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Address correspondence to Yanhua Xuan, M.D., Ph.D., Department of Pathology, Yanbian University Medicine College, No. 977, Gongyuan Rd., Yanji 133002, China.
Although glycolysis plays a pivotal role in breast cancer stem-like cell (BCSC) reprogramming, the molecular mechanisms that couple glycolysis to cancer stem-like cells remain unclear. SETD5 is a previously uncharacterized member of the histone lysine methyltransferase family. The goal of this study was to explore the mechanisms underlying the promotion of stem-like and glycolysis activation traits by SETD5. Previous studies have shown that overexpression of SETD5 in breast cancer tissues is associated positively with progression. The present study showed that SETD5 expression was enriched in BCSCs. Down-regulation of SETD5 significantly decreased BCSC properties and glycolysis in vitro and in vivo. Interestingly, SETD5 and glycolytic enzymes were accumulated in the central hypoxic regions of subcutaneous tumor tissues. Bioinformatic analysis predicted SETD5 binding to E1A binding protein p300 (EP300), and subsequently to hypoxia-inducible factor 1α (HIF-1α). The mechanistic study found that SETD5 is an upstream effector of EP300/HIF-1α. SETD5 knockdown reduced the expression of HIF-1α, hexokinase-2, and 6-phosphofructo-2-kinase in the nucleus after treatment with cobalt chloride, a chemical hypoxia mimetic agent that activates HIF-1α to accumulate in the nucleus. Therefore, SETD5 is required for glycolysis in BCSCs through binding to EP300/HIF-1α and could be a potential therapeutic target for breast cancer patients.
Breast cancer (BC) is one of the most common incident cancers. Approximately 2 million new cases have been reported worldwide and that number is increasing, pertaining to population growth, change in the age structure of the population, and an increase in age-specific incidence rates.
Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 1990 to 2017.
Even when oxygen is present, tumor cells metabolize glucose to generate sufficient ATP and produce lactate, which is much faster than oxidative phosphorylation, and does not require the process of mitochondrial oxidative phosphorylation.
This is known as the Warburg effect (glycolysis). Identifying novel genes involved in BC metabolism and elucidating the potential molecular mechanism of BC are essential strategies for the development of effective therapeutic agents.
Cancer stem-like cells (CSCs) represent small heterogeneous undifferentiated cell populations with a hierarchical organization. Breast cancer stem-like cells (BCSCs), a small population of CSCs with BC, contribute to poor prognosis by malignant progression. Recent studies have suggested that CSCs undergo metabolic alterations that include low mitochondrial respiration and high glycolytic activity.
Hypoxia, nutrient starvation, and low pH within the tumor microenvironment play a central role in regulating CSC progression, metastasis, and patient mortality.
some oncogenes cooperate with the EP300/HIF-1α pathway to promote hypoxic tumor cell metabolic adaptation and proliferation. Therefore, exploring regulators of EP300/HIF-1α activity will advance our understanding of basic biological processes and suggest future therapeutic strategies.
As a member of the SET domain containing gene family, the SETD5 gene encodes histone-modifying proteins.
The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.
found that genetic alteration of histone lysine methyltransferases is linked closely to overall survival of BC patients. SETD5 is also involved in the DNA damage response. Glycolytic regulators lead to DNA damage responses, contributing to drug resistance.
The current study showed that SETD5 plays a positively critical role in BC progression and identified the regulatory role of SETD5 in BCSCs. It also showed that SETD5 is required for activating glycolysis in BCSCs. Mechanistically, SETD5 worked as an upstream effector, and binding to EP300/HIF-1α regulated the expression of glycolytic enzymes after treatment with cobalt chloride (CoCl2), a chemical inducer of hypoxia. The purpose of this research was to examine the feasibility of SETD5 as a treatment strategy for BC.
Materials and Methods
Tissue Specimens
Tissue microarray analysis containing 117 formalin-fixed, paraffin-embedded human BC tissue specimens was obtained from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
Animals
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All mouse experiments were approved by the Yanbian University Animal Care and Use Committee. Spheroid cell suspension (100 μL) of MDA-MB-231 cells (American Type Culture Collection, Manassas, VA) [1 × 105 in 100 μL phosphate-buffered saline containing 50% Matrigel (Corning Inc., Corning, NY)] were injected subcutaneously into the left or right flank of 5-week-old BALB/c nude female mice (Vital Rivers, Beijing, China) to establish a tumor model. Tumors were harvested 6 weeks after inoculation.
Cell Culture, Reagents, and Transfection
MCF-7 and MDA-MB-231 cells (American Type Culture Collection) were cultured under conditions specified by the supplier. CoCl2 was from Sigma-Aldrich (St. Louis, MO). siRNA universal negative control (Sigma-Aldrich) and SETD5 endoribonuclease-prepared short interfering RNAs (Sigma-Aldrich) were transfected into cells with Lipofectamine 3000 (Invitrogen, Carlsbad, CA) as per the methods described by the manufacturer. Table 1 lists the sequence of SETD5 endoribonuclease-prepared short interfering RNAs.
Gli1, a potential regulator of esophageal cancer stem cell, is identified as an independent adverse prognostic factor in esophageal squamous cell carcinoma.
Gli1, a potential regulator of esophageal cancer stem cell, is identified as an independent adverse prognostic factor in esophageal squamous cell carcinoma.
Measurement of Glucose Uptake and Lactate Production
Intracellular glucose was measured according to the Glucose Colorimetric Assay Kit (BioVision, San Francisco, CA) protocol, and extracellular lactate was measured according to the Lactate Colorimetric Assay Kit (BioVision) instructions.
Co-Immunoprecipitation
Co-immunoprecipitation was performed using the Thermo Scientific Pierce co-immunoprecipitation kit (Thermo Scientific, Rockford, IL) following the manufacturer's protocol.
Preparation of Cytoplasmic and Nuclear Extract Analysis
Cytoplasmic and nuclear extracts for Western blot analysis were prepared with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the manufacturer's protocol.
BC Patient Cohort Analysis Based on Public Data Sets
The Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo; accession number GSE103091) was analyzed using gene set enrichment analysis software v2.2.4 (http://www.broadinstitute.org/gsea, last accessed September 10, 2019) to identify the association of SETD5 expression with hypoxia and glycolysis genes. Gene set enrichment analysis includes three key statistics: false discovery rate, normalized enrichment score, and nominal P value. Based on the Clinical Proteomic Tumor Analysis Consortium, UALCAN (http://ualcan.path.uab.edu, last accessed July 21, 2020) was used for clinicopathologic analysis of SETD5. The Kaplan-Meier online plotter tool (http://www.kmplot.com, last accessed July 17, 2020) was performed to evaluate the correlation between the presence of SETD5 and post-progression survival of BC patients. The STRING database (version 11.0) (https://string-db.org, last accessed July 17, 2020) was used for mapping protein interaction networks. Based on The Cancer Genome Atlas, a Spearman correlation analysis was performed using the online Gene Expression Profiling Interactive Analysis web portal (http://gepia.cancer-pku.cn, last accessed July 17, 2020).
Statistical Analysis
Correlations were tested using Spearman correlation analysis as appropriate. Comparisons between groups were performed using the t-test with SPSS version 25.0 (SPSS, Inc, Chicago, IL) and GraphPad Prism version 5.01 (GraphPad, Inc., San Diego, CA). All tests were two sided. Results are expressed as means ± SD as indicated, and P < 0.05 was considered significant.
Results
SETD5 Is a Positive Regulator of BCSCs
Previous data provided evidence that SETD5 expression was predictive of poor prognosis and contributed to BC progression.
Su(var)3–9, enhancer of zeste, and trithorax domain-containing 5 facilitates tumor growth and pulmonary metastasis through up-regulation of AKT1 signaling in breast cancer.
Herein, the clinical pathologic diagnosis of SETD5 expression was analyzed in normal breast (n = 18) and BC tissues (n = 125) in the Clinical Proteomic Tumor Analysis Consortium database through the UALCAN web portal. As shown in Supplemental Figure S1A, SETD5 protein expression significantly increased in BC tissues relative to normal tissues (P < 0.001). Clinical Proteomic Tumor Analysis Consortium data also showed that SETD5 was overexpressed in BC tissues with major subclass (luminal versus normal, P < 0.001; human epidermal growth factor receptor 2 positive versus normal, P < 0.01; triple negative breast cancer versus normal, P < 0.05), patient age (21 to 40 years versus normal, P < 0.01; 41 to 60 years versus normal, P < 0.001; 61 to 80 years versus normal, P < 0.001; and 81 to 100 years versus normal, P < 0.05), and individual cancer stages (stage 1 versus normal, P < 0.001; stage 2 versus normal, P < 0.01; and stage 3 versus normal, P < 0.001) (Supplemental Figure S1, B–D). A poor post-progression survival was considered as the median overall survival minus the median progression-free survival for each trial arm. Furthermore, the Kaplan-Meier online plotter tool analysis showed that SETD5 expression was related positively to a poor post-progression survival in BC patients using all Affymetrix ID including 1569105_at, 1569106_at, 222575_at, and 221806_s_at (Supplemental Figure S1E). Next, the effect of SETD5 on the properties of CSCs was explored. The protein levels of SETD5 in adherent monolayers and spheroid body-forming cells were determined by Western blot analysis. SETD5 was higher in BC spheroid body-forming cells than in adherent monolayer cells (Figure 1A). The efficiency of the knockdown approach for SETD5 protein expression was indicated using SETD5 endoribonuclease-prepared short interfering RNA in both CSC types by Western blot analysis (Figure 1B). The role of SETD5 in BC spheroid body-forming cells was investigated further. Knockdown of SETD5 expression significantly decreased the number and size of BC spheroid body-forming cells (Figure 1C). IF analysis showed that knockdown of SETD5 resulted in a significant reduction in leucine rich repeat containing G protein-coupled receptor 5 (LGR5) staining in BC spheroid body-forming cells (Figure 1D).
Figure 1SETD5 promotes breast cancer stem-like cell (BCSC) properties. A: The protein expression of SETD5 in spheroid cells compared with those in adherent monolayer counterparts was analyzed using Western blot. B: Effects of silencing SETD5 in MCF-7 and MDA-MB-231 spheroid body-forming cells evaluated by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. C: Analysis of spheroid formation ability in control and SETD5-silenced MDA-MB-231 and MCF7 cells. D: Immunofluorescence examined the expression of LGR5 in control and SETD5-silenced spheroid cells. Immunofluorescence intensity of LGR5 staining in control and SETD5-silenced spheroid cells is shown. ∗∗P < 0.01, ∗∗∗P < 0.001 versus controls. Original magnification: ×200 (A); ×400 (C and D). Scale bars: 100 μm (A); 20 μm (C and D). Con, control; esi-SETD5, SETD5 endoribonuclease-prepared short interfering RNA.
To further explore how SETD5 acted as a crucial regulator of BCSC maintenance in vivo, subcutaneous injections of MDA-MB-231 cells dissociated from the spheroid body were used. Tumors were harvested at 6 weeks after inoculation of cells. The xenograft tumor size and tumor volume per week were measured by visual analysis (n = 4 tumors/group). Xenograft tumor growth indicated that inhibition of SETD5 expression could reduce the weight and volume significantly in vivo (Figure 2A). IHC staining of CSC markers was performed in BC xenograft tumor samples. SETD5 endoribonuclease-prepared short interfering RNAs down-regulated the IHC score of CSC markers, prominin 1 (CD133), and LGR5 (Figure 2B). In addition, IF was used to examine the correlation between SETD5 expression and CSC markers. Figure 2C shows that SETD5 was co-located with CD133 or LGR5 in BC patient tissues. These findings support a positive functional role of SETD5 in the maintenance of BCSCs.
Figure 2SETD5 endoribonuclease-prepared short interfering RNA (esi-SETD5) suppresses breast tumorigenicity in vivo. A: Control MDA-MB-231 spheroid cells and esi-SETD5 MDA-MB-231 spheroid cells (1 × 105 cells per mice) were injected subcutaneously into immunodeficient mice (n = 4), respectively. Tumor weights and tumor volumes were monitored at the indicated times after injection. B: Expression of CD133 and LGR5 in xenograft tumors transfected with esi-SETD5 were detected by immunohistochemistry (IHC). C: Immunofluorescence analyzed the co-localization of SETD5 and cancer stem-like cell markers CD133 and LGR5 in breast cancer tissues. ∗∗∗P < 0.001 versus controls. Scale bars = 100 μm. Original magnification: ×100 (B); ×200 (C). Con, control.
There was a positive correlation between SETD5 expression and five key glycolytic genes detected in BC samples by the Gene Expression Profiling Interactive Analysis web portal through the Cancer Genome Atlas database, including HK2, PFKFB3, PKM2, LDHA, and PDK1 (Supplemental Figure S2). To test the effect of SETD5 on the cellular metabolic reprogramming of BCSCs, glucose uptake and lactate production of BC spheroid body-forming cells were assessed. SETD5 depletion inhibited glucose uptake and lactate production in BC spheroid body-forming cells (Figure 3, A and B ). Consistently, blocking SETD5 expression significantly reduced the glycolytic gene expression of HK2 and PFKFB3 in BC spheroid body-forming cells, which was assessed using Western blot and IF (Figure 3, C and D). A similar result was obtained in vivo by IHC examination (Figure 3E). SETD5 depletion also down-regulated HK2 and PFKFB3 protein expression in xenograft tumor samples. Thus, SETD5 may be a potential regulator of glycolysis in BCSCs.
Figure 3SETD5 positively regulates glycolysis in breast cancer stem-like cells (BCSCs). A and B: Comparison of glucose uptake (A) and lactate production (B) of breast cancer (BC) spheroid cells transfected with SETD5 endoribonuclease-prepared short interfering RNAs (esi-SETD5) with control. C: The protein expression of hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in BC spheroid cells transfected with esi-SETD5 analyzed using Western blot. D: Immunofluorescence showing the protein expression of HK2 and PFKFB3 in BC spheroid cells and transfected with esi-SETD5. Immunofluorescence quantitation for HK2/PFKFB3 in BC spheroid cells is presented. E: Expression of HK2 and PFKFB3 in xenograft tumors transfected with esi-SETD5 was detected by immunohistochemistry. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus controls. Scale bars: 20 μm (D); 100 μm (E). Original magnification: ×400 (D); ×100 (E). Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
reported that histone methyltransferase SETD8 contributes to HIF-1α regulation and mediates the metabolism process. Herein, the central (hypoxic) and peripheral (normoxic) regions from xenograft tumor tissues were analyzed using hematoxylin and eosin staining (Supplemental Figure S3A). IHC staining results showed that SETD5-positive cells mostly gathered centrally with highly hypoxic cells, as shown by HIF-1α, when compared with the peripheral regions (Supplemental Figure S3B). HK2 and PFKFB3 were highly expressed in the central positions compared with the peripheral positions (Supplemental Figure S3C). Gene set enrichment analysis of BC patients and the Gene Expression Omnibus database were used to find an enriched expression of the SETD5 gene. A positive correlation was found between RNA levels of SETD5 and the hypoxic gene (normalized enrichment score, 2.07; false discovery rate, q = 0.14; P < 0.001) (Figure 4A), as well as positive regulatory effects of SETD5 on glycolysis (normalized enrichment score, 1.39; false discovery rate, q = 0.23; P = 0.04) (Figure 4B). These results were used to test the hypothesis that SETD5 regulates HIF-1α protein stability to activate glycolysis. First, BC cells were exposed to CoCl2 (100, 200, and 400 μmol/L) for 6 or 12 hours each. CoCl2 significantly induced the expression of HIF-1α protein in a time- and dose-dependent manner (Supplemental Figure S4). HIF-1α protein expression was up-regulated most effectively after 200 μmol/L CoCl2-induced hypoxia for 12 hours. This treatment condition was then used in the ensuing hypoxia mimetic experiments. In addition, knocking down SETD5 reduced the glycolytic gene expression of HK2 and PFKFB3 when exposing BC spheroid body-forming cells to CoCl2 (Figure 4, C and D). Thus, these results indicate that SETD5 activates BCSC glycolysis, which is a hypoxia stimulus.
Figure 4SETD5 is associated positively with hypoxia and glycolysis in breast cancer (BC). A and B: Based on SETD5 expression levels, gene set enrichment analysis was performed on BC data sets from the Gene Expression Omnibus database using the gene sets regulated by hypoxia and glycolysis. C: The protein expression of hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in BC spheroid cells and transfected with SETD5 endoribonuclease-prepared short interfering RNAs (esi-SETD5) was analyzed using Western blot when exposed to 200 μmol/L CoCl2. D: Immunofluorescence showing the expression of HK2 and PFKFB3 in BC spheroid body-forming cells when exposed to 200 μmol/L CoCl2. Immunofluorescence quantitation for HK2/PFKFB3 in MDA-MB-231 cells when exposed to CoCl2 is shown. ∗∗P < 0.01 versus controls. Scale bars = 20 μm. Original magnification: ×400. Con, control; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NES, normalized enrichment score.
To explore the functional connection of SETD5–HIF-1α and glycolysis, the protein–protein interaction network was constructed using the STRING database. Nine of these genes comprised a protein–protein interaction network (protein–protein interaction enrichment P value < 0.001) (Supplemental Figure S5A). Furthermore, tissue microarray analysis was performed for the association between SETD5 and EP300/HIF-1α protein expression in 117 cases of human BC specimens by IHC staining (Figure 5A). Of the 90 cases of the EP300-positive group, 76 cases (76 of 90; 84.4%) were positive for the SETD5 gene (P = 0.008) (Figure 5B). Of the 76 cases of the HIF-1α–positive group, 63 cases (63 of 76; 82.9%) were positive for the SETD5 gene (P = 0.018) (Figure 5B). There was a positive correlation between SETD5 expression and EP300 and HIF-1α detected in BC samples by the Gene Expression Profiling Interactive Analysis web portal (Supplemental Figure S5B). Importantly, knocking down SETD5 expression dramatically decreased the expression of EP300 and HIF-1α in BC cells (Figure 5, C and D). Furthermore, knocking down SETD5 expression decreased the signaling of EP300 and HIF-1α when BC cells were exposed to CoCl2 (Figure 5D). Therefore, SETD5 expression strongly correlated with EP300/HIF-1α and worked as an upstream effector in BC cells.
Figure 5SETD5 bound to EP300/hypoxia-inducible factor 1α (HIF-1α) in breast cancer (BC) cells. A: Immunohistochemical staining for SETD5, EP300, and HIF-1α in 117 BC tissue microarrays. B: The protein expression ratio of SETD5 in EP300 positivity and HIF-1α positivity was examined in 117 BC tissue microarrays using immunohistochemistry staining. C: The expression of SETD5, EP300, and HIF-1α was analyzed in BC cells transfected with SETD5 endoribonuclease-prepared short interfering RNAs (esi-SETD5) using Western blot. D: The expression of EP300 and HIF-1α was analyzed in BC cells transfected with esi-SETD5 using Western blot when exposed to 200 μmol/L CoCl2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. ∗P < 0.05, ∗∗P < 0.01 versus controls. Scale bars = 100 μm. Original magnification: ×100.
SETD5 Facilitates Glycolysis via HIF-1α in the Nucleus
To gain insight into the molecular mechanism of SETD5 in contributing to glycolysis in BC cells, the subcellular localization of SETD5 putative target genes HIF-1α involved in hypoxia-induced glycolysis was investigated using cytoplasmic and nuclear extract assays. The nuclear expression of HIF-1α was stronger than its cytoplasmic expression after treatment with CoCl2 (Figure 6A). Interestingly, IF staining showed that SETD5 knockdown reduced HIF-1α protein expression in the nuclear extracts of BC cells (Figure 6B). Immunoprecipitation confirmed that SETD5 could bind HIF-1α in the nuclear extracts of BC cells under chemically induced hypoxia (Figure 6C). Glycolytic enzymes were examined further in the nucleus of BC cells transfected with SETD5 endoribonuclease-prepared short interfering RNAs under chemically induced hypoxia. SETD5 silencing decreased the expression of HIF-1α, HK2, and PFKFB3 proteins (Figure 6D).
Figure 6SETD5 binds hypoxia-inducible factor 1α (HIF-1α) to facilitate glycolysis of breast cancer (BC) cells. A: The expression of HIF-1α in the cytoplasm (CE) or nuclear extracts (NE) of BC cells was examined by Western blot under normoxia and hypoxia. B: Immunofluorescence showing the location of HIF-1α in BC cells under normal conditions, treatment with 200 μmol/L CoCl2 for 12 hours, and transfection with SETD5 endoribonuclease-prepared short interfering RNAs (esi-SETD5) for 24 hours and treatment with 200 μmol/L CoCl2 for 12 hours, respectively. Immunofluorescence intensity of HIF-1α staining in two cell lines is presented. C: Co-immunoprecipitation of SETD5 and HIF-1α in the nuclear fraction of BC cells under hypoxia. D: The protein expression of HIF-1α, hexokinase 2 (HK2), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in the cytoplasmic and nuclear fractions of BC cells, transfected with esi-SETD5 under hypoxia, was examined by Western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA) were used as loading controls. ∗∗∗P < 0.001 versus controls. Scale bars = 10 μm. Original magnification, ×600. Con, control; IB, immunoblot; IP, immunoprecipitation.
Cancer cells can adjust their metabolic phenotypes to adapt to the microenvironment. Indeed, glycolysis is considered the dominant metabolic phenotype in cancer. The current study shows, for the first time, a correlation between SETD5 expression in BCSCs and glycolysis, which in turn boosts tumor progression and poor prognosis.
Activation of an epithelial stem cell–like transcriptional program in differentiated adult cells may induce pathologic self-renewal, a characteristic of CSCs.
reported that SETD5 plays a vital role in mammalian embryonic development, cell-cycle progression, and the co-transcriptional regulation of histone acetylation. Recently, Sessa et al
found that SETD5 silencing alters the dynamics of proliferation and differentiation of cortical progenitor cells. The current study showed that SETD5 may positively regulate the stem-like properties of BC cells. Furthermore, SETD5 knockdown in BC spheroid body-forming cells significantly down-regulated spheroid diameters, the number of cells, and CSC marker expression in vitro. Inhibition of SETD5 expression in MDA-MB-231 spheroid cells markedly suppressed tumor growth in vivo. Inhibiting SETD5 expression led to down-regulation of CD133 and LGR5 proteins in tumor xenograft tissues. Specifically, as shown in Figure 2, the SETD5-positive cell populations interacted with CSC markers. Moreover, SETD5 depletion inhibited glucose uptake while favoring lactate production. The expression of key glycolytic enzymes in BC spheroid body-forming cells were down-regulated in vitro and in vivo. Thus, this study indicated that SETD5 is a positive regulator of glycolysis in BCSCs. Accordingly, we speculated that SETD5 expression is involved in regulating BCSC properties, as well as glycolysis of BCSCs.
EP300 works as a histone acetyltransferase and regulates transcription via chromatin remodeling. Histone acetylation gives an epigenetic tag for transcriptional activation. SETD5 is a probable transcriptional regulator that acts via the formation of large multiprotein complexes that modify and/or remodel chromatin, which acts as a regulator of histone acetylation during gene transcription. The current data show that SETD5 was associated positively with EP300. A series of epigenetic factors has been shown to exert regulatory roles in HIF-1α protein stability control.
have shown that regulation of HIF-1α protein stability is necessary for the maintenance of glycolysis and malignant properties in BC. These results are highly consistent with the Huang et al
report that histone methyltransferase SETD8 reprograms BC cell metabolism through a HIF-1α–mediated process. Gene set enrichment analysis results showed a positive correlation between SETD5, glycolysis, and hypoxia. In vivo, HIF-1α, SETD5, HK2, and PFKFB3 expression was higher in tumor hypoxic regions than in normoxic regions, which further corroborated the results in vitro. Figure 5 verifies SETD5 as an upstream effector of EP300/HIF-1α in BC tissues. Therefore, this study suggests that SETD5 is a positive regulator of EP300/HIF-1α. Moreover, because Figure 4 shows that SETD5 silencing decreased the expression of HK2 and PFKFB3 proteins in BC spheroid body-forming cells when exposed to CoCl2, the current findings suggest that SETD5 expression induces glycolysis by binding HIF-1α in the nucleus of BC cells under hypoxia stimulants.
The function of HIF-1α depends on its protein stability and subcellular localization. Under hypoxic conditions, ubiquitination of HIF-1α is suppressed, and more HIF-1α binds to HIF-1β to form the HIF-1 transcription complex.
The current results confirm that the hypoxia mimetic agent CoCl2 induces HIF-1α nuclear translocation. Co-immunoprecipitation showed that hypoxic stress enhanced HIF-1α and SETD5 interactions in the nucleus of BC cells. More importantly, SETD5 knockdown decreased HIF-1α expression in the nucleus of BC cells and the expression of key glycolytic enzymes under hypoxia mimetic agent treatment. SETD5 indirectly binds HIF-1α in the nucleus and promotes BC cell glycolysis under hypoxic conditions.
Conclusions
SETD5 expression was high in BC tissues and had a strongly positive relationship with poor survival. SETD5 regulated glycolysis in BCSCs, which in turn contributed to BCSC maintenance through binding to EP300/HIF-1α. Thus, targeting SETD5 may be a potential therapeutic strategy for BC aberrant metabolism treatment.
Acknowledgment
We thank Editage (Beijing, China) for English-language editing.
Supplemental Data
Supplemental Figure S1SETD5 was overexpressed in clinical breast cancer (BC) patients and correlated positively with the survival time. A–D:SETD5 expression was detected in the following normal tissues (n = 18) and BC tissues (n = 125) (A); in normal tissues (n = 18) and major subclasses of BC tissues [luminal, n = 64; human epidermal growth factor receptor 2 (HER2)-positive, n = 10; triple negative breast cancer (TNBC), n = 16] (B); in normal (n = 18) and different patient age groups (21 to 40 years, n = 10; 41 to 60 years, n = 50; 61 to 80 years, n = 53; and 81 to 100 years, n = 7) (C); and in normal (n = 18) and different individual cancer stages (stage 1, n = 4; stage 2, n = 74; and stage 3, n = 32) (D) using the online web portal UALCAN. E: Survival after progression analysis of the correlation between survival time and SETD5 in BC was performed using the online Kaplan–Meier plotter tool at four Affymetrix ID including 1569105_at, 1569106_at, 222575_at, and 221806_at. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus controls. HR, hazard ratio.
Supplemental Figure S2SETD5 is required for activating glycolysis. A–E: The correlation between SETD5 and glycolytic genes was analyzed in breast cancer (BC) tissues by the Gene Expression Profiling Interactive Analysis web portal. Hexokinase 2 (A); ; PFK-2, 6-phosphofructo-2-kinase (PFKFB3) (B); pyruvate kinase M1/2 (PKM2) (C); lactate dehydrogenase A (LDHA) (D); pyruvate dehydrogenase kinase 1 (PDK1) (E). TPM, transcripts per million.
Supplemental Figure S3A: Central and peripheral regions of xenograft tumors were analyzed by hematoxylin and eosin (HE) staining. B and C: Immunohistochemical staining for hypoxia-inducible factor 1α (HIF-1α) and SETD5 in the central and peripheral regions of xenograft tumor tissues (B), as well as glycolysis enzymes, hexokinase 2 (HK2) and PFK-2, 6-phosphofructo-2-kinase (PFKFB3) (C). Scale bars =100 μm. Original magnification: ×100.
Supplemental Figure S4The expression of hypoxia-inducible factor 1α (HIF-1α) protein in breast cancer (BC) cells with CoCl2-induced hypoxia treatment. MCF-7 (left) and MDA-MB-231 (right) cells were treated with CoCl2 (100, 200, and 400 μmol/L) for 6 and 12 hours each, respectively, and maintained in a humidified 5% CO2 atmosphere at 37°C. The expression of HIF-1α protein was analyzed by Western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control.
Supplemental Figure S5The correlation of SETD5, EP300, and hypoxia-inducible factor 1α (HIF-1α) in breast cancer (BC). A: STRING analysis of SETD5 genes indicating the protein–protein interaction (PPI) network. Colored nodes: query proteins and first shell of interactors: blue-green line, known interactions from curated databases; purple line, known interactions from experiments; green line, predicted interactions form gene neighborhood; red line, predicted interactions form gene fusions; dark blue, predicted interactions form gene co-occurrence; yellow line, interactions form text mining; black line, interactions form co-expression; light blue line, interactions form protein homology. B: Gene Expression Profiling Interactive Analysis analyzed the correlation between SETD5 and HIF-1α mRNA levels in BC tissues. EGLN1, Egl-9 family hypoxia inducible factor 1; HK2, hexokinase 2; PFKFB3, PFK-2, 6-phosphofructo-2-kinase; TCEB1, transcription elongation factor B polypeptide 1 ; TPM, transcripts per million; VHL, Von Hippel-Lindau .
Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 1990 to 2017.
The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.
Gli1, a potential regulator of esophageal cancer stem cell, is identified as an independent adverse prognostic factor in esophageal squamous cell carcinoma.
Su(var)3–9, enhancer of zeste, and trithorax domain-containing 5 facilitates tumor growth and pulmonary metastasis through up-regulation of AKT1 signaling in breast cancer.
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