Research ArticlePharmacology

The transcription factor SP3 drives TNF-α expression in response to Smac mimetics

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Science Signaling  29 Jan 2019:
Vol. 12, Issue 566, eaat9563
DOI: 10.1126/scisignal.aat9563
  • Fig. 1 A genome-wide siRNA screen identifies SP3 as a sensitizer to SMC efficacy in cancer cells.

    (A) Alamar blue viability assay of MDA-MB-231 cells treated with vehicle or SM-164 (100 nM) for 48 hours. (B) Quantile-quantile plot showing Z score for siRNA pools (ranks) in the genome screen. The siRNA pool targeting SP3 is indicated by the blue circle. The dashed line indicates the proximity of the lowest Z score of the positive control (siRNA targeting RIP1). (C) Alamar blue viability assay of MDA-MB-231 cells transfected with control (NT) siRNA, pooled SP3 siRNA, or deconvoluted SP3 siRNA for 72 hours and subsequently treated with vehicle or SM-164 (100 nM) for 48 hours. (D) Knockdown efficacy of siRNA targeting SP3 from the experiment depicted in (C). (E) Alamar blue viability assay of MDA-MB-231 cells transfected with plasmids encoding wild-type (WT) or siRNA-resistant (RiR) SP3 cDNA and NT or SP3 #9 siRNA and for 48 hours, and subsequently treated with vehicle or 100 nM SMC for 48 hours. (F) Alamar blue viability assay of cells transfected with NT or SP3 siRNA for 48 hours and subsequently treated with vehicle or LCL161 (500 nM). Bottom, Western blots showing efficacy of SP3 knockdown for the indicated cancer cell lines. Data are means ± SD from n = 3 or 4 experiments (individual data indicated); *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test (A) or two-way analysis of variance (ANOVA) using Dunnett’s multiple comparison test (C, E, and F).

  • Fig. 2 cIAP1, cIAP2, and XIAP cooperatively protect cancer cells from SP3-mediated cancer cell death.

    (A) Alamar blue viability assay of MDA-MB-231 and M059J cells transfected with control (NT), SP1, or SP3 siRNA for 48 hours and subsequently treated with vehicle or LCL161 (500 nM) for 48 hours. (B) Knockdown efficacy of NT, SP1, or SP3 siRNA from the experiment described in (A). Blots are representative of three independent experiments. (C) Alamar blue viability of cells transfected with NT, SP3, cIAP1, cIAP2, or XIAP for 48 hours. (D) Representative siRNA efficacy Western blots for the experiment depicted in (C). Blots are representative of two independent experiments. (E) Viability of cells transfected with NT or SP3 siRNA and treated with vehicle or the indicated SMC (1 μM) for 48 hours. Viability was assessed by Alamar blue. Data (A, C, and E) are means ± SD from n = 3 experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA using Dunnett’s multiple comparison test.

  • Fig. 3 Down-regulation of SP3 rescues cancer cells from SMC-mediated apoptosis.

    (A) Cells were transfected with control (NT) siRNA or siRNA targeting SP3 for 48 hours and then treated with vehicle or LCL161 (500 nM). Cells were harvested for Western blotting at the indicated posttreatment times. Blots are representative of two independent experiments. (B) Cells were treated with vehicle or LCL161 (500 nM) for 24 hours, and endogenous caspase-8–associated complexes were isolated by immunoprecipitation (IP), resolved by SDS–polyacrylamide gel electrophoresis, and probed for the presence of proteins by the indicated antibodies. Blots are representative of two independent experiments. (C and D) Cells were transfected with NT or SP3 siRNA for 48 hours, treated with vehicle or LCL161 (500 nM) for 18 hours, and then processed for flow cytometry with fluorescein isothiocyanate–conjugated annexin V (ANXA5-FTIC) and 7-aminoactinomycin D (7-AAD). Representative flow cytometry plots (C); data (D) are means ± SD from n = 3 experiments. (E) MDA-MB-231 cells were transfected with NT or SP3 siRNA for 48 hours, reseeded in equal numbers, treated with vehicle or SM-164 (100 nM) for 7 days, and stained with crystal violet. Scale bars, 5 mm. Images are representative of two independent experiments.

  • Fig. 4 SP3 promotes the production of TNF-α.

    (A) Western blotting of cells transfected with control (NT) or SP3 siRNA for 48 hours and then treated with vehicle or LCL161 (SMC; 500 nM) for 8 hours. Blots are representative of two independent experiments. (B) Detection of TNF-α at the mRNA level, assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in cells transfected with NT or SP3 siRNA for 48 hours and then treated with vehicle or LCL161 (500 nM). (C) Cells were transfected with NT or SP3 siRNA and vehicle or LCL161 (500 nM) for 24 hours. TNF-α in supernatants was measured by enzyme-linked immunosorbent assay (ELISA). (D) Cells were transfected with NT or SP3 siRNA for 48 hours and then treated with vehicle or LCL161 (500 nM) for 9 hours, and cellular abundance of TNF-α was measured in viable cells (Zombie Green negative) by flow cytometry. Data are representative of two independent experiments. (E) Alamar blue viability assays of cells transfected with NT or SP3 siRNA for 48 hours and treated with combinations of vehicle, 100 nM SM-164, 0.01% bovine serum albumin (BSA), and interleukin-1β (IL-1β; 10 ng/ml). (F) Alamar blue viability assay of cells transfected with NT or SP3 siRNA (48 hours for SNB75 and 24 hours for EMT6) and subsequently treated with vehicle or 1 μM LCL161 and 0.01% BSA, lipopolysaccharide (LPS; 1 μg/ml), polyinosinic-polycytidylic acid [poly(I:C)] (1 μg/ml), or interferon-β (IFN-β; 250 U/ml). (G) Cells were transfected with NT or SP3 siRNA for 48 hours and subsequently treated with vehicle or 500 nM LCL161 and 0.01% BSA, IL-1β (10 ng/ml), or IFN-β (250 U/ml) for 24 hours. Supernatants were processed for the presence of TNF-α by ELISA. (H) Efficacy of siRNA transfections from the experiments depicted in (E) and (F). Data are means ± SD from n = 3 experiments; *P < 0.05 and ***P < 0.001 by one-way ANOVA using Dunnett’s multiple comparison test.

  • Fig. 5 SP3 positively controls the activity of the TNF-α promoter.

    (A) MDA-MB-231 cells were transfected with control NT siRNA or siRNA targeting SP3 for 48 hours and then treated with vehicle or LCL161 (500 nM) for 8 hours. Cells were processed for quantitation of 176 genes encoding cytokines and chemokines by RT-qPCR. Shown are normalized heat maps of two major groups identified by hierarchical clustering. The complete hierarchical cluster profiles are provided in fig. S5. Data represent means from four independent experiments. (B) MDA-MB-231 cells stably expressing NT short hairpin RNA (shRNA) or shRNA targeting SP3 were transfected with plasmids encoding luciferase under the control of the TNF-α promoter for 24 hours. Cells were processed for luciferase assays at the indicated posttreatment times with LCL161 (500 nM). Data are means ± SD from n = 3 experiments. (C) Schematic of predicted or reported binding sites of the human TNF-α promoter for SP1, NF-κB, and IRF1. (D) Cells were treated with vehicle or 500 nM LCL161 for 6 hours and processed for chromatin immunoprecipitation (ChIP) using primers spanning the indicated regions of the TNF-α promoter. (E) Alamar blue viability assays of cells transfected with NT or SP3 siRNA for 48 hours and subsequently treated with vehicle or 500 nM LCL161 and 0.01% BSA or TNF-α (1 ng/ml) for 24 hours. (F) Cells were transfected twice with siRNAs targeting NT or the indicated genes for 24 and 48 hours and then treated with vehicle or LCL161 (500 nM) for 24 hours. Viability was measured by Alamar blue. (G) Luciferase assays of MDA-MB-231 cells stably expressing NT or SP3 shRNA that were transfected with plasmids encoding luciferase under the control of the TNF-α promoter for 24 hours, treated with vehicle or LCL161 (500 nM) for 2 hours and 0.01% BSA or TNF-α (1 ng/ml) for 24 hours. (H) SNB75 cells stably expressing NT or SP3 shRNA were transfected with plasmids encoding luciferase under the control of the TNF-α promoter for 24 hours and were treated with vehicle or LCL161 (1 μM) and BSA (0.01%) or TNF-α (1 ng/ml). At the indicated times, cells were processed for luciferase assays. Data are means ± SD from n = 3 experiments; *P < 0.05 and ***P < 0.001 by two-way ANOVA using Sidak’s (D) or Dunnett’s (E, F, and G) multiple comparison test.

  • Fig. 6 SP3 stimulates NF-κB activity.

    (A) Alamar blue viability of cells transfected with control NT or the indicated siRNA for 48 hours and subsequently treated with vehicle or LCL161 (500 nM) for 24 hours. (B) Verification of siRNA-mediated efficacy by Western blotting for the experiment depicted in (A). (C) Cells were transfected with NT or SP3 siRNA for 48 hours and subsequently treated with vehicle or LCL161 (1 μM) for 6 hours. Cells were harvested for electrophoretic mobility shift assay (EMSA) using a consensus NF-κB probe. Blots are representative of three independent experiments. (D) Cells were transfected with NT or SP3 siRNA for 48 hours and treated with vehicle or LCL161 (500 nM). Cells were processed at the indicated times for an ELISA to test the ability of the indicated proteins to bind to a consensus NF-κB probe. (E) Cells were transfected with NT or SP3 siRNA for 48 hours and treated with vehicle or LCL161 (500 nM) for 6 hours and processed for ChIP using RNA Pol II and primers spanning the transcriptional start site (TSS) of the TNF-α promoter. (F) Cells were treated as in (E) and processed for ChIP using the indicated NF-κB antibodies. The three NF-κB regions on the TNF-α promoter are denoted by κB1 (−873), κB2a/b (−627 and −598), and κB3 (−98). Data are means ± SD from n = 3 experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA using Dunnett’s (A) or Tukey’s (D, E, and F) multiple comparison test.

  • Fig. 7 SP3 expression correlates with susceptibility of cancer cells to death by SMC treatment.

    (A) Cancer cell lines (MDA-MB-231 and SNB75), cultured normal cells (GM38, HEL299, 1059SK, and HMEC), or primary normal cells [human foreskin fibroblast (HFF) or mouse embryonic fibroblasts (MEFs)] were treated with vehicle or LCL161 (5 μM) and BSA (0.01%) or TNF-α (1 ng/ml). Cell viability was assessed 48 hours after treatment by Alamar blue assay. Data are means ± SD from n = 3 experiments; ***P < 0.001 by two-way ANOVA using Dunnett’s multiple comparison test. (B) Abundance of mRNA-encoding TNF-α, assessed by RT-qPCR, in cells treated as described in (A) for the indicated time (h, hours). Data are means ± SD from n = 3 experiments; ***P < 0.001 by two-way ANOVA using Dunnett’s multiple comparison test. (C) Cancer cell lines (MDA-MB-231 and EMT6) and normal cells (GM38, HEL299, HFF, and MEF) were treated with vehicle or LCL161 (1 μM) for 8 hours and processed for Western blotting using the indicated antibodies. Blots are representative of two independent experiments. (D) SMC-resistant cancer cell lines were treated and processed for Western blotting as in (C). Blots are representative of two independent experiments. (E) Abundance of mRNA encoding SP3 in cancer and patient-matched normal tissues. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangio carcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma. *Log2FC = 0.5, q < 0.01; **Log2FC = 1.0, q < 0.01; and ***Log2FC = 1.5, q < 0.01. (F) The median RNA sequencing (RNA-seq)–based expression of the indicated genes in cancers was analyzed by hierarchical clustering. Shown are the normalized heat maps. (G) Gene expression variances between brain tissue, LGG, and GBM for the genes in (F) displayed as a t-distributed stochastic neighbor embedding (t-SNE) of RNA-seq transcript counts.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/566/eaat9563/DC1

    Fig. S1. Evaluation and validation of a rescue genome-wide siRNA screen.

    Fig. S2. Top hits from a secondary deconvoluted siRNA screen.

    Fig. S3. SP3, and not SP1, is required for SMC efficacy in cancer cells.

    Fig. S4. Knocking down SP3 does not prevent cell death induced by cycloheximide, staurosporine, or VP16.

    Fig. S5. Expression of cytokines and chemokines that are modulated by SP3 in SMC-treated cancer cells.

    Fig. S6. SMC treatment does not affect the DNA binding activity of SP3.

    Fig. S7. SMC-induced cancer cell death is prevented by shRNA-mediated knockdown of SP3.

    Fig. S8. SP3 deficiency does not induce cancer cell death upon cotreatment with SMCs and TNF-α.

    Fig. S9. SP3 is the major transcription factor involved in promoting SMC-mediated cancer cell death.

    Fig. S10. SP3 overexpression in normal cells leads to cytotoxicity.

    Fig. S11. Overexpression of SP3 does not lead to cell death in SMC-resistant cell lines.

    Fig. S12. cFLIP and SP3 are critical mediators for SMC efficacy in resistant cancer cells.

    Fig. S13. Full-length Western blots.

    Table S1. Data from siRNA screen.

    Table S2. Primer sequences.

  • The PDF file includes:

    • Fig. S1. Evaluation and validation of a rescue genome-wide siRNA screen.
    • Fig. S2. Top hits from a secondary deconvoluted siRNA screen.
    • Fig. S3. SP3, and not SP1, is required for SMC efficacy in cancer cells.
    • Fig. S4. Knocking down SP3 does not prevent cell death induced by cycloheximide, staurosporine, or VP16.
    • Fig. S5. Expression of cytokines and chemokines that are modulated by SP3 in SMC-treated cancer cells.
    • Fig. S6. SMC treatment does not affect the DNA binding activity of SP3.
    • Fig. S7. SMC-induced cancer cell death is prevented by shRNA-mediated knockdown of SP3.
    • Fig. S8. SP3 deficiency does not induce cancer cell death upon cotreatment with SMCs and TNF-α.
    • Fig. S9. SP3 is the major transcription factor involved in promoting SMC-mediated cancer cell death.
    • Fig. S10. SP3 overexpression in normal cells leads to cytotoxicity.
    • Fig. S11. Overexpression of SP3 does not lead to cell death in SMC-resistant cell lines.
    • Fig. S12. cFLIP and SP3 are critical mediators for SMC efficacy in resistant cancer cells.
    • Fig. S13. Full-length Western blots.
    • Table S1. Data from siRNA screen.
    • Legend for table S2.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Primer sequences.

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