Transcriptional positive cofactor 4 promotes breast cancer proliferation and metastasis through c-Myc mediated Warburg effect
The human positive cofactor 4 (PC4) is initially identified as a transcriptional cofactor and has an important role in embryonic development and malignant transformation. However, the clinical significance and the molecular mechanisms of PC4 in breast cancer development and progression are still unknown.
We investigated PC4 expression in 114 cases of primary breast cancer and matched normal breast tissue specimens, and studied the impact of PC4 expression as well as the molecular mechanisms of this altered expression on breast cancer growth and metastasis both in vitro and in vivo.
PC4 was significantly upregulated in breast cancer and high PC4 expression was positively correlated with metastasis and poor prognosis of patients. Gene set enrichment analysis (GSEA) demonstrated that the gene sets of cell proliferation and Epithelial-Mesenchymal Transition (EMT) were positively correlated with elevated PC4 expression. Consistently, loss of PC4 markedly inhibited the growth and metastasis of breast cancer both in vitro and in vivo. Mechanistically, PC4 exerted its oncogenic functions by directly binding to c-Myc promoters and inducing Warburg effect.
Our study reveals for the first time that PC4 promotes breast cancer progression by directly regulating c-Myc transcription to promote Warburg effect, implying a novel therapeutic target for breast cancer.
KeywordsBreast cancer PC4 Warburg effect C-Myc Metastasis Proliferation
Cell counting kit-8
Extracellular acidification rate
Gene set enrichment analysis
invasive ductal carcinoma
Invasive lobular carcinoma
Positive cofactor 4
quantitative polymerase chain reaction
Breast cancer is one of the most common cancers and a leading cause of tumor-related death in females worldwide. Despite advances in treatments, many patients still develop into metastatic disease, which is the leading cause of breast cancer death [1, 2, 3]. Thus, there is an urgent need to characterize the underlying molecular mechanisms and identify novel therapeutic targets to improve the outcomes for breast cancer .
Recent studies have shown that metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression . The Warburg effect , as known as aerobic glycolysis, is the best-characterized metabolic change in cancer cells, which facilitates cancer growth and progression by increasing glucose uptake, elevating lactate production, and supporting the energy demands . Emerging evidence has indicated the crucial role of the Warburg effect in cancer therapy as a novel target . In the process of glycolysis, the c-Myc oncogenic transcription factor plays a key role by directly regulating the glycolytic genes, such as GLUT1, HK2, LDHA, PDK1, etc. [9, 10]. Although a few transcription factors have been reported to regulate glycolysis , the transcriptional regulation of Warburg effect and the upstream regulatory mechanism of c-Myc are still largely unknown.
The human positive cofactor 4 (PC4) and its yeast ortholog SUB1 (also named as coactivator p15) are initially identified as a coactivator of basal transcription [12, 13]. PC4 is located on chromosome 5p13 and encodes a 127-amino acid protein that has an important role in various cellular processes including transcription [14, 15, 16, 17], DNA replication [18, 19, 20, 21, 22], DNA repair [23, 24, 25, 26, 27, 28, 29, 30, 31] and chromatin organization [32, 33]. By the model of PC4 knockout mouse, we have found that loss of PC4 results in early embryo lethality, highlighting an essential role of PC4 in embryonic development . And our previous studies have shown that overexpression of PC4 is involved in the malignant transformation of normal dermal multipotent fibroblasts, indicating the crucial role of PC4 in tumorigenesis . Besides, PC4 is also found to be upregulated in lung cancer , astrocytoma , prostate cancer  and esophageal squmaous cell carcinoma  and positively related with tumor lymphatic metastasis  and chemo-radiosensitivity [26, 40, 41]. However, the clinical significance and the molecular mechanisms of PC4 in breast cancer development and progression are still unknown.
In this study, we reported firstly that PC4 was highly expressed in breast cancer and positively correlated with metastasis and poor prognosis of patients. Through gene set enrichment analysis (GSEA) in breast cancer specimens and experimental verification, we demonstrated that PC4 promoted the growth and metastasis of breast cancer both in vitro and in vivo. Furthermore, our findings revealed that PC4 exerted its oncogenic functions by directly binding to c-Myc promoters and activating glycolysis. Therefore, our study provided novel insights into the functions and mechanisms of PC4 in breast cancer, and suggested that PC4 might be a promising therapeutic target for breast cancer.
Cell lines and clinical samples
The human breast cancer cell lines (MDA-MB-231 and MCF-7) were purchased from the American Type Culture Collection (ATCC, USA) and the Cell Bank of the Chinese (Shanghai, China). All cells were cultured in the recommended medium (Hyclone, USA), supplemented with 10% FBS (Gibco, USA) and 1% streptomycin/penicillin (Beyotime, Shanghai, China), and incubated in 5% CO2 at 37 °C. A total of 114 paraffin-embedded breast cancer samples, with paired adjacent tissue and normal tissues were obtained at Southwest Hospital of Third Military Medical University. All breast cancer patients were diagnosed independently by at least two experienced pathologists, according to the Union for International Cancer Control classification system. The study was approved by the Ethics Committee of Third Military Medical University.
Cell proliferation assay and colony formation assay
Cell proliferation was measured with the Cell Counting Kit-8 (Dojindo, Japan). Cells were seeded into 96-well plates with a density of 2000 cells each well and 100 ul medium. Cellular proliferation was measured according to the readout at the wavelength of 450 nm. Data were read by a microplate reader (Multiskan Go Multimode Reader; Thermo Scientific). To determine their clonogenic ability, MDA-MB-231 and MCF-7 cells were trypsinized and seeded in 6-well plates. The medium was changed every three days, and cells were cultured for up to 14 day until colonies were clearly visible. At the endpoint, cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with crystal violet (Beyotime, China) for 30 min, and then colonies with > 50 cells were counted.
Wound healing assay
After transfection with the respective shRNAs, cells were tripsinized and seeded into 6-well plates to allow the cells to form a complete monolayer. After they became confluent, wounds were carefully introduced across the cell monolayer with a 10ul pipette tip. The dead cells were removed by PBS washes. Cells were treated with serum-free medium for 24 h. Images were taken at the indicated time and then migration distance was measured.
Transwell chamber assay
Cell inserts pre-coated with matrigel (8.0 μm pore size membrane; Corning) were used. Breast cancer cells were deprived of serum for 12 h and seeded onto the upper chamber surface in serum-free medium at a density of 1 × 105 cells per well, and 20% FBS was placed in the lower chamber and incubated at 37 °C for 24 h. At the end of incubation, nonmotile cells on the upper surface of the filter were wiped off and the cells on the underside chamber were fixed with methanol and stained with Crystal Violet (Beyotime) and counted using bright-field microscopy in 5 random fields in triplicate inserts.
ECAR, glucose uptake, lactate and ATP assays
Extracellular acidification rate (ECAR) was measured using extracellular flux analyzer (XFp) analyzer (Seahorse Bioscience) as described by the manufacturer. Lactate production (Lactate Assay Kit) was measured as described by the manufacturer (BioVision). To measure glucose uptake, cells were incubated with a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] -2-deoxy-D-glucose (2-NBDG; APExBIO) for 20 min at 37 °C. The fluorescence intensity of 2-NBDG was measured using by flow cytometry (BD FACSCanto II™), and data were analyzed with FlowJo software (Treestar). ATP production (Enhanced ATP Assay Kit) was measured according to the protocol recommended by manufacturer (Beyotime).
The shRNA targeting human PC4(shRNA1: 5′- GACAGGUGAGACUUCGAGATT -3′; 5′- UCUCGAAGUCUCACCUGUCTT -3′. shRNA2: 5′- ACAGAGCAGCAGCAGCAGATT -3′; 5′- UCUGCUGCUGCUGCUCUGUTT -3′) and negative control shRNA (5′-UUCUCCGAACGUGUCACGUTT-3′; 5′-ACGUGACACGUUCGGAGAATT-3′) were constructed by GenePharma (Shanghai, China). The human PC4 plasmid and control were purchased from GeneChem (Shanghai, China). The human c-Myc plasmid and control were purchased from GenePharma (Shanghai, China). According to the manufacturer’s protocol, MDA-MD-231and MCF-7 cells were transfected with shRNA or plasmid according to the manufactures’ instructions.
Western blotting analysis
MDA-MB-231 and MCF-7 cells were harvested, washed, and lysed with RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Roche) for 30 min on ice. Total protein was extracted, and quantitated by a BCA kit (Beyotime, China) according to the manufacturer’s instruction. The protein samples were separated by electrophoresis in 10%~ 12% gel, and then transferred onto PVDF membranes (Millipore). Blotted membranes were blocked and incubation with primary antibodies overnight at 4 °C. The membranes were washed 5 min for 3 times with TBST, and subsequently incubated 1 h with HRP-linked secondary antibody (Cell Signaling Technology, USA) at room temperature. The band intensities were visualized and detected by an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Primary antibodies against c-Myc, E-cadherin, N-cadherin, Vimentin, GLUT1, LDHA and β-actin were obtained from Cell Signaling Technology. Primary antibodies against PC4 were obtained from Sigma.
Total RNA was extracted from breast cancer cells using Trizol (Cwbiotech, China). 1 μg RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (#K1622, Thermo Fisher Scientific,Inc.) according to the manufacturer’s protocol. As described in our previous work, qRT-PCR was performed using a SYBR Green qPCR master mix (Takara) according to the manufacturer’s protocol. After the reactions were completed, the comparative threshold cycle (Ct) method was used to calculate the relative gene expression. GAPDH expression was used as the internal control. Human-specific primers were synthesized, and their sequences are shown in Additional file 1: Table S1.
Chromatin immunoprecipitation (ChIP)
MDA-MB-231 cells were conducted ChIP assay with the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003; Cell Signaling Technology) according to the manufacturer’s instructions. Chromatin fragments were immunoprecipitated with anti-PC4 (NB10059774; Novus Biologicals), or rabbit IgG (#2729; Cell Signaling Technology) coupled with ChIP Grade Protein G Magnetic Beads (#9006; Cell Signaling Technology). After DNA purification, quantitative PCR was performed using primers (Additional file 1: Table S2). The fold enrichment was calculated by normalizing samples of anti-TCF-1 or p-STAT3 to normal rabbit IgG controls.
Tumor model in vivo was established using 4-week-old female athymic nude mice from the Laboratory Animal Center of the Third Military Medical University (Chongqing, China) in a specific pathogen-free condition. Animal protocols were followed the Animal Care and Use Committee Guidelines of the Third Military Medical University. Mice were inoculated subcutaneously with 3 × 106 respective MDA-MB-231 cells in 100 ul PBS at one dorsal site. Tumor growth was grossly monitored and measured with sliding calipers every 2 days. Volume of tumors were calculated according to the formula: volume (mm3) = (width2 X length)/2. When mice were killed, tumors were dissected and weighed. For the growth test, mice were sacrificed at the endpoint.
Experimental metastasis assay
Tumor metastasis assays were performed using an intravenous breast cancer mouse model as previous described . Briefly, respective MDA-MB-231 cells were digested in PBS. Single-cell suspensions of 3 × 105 cells were injected into the tail vein of female NOD/SCID mice. Four weeks after injection, animals were sacrificed, lungs collected, and fixed in 10% buffered formalin for subsequent hematoxylin and eosin (H&E) staining.
The paraffin-embedded sections were dewaxed, rehydrated and incubated with human PC4 antibody (1: 500; Sigma, St. Louis, Missouri, USA) at 4 °C overnight. Subsequently, the slides were sequentially incubated with biotinylated secondary antibody at 37 °C for 30 min, and positive staining was visualized by using DAB. PC4 expression in breast cancer was evaluated by percentage of positive-staining cells and staining intensity. The percentage of positive cells was evaluated quantitatively and scored as: 0 (< 5% positive tumor cells), 1 (5–25% positive tumor cells), 2 (26–50% positive tumor cells), 3 (51–75% positive tumor cells) and 4 (> 75% positive tumor cells). Intensity was graded as follows: 0, no signal; 1, weak (light yellow); 2, moderate (brown); and 3, strong staining. The final quantification of each staining was obtained by multiplying these 2 scores. A total staining score of 0–12 was calculated and graded as negative (−, score 0–1), weak (+, score 2–4), moderate (++, score 5–8), or strong (+++, score 9–12). All tissue samples were examined and independently evaluated by two pathologists.
Statistical analysis was carried out using SPSS 13.0 software (SPSS Inc., Chicago, USA), and all data are presented as means ± SD. Comparisons between two groups were performed using the Student’s t-test. Comparisons among three or more groups were performed using a one-way analysis of variance (ANOVA). The survival data was performed using the Kaplan-Meier method. Correlation between PC4 expression and clinical parameters was determined using the Pearson’s χ2 method. P < 0.05 indicated a statistically significant difference.
PC4 is significantly upregulated in breast cancer and is closely correlated with metastasis and poor prognosis of patients
Meanwhile, through analyzing 3951 cases of breast cancer patients from public cancer databases (Kaplan–Meier plotter database), the higher PC4 expression group (n = 1976) had poorer overall survival compared with lower PC4 expression group (n = 1975) (P < 0.0001, Additional file 1: Figure S1). Data from Vant Veer breast cancer databases  and GSE9893  also demonstrated that overall survival periods were shorter among patients with higher PC4 levels in the tumor (P < 0.05, Fig. 1e-f). Taken together, these results indicate that PC4 is commonly upregulated in breast cancer and may represent a significant molecular feature in breast cancer pathogenesis.
PC4 knockdown suppresses breast cancer cell growth both in vitro and in vivo
Furthermore, we established a subcutaneous xenograft model to determine the effect of PC4 on breast cancer cell growth in vivo. The MDA-MB-231 cells with or without stable PC4-knockdown were inoculated subcutaneously into the right flank of female nude mice, and tumor sizes were measured every 2 days. During the whole experiment, the growth of xenografts in shPC4–1 and shPC4–2 group was inhibited compared with the control group (Fig. 2f). And the average tumor size and tumor weight at the endpoint was reduced by PC4 depletion (Fig. 2g-h). Taken together, these data suggest that PC4 promotes breast cancer cell growth both in vitro and in vivo.
Loss of PC4 inhibits metastasis of breast cancer via reducing epithelial-mesenchymal transition both in vitro and in vivo
Next, lung metastasis model, which was established by tail vein injection of PC4-knockdown and control breast cancer cells, was used to evaluate the role of PC4 in tumor metastasis in vivo. Female NOD/SCID mice injected with PC4-knockdown MDA-MB-231 cells showed a significant decrease of lung metastatic lesions in comparison with control group (Fig. 3 h-i). And the hematoxylin and eosin (H&E) staining of lung tissues confirmed that depletion of PC4 resulted in greatly decreased lung metastasis of breast cancer cells in vivo (Fig. 3j-k). Moreover, Fig. 3 l showed that downregulation of PC4 decreased the expression of Vimentin and increased the expression of E-cadherin, indicating that PC4 could induce EMT in vivo. Collectively, PC4 plays an important role in breast cancer metastasis via inducing EMT.
Glycolysis is critically involved in the oncogenic functions of PC4
Owing to the important role of Warburg effect in cancer development and progression , we next confirmed whether the oncogenic functions of PC4 were dependent on glycolysis. The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) was used to block glycolysis in MDA-MB-231 cells, and the specific plasmid was used to overexpress PC4 before 2-DG treatment (Fig. 4k). Then, we found that overexpression of PC4 increased the proliferation (Fig. 4o), migration (Fig. 4j-l) and invasion (Fig. 4m-n) of breast cancer cells, which was inhibited by 2-DG. These results demonstrate that PC4 exerts its oncogenic functions by promoting glycolysis in breast cancer.
PC4 promotes glycolysis by regulating c-Myc transcription
Given the important role of PC4 in transcription regulation , we subsequently examined whether PC4 could directly activate c-Myc transcription to promote its expression. To confirm the relationship between PC4 and c-Myc, we performed a bioinformatic analysis for the presence of putative PC4-binding sites (BS) in the C-MYC promoter, and found two putative PC4 BS in a region encompassing 0.1 kb upstream the transcription start site (TSS). Chromatin Immunoprecipitation (ChIP) showed that PC4 binds to the C-MYC promoter in that region (Fig. 5q), indicating that C-MYC is a direct downstream target of PC4.
Recurrence and metastasis are the two leading causes of death in breast cancer, especially in Oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) triple-negative breast cancer (TNBC) . Clarifying the underlying mechanisms of TNBC progression and developing novel therapeutic targets are crucial for breast cancer treatment [49, 50]. Here, we report a novel oncogene, PC4, which plays an important role in both ER-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell growth and metastasis by enhancing c-Myc mediated Warburg effect.
PC4 is initially identified as a transcription coactivator and contains highly conserved sequence among human, rat, mouse and yeast [12, 13]. As a nuclear protein, PC4 plays important roles in various cellular processes including basal transcription [14, 15, 16, 17], DNA replication [18, 19, 20, 21, 22], DNA repair [23, 24, 25, 26, 27, 28, 29, 30, 31] and chromatin organization [32, 33]. More specifically, PC4 interacts with distinct domains of transcription activators such as GAL4-VP16 , AP2  and SMYD3  to regulate their functions. Based on the studies of its biological function, we have reported that PC4 was significantly upregulated in the malignant transformation of normal dermal multipotent fibroblasts, implying the crucial role of PC4 in tumorigenesis . We and other researchers also showed that PC4 was highly expressed in several tumors [36, 37, 38], and regulated lymphatic metastasis  and chemo-radiosensitivity [26, 40, 41]. Although some studies showed the potential oncogenic role of PC4 in tumor, the moleculer mechanisms of PC4 in tumorigenesis and cancer progression are still unclear, especially in breast cancer. In this study, we specifically characterized PC4 expression in breast cancer. From immunohistochemistry results in both IDC and ILC, PC4 was demonstrated to be significantly upregulated and closely correlated with metastasis. The Kaplan-meier analysis of clinical data showed that PC4 was a potential predictor of poor prognosis in breast cancer. These findings led us to further explore the functional significance of increased PC4 expression in breast cancer. Through GSEA on 155 cases of breast cancer patients, we found that the gene sets of proliferation and EMT were positively correlated with high expression of PC4 in breast cancer. In both in vitro and in vivo experiments, PC4 knock-down obviously inhibited the growth and metastasis of breast cancer. Taken together, PC4 may be a promising therapeutic target in breast cancer.
As a hallmark of cancer cells, metabolic reprogramming is a key process during tumorigenesis and cancer progression . Cancer cells frequently exhibit high rates of aerobic glycolysis, which facilitates cancer growth and progression by increasing glucose uptake, elevating lactate production, and supporting the energy demands . In the process of glycolysis, c-Myc plays a key role by directly regulating the glycolytic enzyme genes . By GSEA, we found that glycolysis-related genes and c-Myc target genes were positively correlated with high expression of PC4 in breast cancer. These findings led us to explore whether the oncogenic functions of PC4 were dependent on c-Myc mediated glycolysis. Indeed, PC4 knock-down dramatically inhibited the expression of c-Myc, as well as glucose uptake, lactate production, ATP generation, ECAR and the protein level of GLUT1 and LDHA. And Overexpression of c-Myc could reverse the decreased glycolysis and malignant phenotypes in PC4-knockdown cells, implying that PC4 exerted its oncogenic roles by c-Myc mediated glycolysis. Finally, the ChIP assays revealed that PC4 could directly bind to c-Myc promoters and transcriptionally activate c-Myc to promote its expression. Given the central role of c-Myc in many human cancers especially in triple negative breast cancer , c-Myc remains a promising target for effective antitumor therapy . Unfortunately, c-Myc presents specific, significant obstacles to develop a strategy for its direct inhibition . Thus, it is critical that the c-Myc transcription regulation be well-understood, particularly in its role in cancer progression and development.
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This work was supported by National Key Research and Development Program (2016YFC1000805), Natural Science Foundation Programs (81372727 and 81773352) and University Innovation Team Building Program of Chongqing (CXTDG201602020).
Availability of data and materials
The data regarding to the expression of PC4 in breast cancer were from an open database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9893).
PL and CZ performed the project, analyzed research data, and wrote the manuscript. FL, LC and ZL took part in animal experiments and immunohistochemistry. YW, ZW and ZJ participated in cellular and molecular biology experiments (RNAi, qPCR, western blotting analysis and ChIP). LL and ZL contributed to the collection of human tissues. HM contributed to helpful discussion and reviewed the manuscript. CS designed the study, supervised the experiments and revised the manuscript. All authors have read and approved the final manuscript.
All the mouse experiments were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University and carried out in accordance with the “Guide for the care and use of laboratory animals” published by the US National Institutes of Health (Publication no.85–23, revised 1996).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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