Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Niraparib promotes SPH accumulation and activates SPHK1 signaling. ( A , B ) Relative proportions of different lipid types (SPH, SM, PS, PI, PE, PC, PA, CL, ChE) in MDA-MB-231 cells treated with Niraparib. In the figure A, the bubbles represent significantly different lipid molecules; the y-axis shows the lipid subclasses, distinguished by different colors. The size of each bubble indicates the level of significance: smaller bubbles denote significant differences (0.01 < p value < 0.05), while larger bubbles denote highly significant differences ( p value < 0.01). ( C ) Statistical plots of the total content of four SPH differential lipid molecules and the total content of SPH with p < 0.05 in the Niraparib-treated group and the negative control group. ( D ) Volcano plot showing SPH lipid molecules with p < 0.05 in the lipid metabolism data. The vertical lines indicate Fold change > 1.5 or Fold change < 0.67; the horizontal line denotes p -value < 0.05. ( E ) Correlation network analysis between differential genes and differential sphingolipids. ( F ) Volcano plot showing SPHK1 in the transcriptomics data. ( G ) SPHK1 protein expression in MDA-MB-231 cells treated with PARPi (Niraparib, Olaparib, Talazoparib, Rucaparib) vs. control (Western blot). ( H ) RT-qPCR validation of whether Niraparib significantly upregulates SPHK1 in four TNBC cell lines. Data are presented as the mean ± SEM from three independent experiments. Statistical significance between groups is indicated (* p < 0.05; *** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Negative Control, Expressing, Control, Western Blot, Quantitative RT-PCR, Biomarker Discovery

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Niraparib sensitizes TNBC cells to ferroptosis by inhibiting SPHK1 activity. ( A ) Venn diagram showing the intersection of differentially expressed genes from lipid metabolism transcriptomic data and anti-ferroptosis-related genes from the GeneCards database. ( B ) Volcano plot of differentially expressed genes generated using GraphPad Prism software (version 10.1.2), with the intersecting genes highlighted. The dashed lines in the figure represent Fold change = 2 and Fold change = −2. ( C ) Measurement of MDA levels in four groups of cells after pharmacological inhibition of SPHK1 (FTY720) or siRNA-mediated knockdown of SPHK1. ( D ) Measurement of GSH levels in four groups of cells after pharmacological inhibition of SPHK1 (FTY720) or siRNA-mediated knockdown of SPHK1. Data are presented as the mean ± SEM from three independent experiments. ( E ) The level of lipid peroxides was quantified by C11 BODIPY 581/591 labeling and flow cytometry. Statistical significance between groups is indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Activity Assay, Generated, Software, Inhibition, Knockdown, Labeling, Flow Cytometry

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Niraparib-induced activation of PI3K-AKT and MAPK pathways is rescued by SPHK1 knockdown. ( A ) KEGG pathway enrichment analysis of transcriptomic sequencing data from MDA-MB-231 cells treated with Niraparib. ( B ) Western blot analysis of the expression levels of Erk and p-Erk pathway proteins in TNBC cell lines (MDA-MB-231 and MDA-MB-468) treated with Niraparib. ( C ) Western blot validation showing that knockdown of SPHK1 reverses the Niraparib-induced upregulation of SPHK1 and the activation of the PI3K/AKT and MAPK signaling pathways in TNBC cells. Statistical significance between groups is indicated (*** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Activation Assay, Knockdown, Sequencing, Western Blot, Expressing, Biomarker Discovery, Protein-Protein interactions

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: The transcriptional regulation of SPHK1 is governed by a super-enhancer in TNBC. ( A ) Distribution map of H3K27ac signals for 331 TNBC-specific SEs across the genome, drawn using the MicroSignal website; ( B ) Venn diagram of target genes of 331 TNBC-specific SEs and differentially expressed genes in the transcriptome; ( C ) Distribution map of H3K27ac signals for 7 genes across the genome, drawn using the MicroSignal website; ( D ) Graph of H3K27ac signals for 331 TNBC-specific SE target genes, drawn using GraphPad Prism software (version 10.1.2); ( E ) IGV display of H3K27ac signal peaks for 7 gene-SEs in 3 TNBC cell lines; ( F ) IGV visualization of H3K27ac/H3K4me1/H3K4me3/BRD4 signals in MDA-MB-231 cells; ( G ) RT-qPCR validation of SPHK1 mRNA levels in TNBC cells treated with 4 μM JQ1/OTX-015 for 24 h, and verification of whether BRD4 inhibitors can reverse the upregulation of SPHK1 expression caused by Niraparib; ( H ) Western blot validation of whether BRD4 inhibitors can reverse the upregulation of SPHK1 protein expression caused by Niraparib. Data are presented as the mean ± SEM from three independent experiments. Statistical significance between groups is indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Software, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Targeted inhibition of super-enhancer activity reverses Niraparib-induced SPHK1 activation. ( A ) IGV software visualization of the SE peak corresponding to SPHK1 and the genomic location of the SE sequence. ( B ) NCBI database mapping of the sequences corresponding to SPHK1-SE1 and SE2. ( C ) Western blot analysis to determine whether the introduction of siRNA-SE in TNBC cells leads to downregulation of SPHK1. ( D ) Western blot analysis to determine whether the introduction of siRNA-SE in TNBC cells reverses the Niraparib-induced upregulation of SPHK1. Statistical significance between groups is indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Inhibition, Activity Assay, Activation Assay, Software, Sequencing, Western Blot

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Niraparib facilitates SP1-mediated super-enhancer activation and subsequent SPHK1 transcription. ( A ) Prediction of transcription factor binding sites on SPHK1 -SE using the PROMO and JASPAR websites. Top four transcription factors ranked by prediction scores. ( B , C ) PyMOL visualization of the docking model between the transcription factor SP1 and the PARP1 protein. ( D ) Western blot analysis to determine whether the introduction of siRNA-SP1 in TNBC cells reverses the Niraparib-induced upregulation of SPHK1. ( E ) Design of specific primers (F-SE/R-SE) targeting the blue SE sequence using SnapGene software. ( F ) ChIP-qPCR analysis to determine whether Niraparib promotes the binding of the SP1 transcription factor to the SE sequence. ( G – I ) The docking results of SP1 protein with SE sequence. Green indicates SP1; all other regions represent SE. ( J ) Co-IP assay to detect whether Niraparib inhibits the PARylation of SP1 in TNBC cells. ( K ) Immunofluorescence assay to determine whether Niraparib promotes the nuclear translocation of SP1 and BRD4 and enhances the fluorescence signals of these two proteins. The BRD4 fluorescence signal was quantified as the nuclear-to-cytoplasmic intensity ratio. The SP1 fluorescence signal was quantified based on its intensity within the nucleus. Statistical significance between groups is indicated (*** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Activation Assay, Binding Assay, Western Blot, Sequencing, Software, ChIP-qPCR, Co-Immunoprecipitation Assay, Immunofluorescence, Translocation Assay, Fluorescence

Journal: Pharmaceuticals

Article Title: The SP1-SuperEnhancer-SPHK1 Axis Mediates Niraparib Resistance in TNBC

doi: 10.3390/ph18111622

Figure Lengend Snippet: Combined inhibition of SP1 and SPHK1 markedly potentiates the anti-tumor efficacy of Niraparib. ( A ) MTT assays were performed to evaluate the cytotoxic effects of Niraparib in TNBC cells following 24 h treatment with siRNA targeting SPHK1, SE, or SP1. ( B ) Molecular docking diagrams illustrating the binding modes of the three candidate compounds to SP1 were generated using PyMOL (Version 2.5.5) and LigPlot+ (Version 2.2.9); ( C ) The binding stability of these complexes was further assessed via molecular dynamics simulations using GROMACS (Version 2023.2); ( D ) Dose-dependent cytotoxicity of the three compounds against TNBC cells was determined by MTT assay after 72 h of exposure; ( E ) The toxicity of Echinatin on normal MCF-10A cells was evaluated using MTT assay following 72 h of treatment. ( F , G ) Following 72 h of treatment with the combination of Niraparib and Echinatin or FTY720 in two TNBC cell lines, cell inhibition rates were measured using the MTT assay. The synergistic effects of the drug combinations were evaluated with SynergyFinder, employing the HSA model for quantitative scoring. Subsequently, the combination index (CI) curves were simulated using CompuSyn software (version 1.0.1) to further assess drug–drug interactions. In the CI-Fa curve plot, the dashed line denotes CI = 1. ( H ) Evaluation of the anti-tumor activity of combination regimens in a 4T1 tumor-bearing mouse model. Representative image of tumors from each group of mice ( n = 5). ( I ) Quantitative analysis of tumor weight in each mouse group. ( J ) Body weight statistics for each group of mice. ( K ) Tumor growth curves and quantitative analysis of tumor volume for each group of mice. Statistical significance between groups is indicated (** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used: Anti-SPHK1(1:1000, proteintech, Lot No: 10670-1-AP); Anti-Erk1/2(1:1000, proteintech, Lot No: 11257-1-AP); Anti- Phospho-ERK1/2 (Thr202/Tyr204) (1:1000, proteintech, Lot No: 80031-1-RR); Anti-AKT (1:1000, proteintech, Lot No: 10176-2-AP); Anti- Phospho-AKT (Ser473) (1:1000, proteintech, Lot No: 66444-1-Ig); Anti-GAPDH (1:1000, proteintech, Lot No: 60004-1-IG); Anti-SP1 (1:1000, proteintech, Lot No: 21962-1-AP); Anti-BRD4 (1:1000, proteintech, Lot No: 67374-2-IG); Anti-PAR (1:1000, cell signaling technology, Lot No: #79109); Anti-mouse IgG, HRP-linked Antibody (1:5000, CST, Cat# 7076, Lot No: 36) and Anti-rabbit IgG, HRP-linked Antibody (1:5000, CST, Cat# 7074, Lot No: 31).

Techniques: Inhibition, Binding Assay, Generated, MTT Assay, Software, Activity Assay