioxidant (46), we speculated that PDSS1 could contribute to cisplatin resistance by buffering ROS. First, we observed that the cellular ROS level decreased when PDSS1-WT, but not PDSS1-D308E, was reintroduced in PDSS1-knockdown MDA-MB-231 cells (Supplementary Fig. S11A). Similarly, overexpression of PDSS1, but not its mutant, significantly reduced cellular ROS levels in the Hs-578t cell line (Supplementary Fig. S11B). Next, we examined the relationship between PDSS1 expression and cisplatin sensitivity in TNBC cell lines. Knockdown of PDSS1 significantly attenuated cell viability and resistance in the presence of cisplatin (Supplementary Fig. S12A). In contrast, ectopic expression of PDSS1 in Hs-578t cells promoted resistance to cisplatin treatment (Supplementary Fig. S12B). In addition, it is well known that cancer stem cells are a main cause of chemotherapeutic resistance and that their ability to buffer ROS is stronger than that of other cells (47, 48). The STAT3-mediated pathway was found to be preferentially active in CD44+/CD24 breast cancer stem cells (49). As expected, the proportion of the CD44+/CD24 subpopulation was enriched by PDSS1 overexpression, while the inverse was true upon PDSS1 depletion (Supplementary Fig. S12C), suggesting that the ectopic expression of PDSS1 promotes, to some extent, the resistance of TNBC cells to the chemotherapeutic drug cisplatin.

Hence, we hypothesized that the PDSS1-mediated signaling pathway could be indirectly targeted by combining cisplatin and Stattic and that this combination therapy could not only inhibit p-STAT3 levels but also induce ROS production. Consistently, there is evidence in the literature supporting the use of this combination (50, 51). To examine the inhibitory effects of Stattic and cisplatin either alone or in combination on TNBC metastasis, mice were administered each of the four treatments (vehicle, cisplatin, Stattic, and cisplatin with Stattic) and evaluated. Compared with vehicle treatment, treatment with cisplatin alone or Stattic alone significantly reduced lung metastasis. Importantly, compared with each monotherapy alone, the combination treatment resulted in a more significant reduction in lung metastasis (Supplementary Fig. S13A and S13B).

Identification of signaling pathways involved in cancer metabolism is important not only for gaining mechanistic insights into tumor progression but also for the development of novel cancer therapy strategies. In the current study, we identified PDSS1 as a prometastatic regulator in TNBC. Moreover, we revealed that PDSS1 promotes CAMK2A-mediated phosphorylation of STAT3, indicating possible therapeutic targets for TNBC (Fig. 7L).

To date, the role of PDSS1 in cancer remains unclear. GSEA revealed a significant positive association between PDSS1 knockdown and STAT3 signaling suppression. Numerous studies have provided strong evidence that STAT3 is critical for metastasis in different cancer models through its participation in multiple steps of metastasis (30, 52, 53). STAT3 is tightly regulated by phosphorylation and transduces signals from various signaling pathways, for example, by transactivating the target genes that mediate metastasis and proliferation (30). STAT3 signaling inactivation caused by PDSS1 knockdown was also evidenced by changes in the phosphorylation status of STAT3 and its downstream target genes. Furthermore, the STAT3 signaling inhibitor Stattic abolished the effect of PDSS1 on cell migration and invasion. Despite these discoveries, how the PDSS1 specifically affects STAT3 phosphorylation needs further investigation. Our mechanistic studies suggested that PDSS1-mediated promotion of metastasis is dependent on its canonical enzymatic activity, as evidenced by its active site (9). PDSS1 catalyzes the formation of all transpolyprenyl pyrophosphates from isopentyl diphosphate during the assembly of polyisoprenoid side chains, the first step in CoQ10 biosynthesis. CoQ10 has been documented to increase cytoplasmic calcium concentrations (12); thus, one possible mechanism is Ca2+ transport across the inner mitochondrial membrane by CoQ10 with the help of cytochrome P450 (54). Consistently, PDSS1 overexpression increased the intracellular calcium concentrations, indicating that PDSS1 is a critical player in modulating cellular calcium that consequently triggers downstream signaling activation in TNBC.

Calcium signaling plays critical roles in various cellular processes and has widely been reported to be associated with cancer progression (55–57). The multifunctional serine/threonine kinase CaMKII is a key calcium signaling effector activated by a surge of cytosolic Ca2+ (35). There are four CaMKII genes (CAMK2A, 2B, 2D, and 2G), which are distributed in different tissues and perform different functions. Here, our findings revealed that knockdown of CAMK2A alone clearly induced a decrease in STAT3 phosphorylation, indicating that CAMK2A has unique functions. Importantly, we are the first to show that CAMK2A expression can induce STAT3 phosphorylation, revealing the potential underlying oncogenic role of CAMK2A. Although recent evidence highlights the important functions of CAMK2A in tumor progression (58–60), the mechanism by which it acts remains to be fully determined. Our study identified the interaction between STAT3 and CAMK2A, which facilitates the phosphorylation of STAT3. In addition, PDSS1 knockdown weakened the CAMK2A–STAT3 interaction. Rescue experiments supported the evidence that PDSS1 facilitates the metastasis of TNBC through CAMK2A-STAT3 signaling. This sequence of events allows STAT3 to enter the nucleus and ultimately promote the oncogenic STAT3 signaling cascade.

Our current study also has potential clinical implications. First, STAT3 activation has been recognized as a mechanism underlying tumor progression and chemotherapeutic drug resistance. Because of the function of PDSS1 in activating STAT3, PDSS1 expression may be a promising marker to stratify patients with breast cancer for STAT3-targeted therapy. Second, increased ROS levels and an altered redox status have been observed in cancer cells compared with their normal counterparts (61). Chemotherapeutic drugs, including cisplatin, often result in further increases in ROS production, and the overproduction of ROS leads to cancer cell death once the ROS level exceeds the toxicity threshold (43, 62). Thus, dysregulation of ROS-regulating enzymes may lead to chemotherapy-resistant tumor growth due to imbalanced redox homeostasis (44). We speculated that the downstream metabolite of PDSS1, CoQ10, can protect tumor cells against excessive ROS production induced by cisplatin. In addition, combination treatment comprising Stattic and cisplatin was reported to further decrease p-STAT3 levels (51). On the basis of the functional role of PDSS1 and its underlying mechanisms, targeting PDSS1-mediated signaling by combining anti-STAT3 therapy with cisplatin may offer additional therapeutic approaches for TNBC.

In summary, this study demonstrated that high expression of PDSS1 is a critical feature of TNBC metastasis. PDSS1 promotes TNBC metastasis in a manner dependent on its catalytic activity and increases the intracellular calcium concentration, leading to phosphorylation of the calcium-dependent kinase CAMK2A. Phosphorylated CAMK2A could further promote activation of STAT3, subsequently upregulating downstream gene expression and ultimately resulting in TNBC metastasis. Our current study provides evidence that this signaling pathway can be exploited to develop novel therapeutic approaches for TNBC.

No disclosures were reported.

T.-J. Yu: Conceptualization, resources, data curation, software, formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing. Y.-Y. Liu: Resources, data curation, software, formal analysis, visualization, methodology, writing–original draft, writing–review and editing. X.-G. Li: Data curation, formal analysis, methodology, project administration, writing–review and editing. B. Lian: Formal analysis, investigation, methodology, writing–review and editing. X.-X. Lu: Software, investigation, methodology, writing–review and editing. X. Jin: Resources, data curation, formal analysis, investigation, methodology, writing–review and editing. Z.-M. Shao: Conceptualization, resources, data curation, supervision, funding acquisition, validation, project administration, writing–review and editing. X. Hu: Conceptualization, data curation, supervision, funding acquisition, validation, investigation, project administration, writing–review and editing. G.-H. Di: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, visualization, project administration, writing–review and editing. Y.-Z. Jiang: Conceptualization, data curation, supervision, funding acquisition, investigation, visualization, methodology, project administration, writing–review and editing.

This work was supported by grants from the National Key Research and Development Project of China (2020YFA0112304 to Z.-M. Shao; 2017YFC0108904 to G.-H. Di; 2018YFE020160 to X. Hu), the National Natural Science Foundation of China (81922048 to Y.-Z. Jiang; 81874112 to Y.-Z. Jiang; 81874113 to Z.-M. Shao; 91959207 to Z.-M. Shao; 82072922 to G.-H. Di; 81902684 to X. Jin), the Program of Shanghai Academic/Technology Research Leader (20XD1421100 to Y.-Z. Jiang), and the Fok Ying-Tong Education Foundation for College Young Teachers (171034 to Y.-Z. Jiang).

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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