Mesenchymal glioma stem cellsに着目した膠芽腫の治療法の提案

Information about Mesenchymal glioma stem cellsに着目した膠芽腫の治療法の提案

Published on June 4, 2016

Author: eisukemiyauchi

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1. 12015年度 文献ゼミ 日時: 2015年9月24日 (木) 時間: 9:30~10:30時間: 9:30~10:30 場所: 中講義室 M h l li ll に着目したMesenchymal glioma stem cellsに着目した 膠芽腫の治療法の提案膠芽腫の治療法の提案 東北大学大学院薬学研究科 薬物送達学分野 博士課程1年 宮内 英輔

2. 2 特 徴:脳腫瘍の中で最も高悪性度で高頻度 高い浸潤性 機能温存のため全摘出困難 余命約1年 膠芽腫(Glioblastoma multiforme: GBM) 高い浸潤性、機能温存のため全摘出困難、余命約1年 Secondary GBM Pediatric GBM Primary GBM(頻度 約90%) 治 療: 外科手術、放射線療法、薬物療法(Temozolomide) Sturm, Dominik, et al. Cancer cell 22.4 (2012): 425-437.

3. 3 Nes-∆TK-GFP transgene GBMの治療成績向上にはGlioma stem cells (GSCs)の根絶が不可欠 hGFAP- Cre;Nf1fl/+;P53fl/fl;Ptenfl/+ (Mut7) glioma-prone mice X glioma-prone mice Combination treatment of TMZ and GCV inhibits glioma progression in cerebrum. a, b, Therapeutic schema targeting both CSCs and their proliferating progeny. Mut7;Nes-DTK mice were treated with TMZ for 5 days, followed 2 days after by GCV. c, Kaplan–Meier survival curve of Mut7;Nes-DTK mice withy , y y , p ; different treatments. GCV-treated (n57; median survival(Med sur.)555 days) andTMZ/GCV-treated (n59; median survival 552.5 days)Mut7;Nes-DTK mice had a similar survival advantage overTMZ- treated (n56; median survival530.5 days) or PBS-treated (n57; median survival521 days) mice. P values were determined using log-rank test. d,GFP and nestin double immunostaining of vestigial tumours in TMZ/GCV-treated Mut7;Nes-DTK mice (right) versus tumour in control (left) demonstrates depletion of CSCs as evidenced by lack of GFP expression. e, Maximal cell density in cortical tumours with different Chen, Jian, et al. Nature 488.7412 (2012): 522-526.http://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination treatment regimens.Untreated Mut7 mice (n56) were used as control. Tumour density of 10-week-old non- symptomatic Mut7 mice (pre-treatment) (n58) was used as a reference starting point. Cell density was significantly lower in the TMZ/GCV-treated Mut7;Nes-DTK mice (n56) compared to untreated tumours (P50.0005), compared to tumours in pre-treatment mice (P50.004), and compared to tumours from mice treated with GCV only (n54). Data are mean6s.e.m.; P50.001, Student’s t-test.

4. 4GSCsは放射線耐性を有する CD1331 tumour cells show radioresistance and lower sensitivity to radiation-induced apoptosis than CD1332 tumour cells dependent on checkpoint kinase activity. a, CD1331 and CD1332 tumour cells were untreated or treated with 5Gy of IR atumour cells were untreated or treated with 5Gy of IR, a Chk1/Chk2 low molecular weight inhibitor (debromohymenialdisine, DBH, 3 mM), or a combination of both. Representative images of colony formation are shown. b, CD1331 and CD1332 cells derived from primary glioblastoma T3590 or D456MG enografts ere ntreated or irradiated ithT3590 or D456MG xenografts were untreated or irradiated with 2Gy or 5Gy of IR. Whole-cell lysates were collected after 24 h and immunoblotted for cleaved caspase-3, an indicator of cell apoptosis. c, CD1331 and CD1332 cells derived from primary glioblastoma T3590 were labelled separately with CFSE (green) and CMTRX (red) fluorescent dyes, mixed in defined ratios (5% CD1331), and visualized by fluorescent microscopy at the indicated time points. d, Mean6s.d. results from c (n5100 cells in three trials; *P,0.002; Bao, Shideng, et al. nature 444.7120 (2006): 756-760.

5. 5 特 徴: 脳腫瘍の中で最も高悪性度で高頻度 膠芽腫(Glioblastoma multiforme: GBM) Secondary GBM Pediatric GBM Primary GBM(頻度 約90%) Sturm, Dominik, et al. Cancer cell 22.4 (2012): 425-437. 治 療: 外科手術、放射線療法、薬物療法(Temozolomide) Today’s topic:グリオーマ幹細胞を標的とした薬物療法

6. 6GSCsには2つのサブタイプが存在する high grade gliomas (HGGs)患者のhigh-grade gliomas (HGGs)患者の 腫瘍組織から確立したGSC cultureを マイクロアレイ解析 Microarray analysis of two distinctive GSC samples. (A) Hierarchical biclustering of genes differentially expressed between PN and Mes cell lines. (B) Heat map with pairwise Pearson correlation for the Phillips HGG dataset and TCGA GBM dataset with the microarray samples. Stronger Mao, Ping, et al. Proceedings of the National Academy of Sciences 110.21 (2013): 8644-8649. correlation is observed among microarray samples of the same type compared among and with TCGA samples of the same subtype. (C) qRT- PCR and microarray of two GSC subtypes (**P < 0.01). Data are representative of three independent experiments with similar results.

7. 72つのGSCsのサブタイプの内、Mesenchymal (Mes)型がより悪性度が高い Phenotypic differences between PN and Mes GSCs. (A) In vitro growth curves of PN GSC samples (n = 4) and Mes GSC samples (n = 5). (B) Repre- sentative H&E staining of various mouse brain sections with tumors established by two PN and two Mes GSCs. (C) Kaplan-Meier survival curves ofmice bearing PN GSC– and Mes GSC–derived tumors (**P < 0.01). Data in A–E are representative of three independent experiments with similar results. Mao, Ping, et al. Proceedings of the National Academy of Sciences 110.21 (2013): 8644-8649.

8. 8放射線治療はProneural (PN)型GSCsの Mes-Associated Markerの発現を誘導する(PN-to-Mes transformation (PMT)) Prominent radioresistance of Mes GSCs and radiation induces transformation of PN GSCs into Mes GSCs (A) DNA microarray analyses and qRT PCR validation of DNA damageinto Mes GSCs. (A) DNA microarray analyses and qRT-PCR validation of DNA damage- repair gene expression in GSCs. Various DNA damage-repair genes are expressed at higher levels in Mes GSCs than that in PN GSCs (*P < 0.05, **P < 0.01). Box: various GSC cells depicted in bar graphs. (B) Effect of radiation treatment on in vitro growth of PN and Mes GSCs (11 samples in total) at indicate doses. (C) Representative PN and Mes gene expressions in PN GSC samples (n = 3) with and without radiation treatment (5 Gy, tested at day 5; **P < 0 01) Data in A–D are representative of three independent experiments Mao, Ping, et al. Proceedings of the National Academy of Sciences 110.21 (2013): 8644-8649. at day 5; P < 0.01). Data in A D are representative of three independent experiments with similar results.

9. 9GBMにおけるMETの活性化がMES transformationを誘導する HGF/MET Signaling Induces an EMT-like Transition in GBM (A) Representative phase contrast microscopy images of VEGFKO and VEGFKO- shMET1 cells. Scale bars, 100 mm. (B) Immunoblot analysis of VEGFKO or VEGFKO-shMET1 cells stimulated with HGF for the indicated times for P-Met, Snail, N-cadherin, and T-cadherin expression. (C)Snail, N cadherin, and T cadherin expression. (C) Immunoblot analysis of T-cadherin expression in VEGFKO-shMET1 cells and parental VEGFKO cells. (D) Immunoblot analysis of VEGFKO cell lysates 6 hr after stimulation with HGF or indicated concentrations of VEGF (E and F)concentrations of VEGF. (E and F) Immunohistochemical staining of intracranial mouse GBMs for P-MET, N-cadherin, and T-cadherin (brown). Sections were counterstained with hematoxylin (blue). Staining at the center of the main tumor mass or at the tumor rim is shown as indicated. (E) Representative images of time-matched (day 27) VEGFKO and VEGFKO-shMET1 tumors. Dashed lines indicate the smooth border of VEGFKO- shMET1 tumors. (F) Mice bearing orthotopic WT-shMET1 tumors. (F) Mice bearing orthotopic WT GBM were treated with B20 or vehicle beginning 3 days after tumor implantation. Representative staining of tumors from mice sacrificed 16 days after tumor implantation are shown. Tumor cells were visualized by staining for SV40 Tag (top) Scale barsvisualized by staining for SV40 Tag (top). Scale bars, 50 mm. See Lu, Kan V., et al. Cancer cell 22.1 (2012): 21-35. EMT markers Snail, T-cadherin to N-cadherin switch B20 the anti-VEGF antibody

10. 10小括 Nakano, Ichiro. Journal of neurosurgery 122.2 (2015): 324-330. •GBMの治療成績向上にはGSCsの根絶が不可欠 •GSCsには少なくともPN型とMES型の2種類が存在するGSCsには少なくともPN型とMES型の2種類が存在する •PN型に比べてMES型がより高悪性度 •PN型への刺激(少なくとも放射線、METの活性化)はPN-to-MES transformationを 誘導する誘導する 治療成績向上のための標的の優先度 MES型GSCs (PN-to-MES transformation)> PN型GSCs

11. 11METはSTAT3を介してMES transformationを誘導する ただし、これはhuman breast carcinomaにおける結果 Positive feedback in the PYK2– STAT3 MET i ( )ThSTAT3–c-MET axis. (c)The c- Met inhibitor PHA-66752 (10mM) reduces the phosphorylation of STAT3 and PYK2 in response to EGF (24h) and also of the -? ( ) mRNA levels of EMT transcription factors as determined by WB analysis (c) and by RT–PCR (d, left panel) or qRT–PCR (d graph)qRT–PCR (d, graph), respectively. The intensity of the RT–PCR bands was assessed by densitometry (Image J). Mean values of qRT–PCR±s.d. were l l t d d t d i thcalculated and presented in the graph as fold of control EGF- untreated values. (e) A scheme depicting the positive feedback loop between EGFR/c-Met, c-Met inhibitor PHA-665752 Verma, Nandini, et al. Nature communications 6 (2015). PYK2 and STAT3. EGFR and c- Met, which can trans- phosphorylate each other, activate PYK2 (PY402), which in turn enhances STAT3 , , ( ) 仮説 turn, enhances STAT3 phosphorylation (pY705). Phospho-STAT3(pY705) translocates to the nucleus, binds to PYK2 prompter and h it t i ti MES型GSCs (PN-to-MES transformation)の根絶には、 mesenchymal genesを発現させる転写因子の阻害が 効果的なのではないか? enhances its transcription. pSTAT3 also enhances the expression of c-Met, possibly indirectly, and of Twist, which is required for EMT. 効果的なのではないか?

12. 12HGGsのmesenchymal signatureの74%以上は 6つの転写因子により制御される The mesenchymal signature of HGGs is controlled by six TFs. a, TFs involved in activation of MGES targets are shown in pink, those involved in repression are in purple. MGES targets controlled by these TFs are in cyan. Overall, the six TFs control74%of the genes in the mesenchymal signature of high-grade glioma. A region between 2 kb upstream and downstream the transcription start site ofin the mesenchymal signature of high grade glioma. A region between 2 kb upstream and downstream the transcription start site of the target genes identified by ARACNe was analysed for the presence of putative binding sites. b–e, Genomic regions of genes containing putative binding sites for specific TFs were immunoprecipitated in SNB75 cells by antibodies specific for STAT3 (b), C/EBPb (c), FOSL2 (d), and bHLH-B2 (e). SOCS3 was included as positive control of STAT3 binding. Total chromatin before immunoprecipitation was used as positive control for PCR. The OLR1 gene was used as negative control f Summary of binding results of the tested TFs to mesenchymal targetscontrol. f, Summary of binding results of the tested TFs to mesenchymal targets. Carro, Maria Stella, et al. Nature 463.7279 (2010): 318-325.

13. 13GliomaのMES transformationおよびMES型の維持の Master regulatorsはSTAT3およびC/EBPβである A hierarchical transcriptional module regulates the MGES. a, b, ChIP for C/EBPb (a) and STAT3 (b). c, Transcriptional network emerging from promoter occupancy analysis. d, qRT–PCR of mesenchymal TFs in glioma cells infected with STAT3 and CEBPB shRNA or control (ctrl) lentiviruses. Error bars are s.d. e, Venn-diagram depicts the proportion of mesenchymal genes identified by ARACNe as targets of only C/EBPb, STAT3 or both TFs. f, Heatmap of MGES gene expression analysis of mouseTFs. f, Heatmap of MGES gene expression analysis of mouse and human cells carrying perturbations of C/EBPb plus STAT3. Samples (columns) were grouped according to species and treatment. Control, control shRNA or empty vector; S2, STAT3 knockdown; S1, STAT3 overexpression; C2, CEBPB knockdown; C1 CEBPB overexpression; S2/ C2 STAT3 and CEBPBC1, CEBPB overexpression; S2/ C2, STAT3 and CEBPB knockdown; S1/C1, STAT3 and CEBPB overexpression. g, GSEA of the MGES on the gene expression profile rank- sorted according to the correlation with theCEBPB3STAT3 metagene. The bar-code plot indicates the position of MGES genes, red and Carro, Maria Stella, et al. Nature 463.7279 (2010): 318-325. blue colours represent positive and negative correlation, respectively. The grey scale bar indicates the Spearman’s rho coefficient used as weighting score for GSEA. LEOR, leading- edge odds ratio; nES, normalized enrichment score; P, sample- permutation-based Pvalue.

14. 14Ionizing radiation (IR)でもSTAT3およびC/EBPβの標的遺伝子の 発現が上昇している IR induces proneural-to-mesenchymal shift in PDGF gliomas. (A) Enrichment plots of CEBP and STAT3 targets in translating mRNA 6 h after 10-Gy IR in PDGF Ink4a/Arf−/− gliomas. (B) Time course of CEBP and STAT3 target up-regulation in PDGF Ink4a/Arf+/− gliomas. Average of total and translating mRNA pools is shown; dotted lines are SE. (C) Enrichment plots of mesenchymal and proneural signature genes in translating mRNA after 10-Gy IR in PDGF-driven Ink4a/Arf−/−,Ink4a/Arf−/−/PTEN−/−,and Ink4a/Arf−/−/p53shRNA gliomas. (D) Enrichment plots of mesenchymal and proneural signature genes in the relative changes of translating mRNA after IR of PDGF Ink4a/Arf−/− tumors vs. PDGF Ink4a/Arf−/−/p53shRNA tumors. Halliday, John, et al. Proceedings of the National Academy of Sciences 111.14 (2014): 5248-5253.

15. 15別の研究からTAZもGliomaのMES transformationおよびMES型の維持の Master regulatorsであることが判明した Supp. FigS5A S l t Fi S5Supplementary Figure S5. A. Western blotting of GSC20 transfected with siRNA against a scrambled sequenceq (control), STAT3 C/EBP-β or TAZ showing expression of respective proteins. TAZ enhances tumor grade reduces survival andTAZ enhances tumor grade, reduces survival, and promotes MES differentiation in the RCAS/N-tva mouse model. (A) Kaplan-Meier survival analysis of PDGF-B-, WT-TAZ+PDGF-B-, 4SA+PDGF-B-, or 4SA-S51A+PDGF-B-injected mice. (B) Stacked b h h i WHO li d ithibar graph showing WHO glioma grades within each group. (C) Representative images of hematoxylin and eosin-stained slides of brains isolated from mice injected with PDGF-B,WT- TAZ+PDGF-B, 4SA+PDGF-B, or 4SA- S51A+PDGF-B. Note that necrosis is observed only in WT-TAZ+PDGF-B and 4SA+PDGF-B mouse tumors. (D) Real-time qPCR analyses of gene expression in RCAS mouse tumors. The gene expression value of PDGF control wasgene expression value of PDGF control was normalized to 1, and the relative expression of CD44, CTGF, and FN1 is shown. Bhat, Krishna PL, et al. Genes & Development 25.24 (2011): 2594-2609.

16. 16治療成績の向上にはC/EBPβ、STAT3(およびTAZ?)の複数阻害が効果的 C/EBPb and STAT3 are essential for glioma tumour aggressiveness in mice and humans. a, Immunofluorescence staining for human i ti CD31 fib ti COL5A1 d YKL40 i t d i d f SNB19 ll i f t d ith l ti i i hRNAvimentin, CD31, fibronectin, COL5A1 and YKL40 in tumours derived fromSNB19 cells infected with lentiviruses expressing shRNA targetingSTAT3,CEBPB,orSTAT3plusCEBPB. B, normal brain; T,tumour. b, Kaplan–Meier survival curve of NOD/SCID mice transplanted intracranially with SNB19 gliomacells transduced with control shRNA(red), STAT3shRNA(black),CEBPBshRNA(green) orSTAT3plusCEBPBshRNA (blue) lentiviruses. c, Immunostaining for human vimentin and Ki67 on representative brain sections from mice injected with BTSC-3408 after silencing of C/EBPb and STAT3. CC, corpus callosum; St,j g striatum. d,Quantificationofhumanvimentin-positive area. a.u., arbitraryunits. Error bars are s.d. e, Quantification of Ki67-positive cells. n55 for each group; error bars are mean6s.d. f, g, Immunostaining for fibronectin (f) and COL5A1 (g) on representative brain sections from mice injected with BTSC- 3408transduced as indicated.h,Kaplan–Meieranalysiscomparing survival of patients carrying tumours double positive for C/EBPb and STAT3 (red) and single- or double-negative tumours (black). *P#0.05, **P#0.01. Carro, Maria Stella, et al. Nature 463.7279 (2010): 318-325.

17. 17現状でSTAT3、C/EBPβおよびTAZの中で標的薬の開発が進んでいるのは STAT3 pathway inhibitor Kim, Jennifer E., et al. Cancers 6.1 (2014): 376-395. •より良い治療効果のためにSTAT3だけではなくC/EBPβやTAZも同時に 課題 阻害できないか。 STAT3、 C/EBPβおよびTAZの上流に共通のシグナルはないか? 解決策

18. 18TNFαによるNFkBの活性化がC/EBPβ、STAT3およびTAZを誘導する NF-kB Controls Master TFs of MES Differentiation in GSCs (A) Western blot analysis of phosphorylated p65 (ser 536) total p65 phosphorylated STAT3 (Tyr 705)(ser 536), total p65, phosphorylated STAT3 (Tyr 705), STAT3, and C/EBPb in GSCs is shown. (B and C) Western blot analysis using indicated antibodies was performed on GSC23 (B) and 11 (C) sorted for CD44high or CD44low subpopulations. (D) Box plots of li d i f STAT3 CEBPB TAZ d NFnormalized expression of STAT3, CEBPB, TAZ, and NF- kB metagene in CD44low (green boxes) or CD44high (red boxes) tumors from multiple data sets as indicated are shown. Boxes show median 25th and 75th percentiles, while whiskers represent the 5th and the 95th percentiles. Outliers are shown as individual points. p value was determined using a nonparametric Wilcoxon test. For the NF-kB metagene, the average expression of 38 NF-kB family members and targets (see Supplemental Experimental Procedures) wasSupplemental Experimental Procedures) was condensed into a metagene and plotted. Wilcoxon signed-rank test was used to test statistical significance. (E) Time course western blot analysis of indicated antibodies after TNF-a treatment in GSC11 transduced ith RFP or IkB SR adeno ir s 24 hr prior to TNF awith RFP or IkB-SR adenovirus 24 hr prior to TNF-a treatment is shown. (F) qRT-PCR analysis of MES signature master TFs STAT3, CEBPB, and TAZ in GSC11 treated with TNF-a with or without pretreatment with RFP or IkB-SR adenovirus is shown. Error bar indicates ± SD. t test was used for statistical significance. *p < 0.05 and **p < 0.005. (G) qRT-PCR analysis of YKL40 and CD44 after knockdown of all three master TFs (STAT3, C/EBPb, and TAZ) in GSC11 is shown. Cells were treated with siRNA 72 hr prior to Bhat, Krishna PL, et al. Cancer cell 24.3 (2013): 331-346. is shown. Cells were treated with siRNA 72 hr prior to treatment with TNF-a for an additional 24 hr. Error bar indicates ± SD. t test was used for statistical significance. *p < 0.05 and **p < 0.005.

19. 19STAT3およびC/BEPβのシグナリングの概要 Shostak, Kateryna, and Alain Chariot. Trends in molecular medicine (2015).IL-6 Kuilman, Thomas, et al. Cell 133.6 (2008): 1019-1031.Yu, Hua, et al. Nature reviews Cancer 14.11 (2014): 736-746.

20. 20Minocycline 特徴 •テトラサイクリン系抗生物質 •Microglia活性およびNF-κB signalingを抑制する 特徴 Microglia活性およびNF κB signalingを抑制する •血液脳関門を通過する Markovic, D. S., et al. Brain, behavior, and immunity25.4 (2011): 624-628., Daginakatte, Girish C., and David H. Gutmann. Human molecular genetics 16.9 (2007): 1098-1112. •FDAの承認薬である(体内動態や毒性がよく分かっている。) Yenari, Midori A., et al. Stroke 37.4 (2006): 1087-1093. •薬価が安い(特許が切れている)

21. 21MinocyclineはMES GSCsに対して抗腫瘍効果を示す Bhat, Krishna PL, et al. Cancer cell 24.3 (2013): 331-346.

22. 22 (J) Western blot analysis of MES markers and master TFs STAT3, C/EBPβ and TAZ after treating indicated MES GSCs with si-p65 for 72 h (left panel) BAY 11 7082 (10 µM) for 96 h (middle panel) and minocycline (50 µM) for 5 days (right panel) We raised the MinocyclineはMES GSCsに対して抗腫瘍効果を示す for 72 h (left panel), BAY 11-7082 (10 µM) for 96 h (middle panel) and minocycline (50 µM) for 5 days (right panel). We raised the possibility that silencing NF- B may impact the MES state of these cells. Consistently, transient knockdown of p65 was sufficient to reduce protein levels of YKL40, TAZ, and C/EBP , but not STAT3, implying that the p65 subunit is not required for maintenance of STAT3 expression. However, treatment of cells with I B kinase inhibitor, BAY 11-7082, caused a decrease in expression of all three TFs suggesting that while the p65 subunit of NF- B alone was not sufficient to alter STAT3, global inhibition of the NF- B pathway had a suppressive effect on STAT3 expression. In vitro treatment of GSC2 and GSC20 also caused dramatic reduction of master TF expression as well as CD44 and FN1 within 4 days. (K) Cell viability was detected using the WST-1 assay. Bar graphs indicate percentage of cells viable after a 5 day minocycline treatment (black bars) against untreated controls (gray bars). Error bar indicates +/- SD. t test was used for statistical significance. We observed a more profound minocycline induced reduction of cell viability of MES GSCs in comparison to PN GSCs.e obse ed a o e p o ou d ocyc e duced educt o o ce ab ty o S GSCs co pa so to GSCs (L) Representative bio-luminescence images of GSC20 (left panel) or GSC23 (right panel) transduced with pCignal lenti-CMV-luc injected into Foxn1nu mice. Cells were imaged 2-3 weeks after implantation as the first timepoint (week 1) after which the radiation group received four cycles of 2.5 Gy IR on consecutive days. We treated mice bearing xenografts with 50 mg/kg minocycline starting four days prior to radiation. Minocycline treatment strongly reduced tumor growth in X-GSC20 and the effect was additive when combined with radiation while radiation alone had modest but not statistically significant reduction in tumor volume compared to thecombined with radiation, while radiation alone had modest, but not statistically significant reduction in tumor volume compared to the control group. To test whether the effect of minocycline was specific for the MES GSCs, we treated implanted GSC23 intracranially into mice and followed the same treatment regimen as GSC20. As expected, radiation caused a decrease in tumor volume compared to control groups but minocycline had no significant effect on tumor size (right panel). Thus, minocycline specifically had antitumor effects only on MES GSCs in the xenograft model. Black bar shows average radiance (photons/s/cm2/sr) with various treatments and time points. Error bar indicates +/- SEM. Color key for luminescence is shown on the right. t test was used to assess statistical significance. NS = not significant. (M) Stacked bar graph shows percentage of mice exhibiting features of AA or GBM within each group. To discern if minocycline treatment influenced tumor grade, we inspected hematoxylin and eosin (H&E) stained sections from all groups. Tumors from GSC20 injected mice predominantly showed features of GBM (with tumor necrosis), whereas those treated with minocycline lacked necrosisj p y ( ), y while still exhibiting features of grade III AAs. (N) IHC analysis of various markers in GSC20 xenograft tumors that were treated with minocycline alone or in combination with IR compared to untreated mice. Upper panel shows the representative H&E staining. Middle and lower panel show the expression of YKL-40 and OLIG2 respectively. A significant reduction in the expression of YKL40 and CD44 (data not shown) was seen in all tumors treated with minocycline A subset of mice treated with minocycline (n = 2 out of 5) and those with both minocycline andtumors treated with minocycline. A subset of mice treated with minocycline (n = 2 out of 5) and those with both minocycline and radiation (n = 2 out of 5) showed OLIG2 staining in about 10-60% of the tumor area, although the induction of PN genes was not seen in vitro. Scale bar: 100 µm. Bhat, Krishna PL, et al. Cancer cell 24.3 (2013): 331-346.

23. 23総括 •GBMの治療成績向上にはGSCsの根絶が不可欠。 •GSCsのサブタイプの内、最も悪性度の高いMES GSCsの根絶を治療成績向上の ために優先すべき。 •GliomaのMES transformationおよびMES型の維持のMaster regulatorsはSTAT3、 C/EBPβおよびTAZである。 STAT3 C/EBPβおよびTAZはTNF によるNF Bの活性化により誘導される•STAT3、C/EBPβおよびTAZはTNFαによるNF-κBの活性化により誘導される。 •NF-κB signaling inhibitorであるminocyclineは血液脳関門を通過しMES GSCsに 対して抗腫瘍効果を示す対して抗腫瘍効果を示す。 結論 MinocyclineのGBM治療へのDrug repositioningにより、迅速に安くこれまでに比べ て効果的な治療をGBM患者さんに提供できるのではないか。

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