The MCF-7 cell line was purchased from the Chinese Academy of Sciences (Shanghai, China), the MDA-MB-231 cell line was donated by Fudan University Cancer Hospital (Shanghai, China), and the 293T cell line was purchased by Shanghai Sixth Hospital Central Laboratory (Shanghai, China). The MCF-7 and 293T cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Wuhan Servicebio Technology Co., Ltd.) at 37°C and 5% CO₂. MDA-MB-231 cell line was cultured in Leibovitz's L-15 medium supplemented with 10% FBS without CO₂ at 37°C. The cells were digested with 0.25% trypsin-EDTA (cat. no. 25200072) (Gibco; Thermo Fisher Scientific, Inc.) and passaged.

The full-length cDNAs of KMT5A and FBP1 were obtained by PCR amplification from human tissue and directly connected downstream of the CMV promoter of the lentiviral expression vector after enzyme digestion through ligase reaction. The overexpression plasmid was Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin (GV358) (Shanghai GeneChem Co., Ltd.), and GV358-Control and GV358-KMT5A (Shanghai GeneChem Co., Ltd.) were constructed by sequencing and identification. The vector with the correct expression sequence of the recombinant FBP1 gene was selected by gene sequencing, and the plasmid GV358-Control and GV358-FBP1 (Shanghai GeneChem Co., Ltd.) were constructed by sequencing. shRNA targets design software (https://rnaidesigner.thermofisher.com/rnaiexpress/sort.do) from human KMT5A (NM_020382) was used. The synthetical oligos were inserted into the lentivirus expression plasmid hU6-MCS-Ubiquitin-EGFP-IRES-puromycin (GV248) (Shanghai GeneChem Co., Ltd.) (additional vector information and the sequences of shRNA are included in Tables SI and SII). Empty vectors were used as negative controls in the aforementioned plasmid construction experiments of overexpression and knockdown expression.

For virus packaging, 293T cells were seeded into a 10-cm culture dish with puromycin (2 µg/ml). When the cells reached a density of 60–70%, KMT5A(GV358-KMT5A) (2.67 µg)/ shKMT5A(GV248-KMT5A) (4 µg)/FBP1(GV358-FBP1) (1.33 µg) plasmid or control vector was transfected into 293T cells along with lentiviral helper plasmid (ps-PAX2, pMD2G tagged in green fluorescent protein) (Shanghai GeneChem Co., Ltd.) using Lipofectamine^® 3000 transfection kit (Invitrogen; Thermo Fisher Scientific, Inc.) at 25°C. The proportions were as follows: target plasmid: ps-PAX2: pMD2G=1:3:4. After transfection for 48 h, the culture media containing recombinant lentivirus were collected, filtered, concentrated, purified and stored at −80°C. The sequence of shRNA was CGCAACAGAATCGCAAACTTA.

The MDA-MB-231 cells and MCF-7 cells were cultured in 6-well plates. Then prepared recombinant lentivirus was added into the culture medium with multiplicity of infection of 20. After 72 h infection, the GFP expression was observed under a fluorescence microscope to evaluate lentivirus infection efficiency. Total RNA and protein were extracted 72 h after infection.

The cells were collected (6-well plate with 80% cell density) and centrifuged at 14,000 × g for 5 min at 25°C, after which total RNA was extracted with TRIzol^® reagent (Shanghai Pufei Biotechnology Co., Ltd.). Total RNA (2.0 µg) was used for complementary DNA (cDNA) synthesis via a Promega M-MLV kit (cat no. M1701; Promega Corporation) following the manufacturer's protocol. RT-qPCR was conducted with SYBR Premix Ex Taq (Takara Bio, Inc.) via a two-step method, and a melting curve was prepared for quantitative data analysis. The thermocycling conditions were as follows: 95°C for 10 min, 95°C for 15 sec, 59°C for 40 sec, 72°C for 45 sec, and the cycles were 40 times. The relative mRNA levels were calculated using the comparative Ct method (2^−ΔΔCq) (21). The primer pairs were designed with Primer Express 3.0 (ABI Inc.). The sequences of primers (Biosune; http://www.biosune.com/) were as follows: KMT5A forward, 5′-GAAGTCCGAGGAACAGAAG-3′ and reverse, 5′-ACAGGGTAGAAATCCGTAA-3′; FBP1 forward, 5′-TCTACCAACGTGACAGGTGA-3′ and reverse, 5′-ATCAAGGGGATCAAAACAGA-3′; and β-actin forward, 5′-GCGTGACATTAAGGAGAAGC-3′ and reverse, 5′-CCACGTCACACTTCATGATGG-3′.

The MTT assay was used to evaluate the cytotoxicity of DTX against BRCA cells. First, the MDA-MB-231 cells were incubated at 37°C after seeding into 96-well plates at 5×10⁴ cells/well for 24 h. Then, the cells were treated with gradient concentrations of DTX at equal concentrations. MTT solution (20 µl, 5 mg/ml) (Shanghai Dingguo Biotechnology Co., Ltd.) was added to each well according to the design time. After 4 h of incubation at 37°C, the culture medium containing unreacted MTT was completely removed from the wells, and dimethyl sulfoxide (100 µl) was added to each well. After the samples were oscillated for 3–5 min, the OD values were detected by a microplate reader at 490/570 nm. Finally, the data were analyzed.

Cell proliferation and cell activities were measured by a CCK-8 assay (Dojindo Laboratories, Inc.). All the MDA-MB-231 or MCF-7 cells were plated at 1×10⁴ cells per well and treated with different concentrations of DTX for 48 h. Then, CCK-8 solution (100 µl/well) was added to all the wells and incubated at 37°C for 4 h, the absorbance was read at 450 nm, and calculated the percentage of cell viability.

BRCA cell lines were treated with KMT5A inhibitor UNC0379 (0, 5 or 10 µM) (MedChemExpress) and DTX (20 µM) for 48 h. A total of 10 µl of diluted CCK-8 was added into each well and cells were incubated in a 5% CO₂ incubator for 2 h at 37°C. Absorbance was measured at 450 nm.

When the cells cultured in 6-cm dishes reached to a confluence rate of ~80% (the cells did not reach the growth plateau stage), they were digested with pancreatic enzymes and collected, and three wells were set in each group (to ensure a sufficient number of cells on the machine, ≥10⁶ cells were used). The cells were centrifuged at 350 × g for 5 min at 4°C, washed with precooled Dulbecco's phosphate-buffered saline (DPBS) (pH=7.2~7.4) at 4°C and precipitated once. The cells were centrifuged at 350 × g for 5 min at 4°C. The cell staining agent used was 40X propidium iodide (PI) (2 mg/ml) (MilliporeSigma), 100X RNase (10 mg/ml) (Thermo Fisher Scientific, Inc.), 1X DPBS and 25X Triton X-100 (MilliporeSigma) (25:10:1,000:40). The cell suspension was added to a certain volume of cell staining solution (0.6–1 ml) to ensure that the cell passage rate was 300 to 800 cells/sec. After machine (CytoFLEX; Beckman Coulter, Inc.) detection, the data analysis was performed by ModFit software (5.0) (Verity Software House, Inc.).

A total of 60 patients (age range, 31–82 years) with BRCA admitted to Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from August 2023 to December 2023 were selected as the present study's subjects. The present study was approved (approval no. YS-2017-006) by the Ethics Committee of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China), and written informed consent was obtained from each patient. The inclusion criteria were as follows: i) Meeting the diagnostic criteria for BRCA; ii) Surgical resection was performed, and samples of BRCA tissues and adjacent tissues were obtained. The diagnosis of BRCA was confirmed by pathological examination, iii) for the first time, no anticancer treatment such as endocrine therapy, radiotherapy and chemotherapy was taken before surgery, iv) women, v) complete clinical data; and vi) signed informed consent for the present study. Exclusion criteria were the following: i) Combined with systemic infectious diseases, cardiovascular and cerebrovascular diseases, liver and kidney insufficiency; ii) combined with other types of tumors; iii) combined with terminal disease; iv) tissue samples that cannot be obtained; and v) ment0al system diseases and suicidal tendencies.

Normal tumor and breast tissues were excised from patients. These tissues were fixed in 10% neutral buffered formalin, embedded in paraffin at 4°C for 12 h, sectioned (5 µm), and stained with haematoxylin-eosin staining. Immunohistochemical staining using KMT5A antibody (1:100; cat. no. PA5-102712; Thermo Fisher Scientific, Inc.; 22°C for 1.5 h) and Rabbit IgG (H + L) cross-Adsorbed Secondary Antibody (1:2,000; cat. no. A-11008; Thermo Fisher Scientific, Inc.; 25°C for 30 min) was performed by the Department of Pathology at Shanghai Sixth People's Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. KMT5A immunohistochemical markers were assessed using light microscopy. The immunostained slides were scored according to the proportion of tumor cells that exhibited nuclear staining. KMT5A expression was considered positive when >25% of the tumor cell nuclei were stained (Table SIII).

MCF-7 or MDA-MB-231 cells in 6-well plates were collected and washed twice with phosphate- buffered saline (PBS) (Wuhan Servicebio Technology Co., Ltd.). Protein was extracted from MCF-7 or MDA-MB-231 cells by RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentration of the proteins was determined with a BCA protein assay kit (Beyotime Institute of Biotechnology). The protein lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The separation gel with 10% polyacrylamide was prepared, and 30 µl protein was loaded to each lane and transferred onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma). The PVDF membranes were blocked with 0.1% TBST solution (Wuhan Servicebio Technology Co., Ltd.) containing 5% skim milk at 25°C for 1 h. After the membranes were incubated with primary antibodies KMT5A, (1:1,000; cat. no. 14063-1-AP; Proteintech Group, Inc.; 37°C for 1 h;) and FBP1 (1:2,000; cat. no. 12842-1-AP; Proteintech Group, Inc.; 25°C for 1.5 h;) and secondary antibody Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (1:5,000; cat. no. RGAR001; Proteintech Group, Inc.; 25°C for 1 h;), an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.) and X-ray (Carestream Health, Inc.) were used to visualize the membranes. The X-rays were removed, the samples were allowed to dry, and the blots were analysed. Because the molecular weights of KMT5A and actin were both 43 KD. At first, the target protein KMT5A was incubated. After KMT5A was stripped, the internal reference protein actin was incubated on the same membrane.

KMT5A 3′-UTR sequence was obtained from KMT5A and twist family BHLH transcription factor 1 (TWIST1) full-length cDNA vector (Shanghai GeneChem Co., Ltd.) and sub-cloned to pcDNA3.1-control vector (Shanghai GeneChem Co., Ltd.) as wild-type vector pcDNA3.1-KMT5A-3′-UTR-WT (KMT5A-WT) and pcDNA3.1-TWIST1-3′-UTR respectively. The mutant vector pcDNA3.1-KMT5A-3′-UTR-Mut (KMT5A-R295G) was obtained by site-directed mutagenesis using QuikChange^® Site-Directed Mutagenesis Kit (Stratagene; Agilent). MDA-MB-231 cells were seeded in a 24-well culture plate in triplicate and were transfected followed by pcDNA3.1-KMT5A-3′-UTR-WT or pcDNA3.1-KMT5A-3′-UTR-Mut or pcDNA3.1-TWIST1-3′-UTR by ExFect Transfection Reagent (Vazyme Biotech Co., Ltd.) according to the manufacturer's procedure.

Cells were seeded in 24-well plates at a density of 500 µl/well and transfected with the target plasmid (Table SIV). After 48 h at 37°C, the luciferase activity of each group was detected by a dual luciferase system (cat. no. E1910; Promega Corporation). The cell lysate was supplemented with firefly luciferase (100 µl), after which firefly luciferase activity was measured (F value). After adding Renilla luciferase (100 µl), the cell lysate was immediately placed into the fluorescence detector to calculate the Renilla luciferase activity (R-value). Based on the F and R values of each group detected previously, the relative luciferase activity was calculated to get promoter activity. The primers for the FBP1 promoter were: forward, 5′-GACAGAAGGGCCAGGTGA-3′ and reverse, 5′-GCCAGAGAGAAAGCTATGACTG-3′; transcription start site: (967–979 bp) AAGCCAGATGA.

To detect glucose consumption and lactate production in BRCA cells, a Glucose Uptake Colorimetric Assay Kit (cat. no. K676-100; BioVision, Inc.; Abcam) and a Colorimetric/Fluorometric Assay Kit (cat. no. K607-100; BioVision, Inc.; Abcam) were used according to the manufacturer's instructions. A total of 2,000 MDA-MB-231 cells were seeded in each well of a 96-well plate and incubated for 48 h at 37°C. After that, based on the Glucose Uptake Colorimetric Assay Kit protocol, absorbance was measured at 412 nm in a microplate reader at 37°C every 5 min until the 100 pmol standard reached 1.5–2.0 OD. An endpoint reading of all samples and standards was taken. The 2-deoxy-D-glucose 6P standard curve was plotted and the glucose intake was calculated. Based on the Colorimetric/Fluorometric Assay Kit protocol, absorbance was measured at 570 nm in a microplate reader. The lactate standard curve was plotted and sample lactate concentration was calculated. The glucose and lactate levels were calculated using a standard calibration curve prepared under the same conditions.

The peptide mixture (100 µg) of each sample was labelled by TMT reagent according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). TMT-labelled peptides (10 µl) were fractionated by peptide fractionation with reversed phase chromatography using the Agilent 1260 infinity II HPLC. Each fraction was injected for nano LC-MS/MS analysis. The peptide mixture was loaded onto the C18-reversed phase analytical column (Thermo Fisher Scientific, Inc.; Acclaim PepMap RSLC 50 µm ×15 cm, nano viper, P/N164943) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min. LC-MS/MS analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Inc.) that was coupled to Easy nLC (Thermo Fisher Scientific, Inc.) for 60 min. MS/MS raw files were processed using MASCOT engine (version 2.6; Matrix Science, Ltd.) embedded into Proteome Discoverer 2.2, and searched against the UniProt database, downloaded from https://www.uniprot.org/, including HomoSapiens_20367_20200226 sequences. Proteins with Fold change >1.2 and adjusted. P<0.05 (Student's t-test) were considered to be differentially expressed proteins. The annotation from gene ontology (GO) terms to proteins was completed by Blast2GO Command Line. After the elementary annotation, InterProScan (https://www.ebi.ac.uk/interpro/) was used to search the EBI database (https://www.ebi.ac.uk/) by motif and then add the functional information of motif to proteins to improve annotation. Then further improvement of annotation and connection between GO terms were carried out (Table SV). Fisher's Exact Test was used to enrich GO terms by comparing the number of differentially expressed proteins and total proteins associated to GO terms. Pathway analysis was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html). Fisher's Exact Test was used to identify the significantly enriched pathways by comparing the number of differentially expressed proteins and total proteins associated to pathways (Table SVI Table VII, Table VIII, Table IX, Table SX) and protein structure (Table SXI).

The partial immunohistochemistry images and interaction network were obtained from The Human Protein Atlas (HPA, http://www.proteinatlas.org/) (22). The gene expression levels in healthy breast and BRCA were obtained from The University of Alabama at Birmingham database (UALCAN, http://ualcan.path.uab.edu/index.html) (23). Some data were downloaded from the TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). The expression data of KMT5A gene in MDA-MB-231 and MCF-7 cell lines was downloaded from the ‘Expression 23Q4 Public’ dataset of the Cancer Cell Line Encyclopedia database (CCLE, http://www.broadinstitute.org/ccle) (24).

The present study was approved (approval no. YS-2017-006) by the Ethics Committee of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China), and written informed consent was obtained from each patient.

SPSS software 26.0 (IBM Corp.) was used for Pearson correlation statistical analyses. Most of the data were analyzed and bar charts were generated with GraphPad Prism 9.5 software (Dotmatics). Volcano map was expressed by R studio (4.3.2) (RStudio, Inc.). KEGG and GO analysis were drawn by bioinformatics (https://www.bioinformatics.com.cn). Data are presented as the mean ± standard deviation (minimum three repeats, depending of the experiments). Comparisons between groups were analyzed using one-way ANOVA followed by Bonferroni's (for comparing within treatment conditions) or Dunnett's (for comparing between treatment conditions) or Multiple Comparison Test. P<0.05 was considered to indicate a statistically significant deference.

The UALCAN database was searched (https://ualcan.path.uab.edu/index.html) (23) to determine the expression of KMT5A in BRCA. KMT5A was upregulated in BRCA (P<0.05). The expression level of KMT5A was the highest in triple-negative BRCA (P<0.05) and the lowest in the luminal subtype (P<0.05) (Fig. 1A) (All data and significance levels were from the UALCAN database). The immunohistochemical results of 60 patients were included in the analysis. Patients ranged in age from 31 to 82 years, with a median age of 60 years. IHC also revealed that KMT5A was highly expressed in BRCA tissues (Fig. 1A).

The expression of endogenous KMT5A was lower in MCF-7 cell lines (5.35), however higher in MDA-MB-231 cell lines (5.74) (Data was from ‘Expression 23Q4 Public’ dataset of the CCLE database). Therefore, lentivirus-mediated KMT5A shRNA silenced KMT5A expression in MDA-MB-231 cell line, and overexpressed KMT5A mRNA in MCF-7 cell line. As demonstrated in Fig. 1B, the stably transfected MDA-MB-231 BRCA cell line was subsequently generated via lentivirus infection and puromycin resistance screening and subsequently divided into three groups for identification. The negative control (NC) group was generated by stably transforming MDA-MB-231 cells with a control lentiviral vector. The shKMT5A-1 and shKMT5A-2 group were stably transformed into MDA-MB-231 cells via the LVpFU-GW-007-mediated transfer of the lentiviral vector. KMT5A knockdown was confirmed at the mRNA and protein levels. The shKMT5A-1 and shKMT5A-2 groups exhibited significantly lower levels of the KMT5A mRNA and protein (P<0.05) (Fig. 1C). Furthermore, the knockdown efficiency of shKMT5A-1 was greater than that of shKMT5A-2. Moreover, RT-qPCR and western blot analysis confirmed that KMT5A was significantly highly expressed in MCF-7 cells transfected with an overexpression lentiviral vector (Fig. 1D).

To further confirm whether KMT5A knockdown affects chemical resistance and the cell cycle, MDA-MB-231 cells were treated with DTX, the half-maximal inhibitory concentration (IC₅₀) was measured, dose-response curves were constructed for each group, and the proportions of cells in different phases were evaluated. After KMT5A knockdown, the IC₅₀ of DTX decreased significantly (Fig. 1E). In the DTX positive KMT5A-OE negative group, the proliferation rate of MCF-7 cells when DTX (20 µM) was added only was 53.9%. However, in the case of high expression of KMT5A, the cell activity increased to 66.5% after DTX addition compared with the DTX positive KMT5A-OE negative group. It was revealed that high expression of KMT5A antagonized DTX and promoted cell proliferation (Fig. 1F). KMT5A expression may mediate the development of DTX resistance in BRCA cells.

In the control group, the proportion of G1-phase cells was 49.51±0.71%, the proportion of S-phase cells was 43.43±1.38%, and the proportion of G2/M-phase cells was 7.06±0.67%. The proportions of G1 phase cells, S phase cells, and G2/M phase cells in the shKMT5A-1 group were 38.91±1.27, 51.60±0.93% and 9.49±0.37%, respectively. After adding DTX, the proportion of cells in the G2/M phase increased, and the proportion of cells in the S phase decreased significantly. In the control group, the proportion of G2/M phase cells increased to 82.82±0.53%, and the proportion of S phase cells decreased to 11.28±0.36%. The proportion of G2/M phase cells in the shKMT5A-1 group was 76.15±1.04%, and the proportion of S phase cells was 19.50±0.96%. After KMT5A knockdown, the proportion of S-phase cells was markedly greater than that of parental cells (Fig. 1G). First of all, DTX is effective against various pathological types of BRCA, including triple-negative BRCA, and remains the first-choice chemotherapy drug for this type. The triple-negative BRCA cell line (MDA-MB-231) is less sensitive to DTX than the hormone receptor-positive MCF-7 cell line. Secondly, the mechanism of DTX is to enhance the polymerization of tubulin and inhibit the depolymerization of microtubules, leading to the formation of stable non-functional microtubule bundles, thus disrupting the mitosis of tumor cells. The present study revealed that MDA-MB-231 cell line was blocked in mitosis and remained in the G2/M phase, which is consistent with its pharmacological effect and previous findings (25).

The levels of monomethylated H4K20 and KMT5A changed dynamically during different phases of the cell cycle, and these dynamic changes regulated the progression of the cell cycle. KMT5A and monomethylated H4K20 are expressed at low levels in the S phase, and KMT5A and monomethylated H4K20 reach their peak expression in the G2/M phase. The normal process of the S phase requires KMT5A (26); therefore, after KMT5A expression was knocked down, cells were arrested in the S phase and could not enter the G2/M phase. However, DTX mainly affects M-phase cells (27); therefore, when DTX is added, the proportion of G2/M phase cells was greater than that of parent cells (negative control without DTX).

To identify the downstream proteins regulated by KMT5A, protein expression levels were evaluated in the BRCA cell line MDA-MB-231 transfected with NC lentivirus (negative control) or KMT5A-targeting lentivirus (shKMT5A). A total of three samples were randomly selected from NC and shKMT5A groups, and the protein was quantitatively analyzed by TMT (Tandem Mass Tag) proteomics technology and the differentially expressed protein was screened out. The analysis of differential gene expression revealed that the expression levels of 261 proteins increased after KMT5A was knocked down. Conversely, the expression of 153 proteins was downregulated (Fig. 2A). As a key gluconeogenic enzyme, FBP1 expression increased significantly (Fold change=1.23, adjusted. P<0.05) after KMT5A was knocked down.

Pathway enrichment analysis demonstrated that KMT5A regulates several pathways, including the extracellular exosome and membrane pathways. In addition to these pathways being enriched, it was revealed that they were tightly connected to chemoresistance and glucose metabolism (Fig. 2B). Moreover, the ‘extracellular exosome’ plate differs most significantly among the cell components, and exosomes have been revealed to lead to chemical resistance in various cancers and regulate chemical resistance in several ways (Fig. 2C). Exosomes transport glycolytic enzymes. According to the proteomic analysis results, exosomes lead to metabolic reprogramming and promote alterations in both glucose metabolism and lactate production.

UALCAN database was searched and was revealed that the expression of FBP1was significantly increased in BRCA, however there were significant differences among the different subtypes. FBP1 was significantly increased in luminal BRCA (P<0.05) but significantly decreased in triple-negative BRCA (P<0.05) (Fig. 2D). IHC also demonstrated that FBP1 was highly expressed in BRCA tissues (Fig. 2D). Inadequately, IHC cannot divide into different subtypes. IHC results of FBP1 in BRCA and adjacent normal breast tissue were obtained from the HPA database and could not be differentiated into different subtypes for analysis.

MDA-MB-231 cells were transfected with the lentivirus LVCON238 (negative control) or LVKL68505-2 (FBP1-OE). RT-qPCR also revealed that the mRNA level of FBP1 was significantly increased in cells transfected with the lentivirus LVKL68505-2 (FBP1-OE) (P<0.05) (Fig. 2E). Western blot analysis revealed that FBP1 was markedly overexpressed in MDA-MB-231 cells transfected with the lentivirus LVKL68505-2 (FBP1-OE) (Fig. 2E).

After overexpression of FBP1, the proportion of G2/M phase cells increased significantly (Fig. 2F). DTX is a G2/M phase cycle-specific drug, and overexpression of FBP1 may enhance the proapoptotic effect of DTX on BRCA cells. Therefore, the effect of overexpression of FBP1 was detected on cell proliferation using a CCK-8 assay and MDA-MB-231 cells were subsequently treated with DTX. After the addition of the DTX (20 µM), cell proliferation was further inhibited in the FBP1 overexpression group (Fig. 2G).

TCGA-BRCA data were downloaded from the TCGA database. The expression of FBP1 was negatively correlated with that of KMT5A [Pearson correlation coefficient (r=−0.131, P<0.05] (Fig. 3A). To verify the relationship between KMT5A and FBP1, the expression of FBP1 was detected after knocking down KMT5A in MDA-MB-231 cell line. The expression of FBP1 was increased in the shKMT5A group (Fig. 3B). A dual-luciferase reporter gene assay was used to determine the regulatory relationship between KMT5A and FBP1. The NC-Luc + KMT5A-WT group and the FBP1-Promoter-Luc + vector group were designed to observe the internal interaction between the reporter vector and the internal reference vector to eliminate the background influence. The luciferase activity of the FBP1-Promoter-Luc + KMT5A-WT group was lower than that of the FBP1-Promoter-Luc+vector group and NC-Luc + KMT5A-WT group. The results revealed that FBP1 demonstrated strong promoter activity when KMT5A was not expressed. Thus, it was found that KMT5A inhibits FBP1 after the exclusion of endogenous influences (Fig. 3C).

To determine whether FBP1 has a reverse regulatory effect on KMT5A, a lentivirus was used to construct a NC group, an FBP1 overexpression group (FBP1-OE), a KMT5A low-expression group (shKMT5A) and an FBP1 overexpression + KMT5A low-expression group (FBP1-OE + shKMT5A). Moreover, RT-qPCR and western blot analysis successfully verified that FBP1 was overexpressed and KMT5A was downregulated in the MDA-MB-231 cell line (Fig. 3D).

When DTX was not added, no matter whether KMT5A was knocked down alone (shKMT5A) or FBP1 was overexpressed (FBP1-OE), the proliferation inhibition rate of MDA-MB-231 cells was not significantly different from that of the NC group. However, the inhibitory rate of cell proliferation in the FBP1-OE + shKMT5A group was 10.3±4.4%. After addition of DTX, the cell proliferation inhibition rate was 26.1±2.6% in the FBP1-OE group alone, 15.2±2.7% in the shKMT5A group alone, however increased to 43.4±1.3% in the FBP1-OE + shKMT5A group. Neither the overexpression of FBP1 nor low expression of KMT5A alone affected the proliferation of BRCA cells. When KMT5A was simultaneously expressed at low levels and FBP1 was overexpressed, the proliferation of BRCA cells was significantly inhibited after DTX (20 µM) was added (Fig. 2G). It was also revealed that the MDA-MB-231 cell activity decreased to ~55.8 and 41.0% respectively after DTX addition without/with FBP1 overexpression. As demonstrated in Fig. 2G, the MDA-MB-231 cell activity decreased to ~91.4 and 73.9% respectively after DTX addition without/with FBP1 overexpression. Therefore, after the addition of DTX, overexpression of FBP1 or/and downregulation of KMT5A jointly inhibited the proliferation of MDA-MB-231 cells.

UNC0379 is a selective inhibitor of KMT5A, however it can also target other histone methyltransferases apart from KMT5A (28). The DTX groups contain both DTX (20 µM) and DTX solvent. UNC0379 (0, 5 or 10 µM) was added respectively the DTX solvent groups and DTX groups. As it can be observed, UNC0379 can also cooperate with DTX to inhibit the proliferation of BRCA cells. Moreover, the change in concentration of UNC0379 can enhance the inhibitory effect on the proliferation of BRCA cells. As the concentration UNC0379 was increased, MDA-MB-231 cell proliferation was more strongly inhibited (Fig. 4A).

As the concentration of the inhibitor UNC0379 increased, the amount of lactic acid produced by the BRCA cells decreased (Fig. 4B). Moreover, after the addition of the inhibitor UNC0379, the glucose uptake of BRCA cells was also significantly reduced (Fig. 4C).

As revealed in Fig. 3C, FBP1 promoter activity decreased when KMT5A-WT was overexpressed. To further verify whether KMT5A methylase activity affects FBP1 transcription, a KMT5A mutant (KMT5A-R295G) was added to the FBP1 promoter. Both KMT5A-WT and KMT5A-R295G inhibited FBP1 gene expression. Therefore, compared with the NC-Luc + Vector (control) group, the relative activity of luciferase in both groups decreased. The results also revealed that when KMT5A lost methylase activity, FBP1 promoter activity increased compared with KMT5A-WT group (Fig. 4D). This suggests that KMT5A has an inhibitory effect on the activation and regulation of FBP1 promoter depending on its methylase activity.

How does KMT5A affect FBP1 expression through methylase activity? Yang et al (29) demonstrated that SET8 and TWIST commonly promote epithelial-mesenchymal transformation and enhance the invasive potential of BRCA cells. In BRCA, KMT5A (SETD8) interacts with TWIST and synergistically upregulates N-Cadherin and downregulates E-Cadherin at the transcriptional level. The HPA database was utilized to search for an interaction network between KMT5A and TWIST and discovered that there was indeed an interaction between the two (Fig. 4E). Based on the prediction of binding sites between FBP1 promoter and TWIST1, dual-luciferase reporter gene experiment was used to verify whether TWIST1 had an effect on promoter activation and regulation. Using empty plasmids as controls, results revealed that overexpression of TWIST1 can increase FBP1 promoter activity by ~5-fold, suggesting that TWIST1 activates FBP1 promoter activity (Fig. 4F).