Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: Adipocyte-specific knockdown of Ifi204 attenuates the type I IFN-induced upregulation of aromatase and E 2 production. a Schematic presentation of the domain structure of human IFI16 and mouse Ifi204 proteins. Both proteins comprise a pyrin domain (PYD) involved in protein–protein interaction, and two hematopoietic interferon-inducible nuclear (Hin) domains, HinA and HinB, which are involved in DNA binding (left top). Primary preadipocytes isolated from the inguinal mammary gland of Ifi204-AKO and Ifi204 f/f mice were treated with vehicle (−), or 1 or 10 ng/ml IFNβ for 48 h. Expression levels of aromatase and Ifi204 were measured by western blotting (left bottom) and qPCR (right). The concentration of E 2 in the culture supernatants was measured by ELISA (right). * P < 0.05 vs vehicle, # P < 0.05 vs Ifi204 f/f ( n = 3). b Schematic representation of the E0771 and BMDM coculture experiments (left top). BMDMs were seeded in the bottom chamber of a transwell and E0771 cells were seeded in the top chamber and allowed to incubate for additional 48 h. Primary preadipocytes were incubated with DMEM (M) or conditioned medium (CM) obtained from the E0771 and BMDM coculture for 48 h. Expression levels of aromatase and Ifi204 were measured by western blotting (left bottom) and qPCR (right). The concentration of E 2 in the culture supernatants was measured by ELISA (right). * P < 0.05 vs DMEM, # P < 0.05 vs Ifi204 f/f ( n = 3). c Mouse BMDMs and primary preadipocytes were seeded in the bottom chamber of the transwell, as indicated in the scheme. E0771 cells were seeded in the top chamber of the transwell insert. The number of viable E0771 cells was counted using a hemocytometer. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni posttest. *** P < 0.001 vs E0771 monoculture, ### P < 0.001 vs E0771 cocultured with Ifi204 f/f preadipocytes with BMDMs (filled square) ( n = 4) (left). Total RNAs were obtained from the E0771 cells at the day 5 and subjected to qPCR analysis. * P < 0.05 vs E0771 monoculture, # P < 0.05 vs E0771 cocultured with Ifi204 f/f preadipocytes with BMDMs (filled square) ( n = 3) (right)

Article Snippet: Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [ ].

Techniques: Knockdown, Binding Assay, Isolation, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: Adipocyte-specific knockdown of Ifi204 suppresses growth of E0777 mouse mammary tumor cells in allograft experiments. a Schematic representation of the mouse allograft experiment. Ifi204-AKO and Ifi204 f/f mice were ovariectomized at the age of 7 weeks. One week after ovariectomy, mice were fed with either high-fat diet (HFD) or normal diet (ND) for 10 weeks, and then E0777 cells were implanted into the mammary fat pad of mice. Mice were continuously fed with either HFD or ND until the end of the experiment. b Tumor volume was measured every 3 days with a caliper. The number of specimen of each group was as follows: Ifi204 f/f -ND ( n = 8), Ifi204 f/f -HFD ( n = 12), Ifi204-AKO-ND ( n = 6), and Ifi204-AKO-HFD ( n = 12). Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni posttest. ** P < 0.01 and *** P < 0.001 vs Ifi204 f/f mice on the same diet at each time point, ### P < 0.001 vs Ifi204 f/f mice with HFD. c Immunohistochemistry staining of aromatase, IFNβ, Cyclin D1, and Ki67 in tumor sections. Representative images from each group are shown (top). Staining intensities were quantified from three tumor samples from each group and five random fields per sample using ImageJ software and the IHC Profiler plug-in (bottom). ** P < 0.01 and *** P < 0.001 vs Ifi204 f/f mice on the same diet, # P < 0.05 and ### P < 0.001 as indicated. Bar represents 100 μm

Article Snippet: Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [ ].

Techniques: Knockdown, Immunohistochemistry, Staining, Software

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: IFI16, HIF1α, and PRMT2 are physically associated in cancer-associated adipocytes of mouse allograft tumors and human BC specimens. a Allograft tumor tissue sections from HFD-fed mice were subjected to proximity ligation assay (PLA) using anti-Ifi204 and HIF1α antibodies (left), and anti-Ifi204 and PRMT2 antibodies (right), respectively. Arrows indicate PLA signals detected in the nucleus of adipocytes adjacent to mammary tumor cells. Quantification of the PLA signals are shown in Fig. S8. Bar represents 50 μm. b Luminal-type BC tissue sections were subjected to PLA using anti-IFI16 and HIF1α antibodies (top), and anti-IFI16 and PRMT2 antibodies (bottom), respectively. Cancer-associated adipocytes (CAAs) and BC cells were analyzed from the same specimens. Arrows indicate PLA signals detected in the nucleus of CAA. The number of cells with PLA-positive signal was counted from ten BC specimens (right). Data are presented as the mean ± SD. *** P < 0.001. Bar represents 50 μm. c Schematic model for the role of type I IFNs in aromatase activation and E 2 -dependent growth of ER-positive BC cells. The level of type I IFNs are elevated through the interaction between immune cells and BC cells, which induces the expression of IFI16 in preadipocytes. IFI16 then binds to the HRE site on the aromatase PI.3/PII promoter together with HIF1α and PRMT2, leading to the activation of aromatase transcription. Abundant estradiol in tumor microenvironment promotes growth of ER-positive BC cells

Article Snippet: Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [ ].

Techniques: Proximity Ligation Assay, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A, B) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05. (C, D) Localization of Nox4 was detected by immunohistochemistry. The expression of Nox4 was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS production was not increased by mannitol (b) compared with normal glucose (a), but was increased by high glucose (c). High-glucose-induced ROS production was decreased by dapagliflozin pretreatment in a dose-dependent manner (d: 0.2 nM; e: 2.0 nM; f: 20.0 nM). (B) Densitometric quantification of ROS production. Data are mean ± SEM. * P <0.05 vs. high glucose; NG: normal glucose; Man: mannitol; HG: high glucose; dapa: dapagliflozin. Quantitative RT-PCR analysis of the expression of Nox4 (C), MCP-1 (D) and osteopontin (E) showed that dapagliflozin inhibited diabetes-induced inflammation in the kidney. mRNA levels were normalized against Atp5f1 expression. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) Effect of ITPKB knockdown on the activity of ROS-regulating enzymes in KB-3-1cisR and A549cisR cells. Cells with or without ITPKB knockdown were treated with cisplatin (KB-3-1cisR, 5 μg/ml; A549cisR, 2 μg/ml), and activities of redox regulatory enzymes were measured after 12 hours. (B) Effect of NOX1/4 inhibitor GKT137831 on ROS level, NOX activity, and cell viability in ITPKB-knockdown cells in the presence of cisplatin. Cells were treated with GKT137831 (10 μM) and cisplatin as in A. (C) Effect of NOX4 knockdown on ROS level, NOX activity, and cell viability in ITPKB-knockdown cells in the presence of cisplatin. (D and E) Bcl-xL expression (D) and Bax localization (E) change upon GKT137831 treatment in ITPKB-knockdown cells. c, cytosol; m, mitochondria. (F–H) The rescue effect of NOX inhibitor on the tumor growth of ITPKB-knockdown KB-3-1cisR xenograft mice treated with cisplatin. Mice were treated with vehicle control, cisplatin (5 mg/kg), and GKT137831 (30 mg/kg) by i.p. injection twice a week from 3 days after xenograft. Tumor size (F), tumor weight (G), H2O2 level (H, top), NOX activity (H, middle), and ITPKB expression in tumors (H, bottom) are shown. Scale bars: 10 mm for F. Error bars represent SEM for F and SD for G (n = 7). Data are mean ± SD from 3 technical replicates of each sample and are representative of 2 independent biological experiments for A–E and H. Statistical analysis was performed by 2-way ANOVA for F and 1-way ANOVA for all other data (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001).

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Activity Assay, Expressing, Injection

Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) Purification of the recombinant NOX4 dehydrogenase active domain (NOX4-DH). Top: Schematic representation of NOX4 domains. TM, transmembrane domain; FAD, flavin adenine dinucleotide binding domain; NADPH, NADPH binding domain. Bottom: Coomassie staining of purified recombinant NOX4-DH. (B) Thermal shift analyses of NOX4-DH incubated with increasing concentrations of IP4. Tm, melting temperature. (C) Thermal shift analyses of NOX4-DH incubated with 80 μM of the inositol metabolites PIP3, IP3, IP4, and IP5. Dissociation constant (KD) values for the interaction are shown. ND, not determined. (D) Interaction between IP4 and NOX4-DH was determined by Biacore surface plasmon resonance analysis and is shown as a KD value. (E and F) The activity of purified NOX4-DH was measured in the presence of increasing concentrations of IP4 (E) or 80 μM of different inositol metabolites (F). (G) Quantification of NADPH bound to NOX4 in the presence of increasing concentrations of IP4. FLAG-tagged NOX4-DH (30 μM) was incubated with NADPH (100 μM) and IP4 (0–100 μM). Amount of NADPH retained on immobilized NOX4-DH was determined by measurement of absorbance at A340 nm. Data are mean ± SD from 3 technical replicates of each sample for E–G. Data shown are representative of 2 (B–D) and 3 (E–G) independent biological experiments. Statistical analysis was performed by 1-way ANOVA (****P < 0.0001).

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Purification, Recombinant, Binding Assay, Staining, Incubation, SPR Assay, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) BAMB-4 inhibits ITPKB activity in vitro (left) and in KB-3-1 cells (right). (B) Effect of BAMB-4 on ROS level, NOX activity, and cisplatin IC50 in cisplatin-treated cells with or without ITPKB knockout. Cells were treated with cisplatin (KB-3-1cisR and A2780cisR, 5 μg/ml; A549cisR, 2 μg/ml) and BAMB-4 (10 μM) for 12 hours, and ROS and NOX activity were examined. (C and D) Effect of BAMB-4 and/or cisplatin treatment on tumor growth (C) and tumor size (D) of ovarian cancer PDX mice. Mice were treated with cisplatin (5 mg/kg) and BAMB-4 (10 mg/kg) by i.p. injection twice a week after 42 days from xenograft. (E) Effect of BAMB-4 and/or cisplatin treatment on ITPKB activity, H2O2 level, and NOX activity in ovarian cancer PDX tumors is shown. Scale bars: 10 mm for C. Error bars represent SEM for C and SD for D (n = 6). Data are mean ± SD from 3 technical replicates of each sample and are representative of 2 independent biological experiments for A, B, and E. Statistical analysis was performed by 2-way ANOVA for C and 1-way ANOVA for all other data (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001). (F) Proposed model of ITPKB-mediated cisplatin resistance in human cancer. The ITPKB product IP4 inhibits NOX4 by competing with its cofactor NADPH for binding. IP4-induced NOX4 inhibition controls cisplatin-induced ROS, which provides a metabolic advantage to cancer cells that leads to cisplatin-resistant cancer cell survival and tumor growth.

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Activity Assay, In Vitro, Knock-Out, Injection, Binding Assay, Inhibition