SETD5 Binding to EP300/HIF1α is Required for Glycolysis in Breast Cancer Stem-Like Cells

Background Glycolysis plays a pivotal role in breast cancer stem-like cell reprogramming. The SET-domain containing 5 (SETD5) is a previously uncharacterized member of the histone lysine methyltransferase family. Yet, the molecular mechanisms underlying the promotion of stem-like and glycolysis activation traits of SETD5 have not been elucidated. Basing on public datasets, we explored clinicopathological and survival analysis of SETD5 on breast cancer (BC) patients. Spheroid formation, transfection experiments and measurement of glucose uptake and lactate production analyzed the regulatory function of SETD5 on glycolysis in breast cancer stem-like cells (BCSC). The impact of SETD5 on tumor growth was studied in a murine xenograft model. Immunohistochemistry, immunouorescence, western blot, preparation of cytoplasmic and nuclear extracts and co-immunoprecipitation were used to determine the molecular mechanisms of SETD5 in cancer cell glycolysis. and could be a potential therapeutic target for BC patients.


Introduction
Breast cancer (BC) is one of the most common incident cancers. Approximately 2.0 million new cases have increased worldwide, pertaining to population growth, change in the age structure of the population, and increase in age-speci c incidence rates [1]. Among various therapeutic approaches, cancer cell metabolism is a promising target [2]. To ensure survival, tumor cells usually select their metabolism to avoid host immune attack and to continue to proliferate [3]. Even when oxygen is present, tumor cells metabolize glucose to generate su cient ATP to produce lactate, which is much faster than oxidative phosphorylation, and does not require the process of mitochondrial oxidative phosphorylation [4]. This is known as the Warburg effect (aerobic glycolysis). Identifying novel genes involved in BC metabolism and elucidating the potential molecular mechanism of BC are essential 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 (BCSC), a small population of CSC with BC, are suggested to contribute to poor prognosis by malignant progression. Recent studies suggest that CSCs undergo metabolic alterations that include low mitochondrial respiration and high glycolytic activity [4]. Exploiting the regulator of CSCs' metabolism may provide new effective therapies and diminish the risk of recurrence and metastasis [5,6].
p300 (also called EP300) is a transcriptional/epigenetic regulator [13], and involved in a series of physiological and pathological processes [13]. In addition, as EP300 is a key co-activator of hypoxiainducible transcription [14], some oncogenes cooperate with EP300/HIF1α pathway to promote hypoxic tumor cell metabolic adaptation and proliferation. Therefore, exploring regulators of EP300/HIF1α 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 SET-domain containing 5 (SETD5) gene encodes histone-modifying proteins [15]. SETD5 is a previously uncharacterized member of the histone lysine methyltransferase family [16]. De novo heterozygous mutations in SETD5 are considered responsible for intellectual disability and autism spectrum disorders [17,18]. Overexpression of SETD5 is positively associated with poor prognosis in lung [19] and esophageal cancer [20]. A meta-analysis by Liu et al. previously found that genetic alteration of histone lysine methyltransferases is closely linked to overall survival of BC patients [21]. Previous studies have shown that glycolytic regulators lead to DNA damage responses, contributing to drug resistance [22]. SETD5 is also involved in DNA damage response.
Here, we demonstrated that SETD5 plays a positively critical role in BC progression. Further, we identi ed the regulatory role of SETD5 in BCSCs. We also found that SETD5 is required for activating glycolysis in BCSCs. Mechanistically, we explored SETD5 binding to EP300/HIF1α through protein-protein associations (PPI) using the STRING database. SETD5 is an upstream effector of EP300/HIF1α. Next, SETD5 knockdown reduced the expression of HIF1α and glycolytic enzymes after treatment with cobalt chloride (CoCl 2 ), a chemical inducer of hypoxia. The purpose of this research was to examine the feasibility of SETD5 as a treatment strategy for BC.

Material And Methods
Tissue specimens TMA containing formalin-xed and para n-embedded 117 human BC tissue specimens were obtained from Shanghai Outdo biotech Co. Ltd. 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 Helsinki declaration 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 Yanbian University Animal Care & Use Committee. Immunohistochemistry, western blot, immuno uorescence, and spheroid formation analysis According to previous protocols, immunohistochemistry staining and evaluation [23], western blot [23], immuno uorescence [24] and spheroid formation assay [24] procedures were performed. The antibodies used are listed in Supplementary table 2.
Measurement of glucose uptake and lactate production

Statistical analysis
Correlations were tested using Spearman correlation analysis as appropriate. Comparisons between groups were performed using Student's t-test with SPSS version 25.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism version 5.01 (GraphPad Inc., San Diego, CA, USA). All tests were two sided, Results are expressed as mean ± standard deviation as indicated, and p < 0.05 was considered signi cant.

SETD5 expression was positively related to BC progression
Firstly, we analyzed the clinical pathological diagnosis of SETD5 expression in normal breast (n = 18) and BC tissues (n = 125) in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database through the UALCAN web portal. As shown in Fig. 1a, SETD5 protein expression signi cantly increased in BC tissues relative to normal tissues (p < 0.001). CPTAC data also showed that SETD5 was over-expressed in BC tissues with major subclass (Luminal vs Normal, p < 0.001; HER2 vs Normal, p < 0.01; TNBC vs Normal, p < 0.05), patient's age (21-40 years vs Normal, p < 0.01; 41-60 years vs Normal, p < 0.001; 61-80 Years vs Normal, p < 0.001; 81-100 years vs Normal, p < 0.05), and individual cancer stages (stage 1 vs Normal, p < 0.001; stage 2 vs Normal, p < 0.01; stage 3 vs Normal, p < 0.001) ( Fig. 1b-d). Post-progression survival (PPS) was considered as the median overall survival minus the median progression-free survival for each trial arm [25]. Furthermore, the Kaplan-Meier Plotter online tool analysis showed that, SETD5 expression was positively related to a poor PPS in BC patients using all Affymetrix ID (Fig. 1e). These data suggest that overexpression of SETD5 in BC tissues could be used as a potential predictor of survival.

SETD5 is a positive regulator of BCSC
Our data provide evidence that SETD5 expression was predictive of poor prognosis and contributed to BC progression. Next, we explored the effect of SETD5 on the properties of CSCs. The protein levels of SETD5 in adherent monolayers and spheroid body-forming cells were determined by western blot analysis. SETD5 were obviously higher in BC spheroid body-forming cells than in adherent monolayer cells (Fig. 2a). The role of SETD5 in BC spheroid body-forming cells was then further investigated. Knockdown of SETD5 expression signi cantly decreased the number and size of spheroid body-forming cells of BC (Fig. 2b). Immuno uorescent (IF) analysis demonstrated that knockdown of SETD5 resulted in a signi cant reduction in LGR5 staining in BC spheroid body-forming cells (Fig. 2c).
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 administered in nude mice for 6 weeks. Then, we measured the xenograft tumor size and tumor volume per week by visual analysis (n = 4 tumors/group). Xenograft tumor growth indicated that inhibition of SETD5 expression could signi cantly reduce the weight and volume in vivo (Fig. 3a). Immunohistochemical (IHC) staining of CSC markers was performed in BC xenograft tumor samples. esi-SETD5 downregulated the IHC score of CSC markers, CD133, and LGR5 (Fig. 3b). Additionally, we used IF to examine the correlation between SETD5 expression and CSC markers. Figure 3c showed that SETD5 was co-located with CD133 or LGR5 in BC patient tissues. These ndings support a positive functional role of SETD5 in the maintenance of BCSCs.

SETD5 activated BCSC glycolysis
To test the effect of SETD5 on the cellular metabolic reprogramming of BCSC, 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 (Fig. 4a, b). Consistently, blocking SETD5 expression signi cantly reduced glycolytic genes HK2 and PFKFB3 expression in BC spheroid bodyforming cells, which was assessed using IF (Fig. 4c). A similar result was obtained in vivo by IHC examination (Fig. 4d). SETD5 depletion also downregulated HK2 and PFKFB3 protein expression in xenograft tumor samples. Thus, SETD5 may be a potential regulator of aerobic glycolysis in BCSCs.

SETD5 binding to EP300/HIF1α in BC cells
Hypoxia is a pervasive environmental stimulus of glucose metabolic reprogramming. Recently, Huang et al. reported that histone methyltransferases SETD8 contribute to HIF1α regulation and mediate the metabolism process [22]. Thus, the central (hypoxic) and peripheral (normoxic) regions from xenograft tumor tissues were analyzed using hematoxylin-eosin staining (Fig. 5a). IHC staining results showed that SETD5-positive cells mostly gathered centrally with highly hypoxic cells as revealed by HIF1α, when compared with the peripheral regions (Fig. 5b). HK2 and PFKFB3 were highly expressed in the central positions compared with the peripheral positions (Fig. 5c). Gene Set Enrichment Analysis (GSEA) and used the GEO database to nd an enriched expression of SETD5 gene in BC tissues. We found a positive correlation between RNA levels of SETD5 and the hypoxic gene (NES = 2.07, FDR q = 0.14, p < 0.001) (Fig. 5d), as well as positive regulatory effects of SETD5 on glycolysis (NES = 1.39, FDR q = 0.23, p = 0.04) (Fig. 5e). Thus, these results indicate that SETD5 activates BCSC glycolysis which is a hypoxia stimulus.
To explore the functional connection of SETD5-HIF1α and glycolysis, we constructed the PPI network using the STRING database. Nine of these genes comprised a PPI network (PPI enrichment p-value < 0.001) (Fig. 6a). Co-expression analysis revealed tight co-expression across the tissue types between SETD5 and EP300 genes (Fig. S1). Confocal microscopy IF experiments also measured co-expression of SETD5 and EP300 in BC tissues (Fig. 6b).

SETD5 facilitates glycolysis via HIF1α in the nucleus
The above-mentioned results prompted us to test the hypothesis that SETD5 regulates HIF1α protein stability to activate glycolysis. Firstly, we exposed BC cells to CoCl 2 (100, 200, and 400 µmol/L) for 6 and 12 hours each. CoCl 2 signi cantly induced the expression of HIF1α protein in a time-and dose-dependent manner (Fig. S3). HIF1α protein expression was most effectively upregulated after 200 µmol/L of CoCl 2induced hypoxia treatment for 12 hours. This treatment condition was then used in the ensuing hypoxia mimetic experiments. To get insight into the molecular mechanism of SETD5 in contributing to glycolysis in BC cells, the subcellular localization of SETD5 and its putative target genes HIF1α involved in hypoxiainduced glycolysis were investigated using cytoplasmic and nuclear extract assays. The nuclear expression of HIF1α was stronger than the cytoplasmic expression after treating with CoCl 2 (Fig. 7a).
SETD5 expression levels increased under chemically-induced hypoxia than under normoxic conditions in the total cell lysate. However, there was no obvious change in SETD5 nuclear and cytoplasmic distribution (Fig. 7a). Immunoprecipitation con rmed that SETD5 could bind HIF1α in the nuclear extracts of BC cells under chemically-induced hypoxia conditions (Fig. 7b). Interestingly, IF staining showed that SETD5 knockdown reduced HIF1 protein expression in the nuclear extracts of BC cells (Fig. 7c). Glycolytic enzymes were further examined in the nucleus of BC cells transfected with esi-SETD5 under chemically-induced hypoxia conditions. Figure 7d showed that SETD5 silencing decreased the expression of HK2 and PFKFB3 proteins.

Discussion
It is considered that cancer cells can adjust their metabolic phenotypes to adapt to the microenvironment. Indeed, aerobic glycolysis is considered the dominant metabolic phenotype in cancer. Here, for the rst time, we correlated SETD5 expression in BCSCs with aerobic glycolysis, which in turn boosts tumor progression and poor prognosis.
Firstly, the association between SETD5 expression and the clinical pathological diagnosis of BC was analyzed with an online tool. The expression of SETD5 in BC was higher than in normal breast tissues, which is consistent with previous results [21]. Furthermore, higher SETD5 expression was associated with worse PPS in BC patients. SETD5 may be an oncogenic factor, as SETD5 was upregulated in non-small cell lung cancer and was a prognostic determinant in prior studies [19].
Activation of an epithelial stem cell (ESC)-like transcriptional program in differentiated adult cells may induce pathologic self-renewal, a characteristic of CSCs [26]. Osipovich et al. reported that SETD5 plays a vital role in mammalian embryonic development, cell cycle progression, and the co-transcriptional regulation of histone acetylation [27]. Recently, Sessa et al. found that SETD5 silencing alters the dynamics of proliferation and differentiation of cortical progenitor cells [28]. This study showed that SETD5 may positively regulate the stem-like properties of BC cells. Furthermore, SETD5 knockdown in BC spheroid body-forming cells signi cantly downregulated 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 downregulation of CD133 and LGR5 proteins in tumor xenograft tissues. Speci cally, as shown in Fig. 3, the SETD5-positive cell populations interacted with CSC markers. Moreover, our results showed that SETD5 depletion inhibited glucose uptake while favoring lactate production. The expression of key glycolytic enzymes in BC spheroid body-forming cells were downregulated in vitro and in vivo. Thus, we suggest that SETD5 is a positive regulator of aerobic glycolysis in BCSCs. Accordingly, we speculated that SETD5 expression is involved in regulating BCSC properties, as well as glycolysis of BCSCs. EP300 works as 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 the chromatin which acts as a regulator of histone acetylation during gene transcription. Our data showed that SETD5 was not only positively associated with EP300 but also co-expressed EP300. A series of epigenetic factors has been shown to exert regulatory roles in HIF1α protein stability control [22].
Moreover, Finley et al. have shown that regulation of HIF1α protein stability is necessary for the maintenance of aerobic glycolysis and malignant properties in BC [29]. These results are highly consistent with Huang's report that histone methyltransferase SETD8 reprograms BC cell metabolism through an HIF1α-mediated process [22]. GSEA results showed a positive correlation between SETD5, glycolysis, and hypoxia. In vivo, HIF1α, SETD5, HK2, and PFKFB3 were discovered to be increasingly expressed in tumor hypoxic regions than in normoxic regions, which further implied the results in vitro. Figure 6 veri es SETD5 as an upstream effector of EP300/HIF1α in BC tissues. Therefore, we suggest that SETD5 is a positive regulator of EP300/ HIF1α. Moreover, as Fig. 7d shows that SETD5 silencing decreased the expression of HK2 and PFKFB3 proteins, our ndings suggest that SETD5 expression induces glycolysis by binding HIF1α in the nucleus of BC cells under hypoxia stress.
The functions of HIF1α depend on its protein stability and subcellular localization. Under hypoxic conditions, ubiquitination of HIF1α is suppressed, and increased concentrations of HIF1α bind to HIF1β to form the HIF1 transcription complex [30]. HIF1 then translocates to the nucleus, where it binds to the hypoxic response element within the promoters of its target genes [30]. Our results con rm that hypoxia mimetic agent CoCl 2 induces HIF1α nuclear translocation. Co-IP showed that hypoxic stress enhanced

Supplementary Files
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