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FOXP3-mediated Transcriptional Regulation of MicroRNAs in Breast Cancer

FOXP3-mediated Transcriptional Regulation of MicroRNAs in Breast Cancer. Department of Genetics University of Alabama at Birmingham Runhua Liu, M.D & Ph.D. 09/2017. Outlines FOXP3 is an X-linked tumor suppressor gene in breast cancer FOXP3-miR-146-NFkB axis and its role in breast cancer

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FOXP3-mediated Transcriptional Regulation of MicroRNAs in Breast Cancer

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  1. FOXP3-mediated Transcriptional Regulation of MicroRNAs in Breast Cancer Department of Genetics University of Alabama at Birmingham Runhua Liu, M.D & Ph.D. 09/2017

  2. Outlines • FOXP3 is an X-linked tumor suppressor gene in breast cancer • FOXP3-miR-146-NFkB axis and its role in breast cancer • FOXP3-regulated micRNA200 family as biomarks in Breast cancer

  3. X-linked FOXP3 (forkhead box P3 ) • FOXP3 is a member of the forkhead-box/winged-helix transcription factor family (Nat Genet, 27: 68–73, 2001). • FOXP3 is responsible for X-linked autoimmune disease IPEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked) syndrome in both mice and humans (Nat Genet, 27:18-20, 2001; Nat Genet, 27:20-1, 2001). • FOXP3 functions as a master regulator in the development and function of regulatory T cells (Nat Immunology, 4: 330-6, 2003).

  4. FOXP3 Xp11.23 FOXP3 is an X-linked gene FOXP3 function in immune system FOXP3 in normal epithelial cells J Immunol. 2008,180:5163-6. FOXP3-GFP (green) and K8 (red)

  5. Foxp3 is an X-linked tumor suppressor gene Scurfy (Foxp3sf/+) female mouse What is Foxp3scurfy mutant mouse? The scurfy mouse has a spontaneous Foxp3 mutation, and this mutation is a frame shift mutation, resulting in loss of Foxp3 function. Homozygous or hemizygous scurfy mice generally die within 2 weeks after birth, but heterozygous scurfy female mice have a normal life span. 1.2 1.0 0.8 0.6 p < 0.0001 Tumor free Survival 0.4 WT (n=35) WT (n=35) 0.2 sf /+ sf /+ (n=18) (n=18) Foxp3 Foxp3 0.0 0.2 10 12 14 16 18 20 22 24 26 spontaneous breast cancer MONTHS Spontaneous breast cancer Foxp3sf/+ female mice One year Cell, 2007; 129:1275-86.

  6. Genetic alterations of FOXP3 in human breast cancers CEPX FOXP3 Heterozygous somatic mutations Heterozygous gene deletions FKH: forkhead LZ: leucine zipper ZF: zinc finger Breast cancer 28/223 (13%) Cell, 2007; 129:1275-86.

  7. FOXP3 is down-regulated in both mouse and human breast cancers A B Spontaneous breast cancer Breast carcinoma Lung metastasis Mouse C Human • FOXP3 (Xp11.23) as a X-linked tumor suppressor genes • in breast cancer (Cell, 129: 1275-1286, 2007)

  8. miRNA expression profiles regulated by FOXP3 GFP GFP-FOXP3 miR-146 family miR-200 family 0.33 0.50 0.66 1.00 1.50 2.00 3.00

  9. Outlines • FOXP3 is an X-linked tumor suppressor gene in breast cancer • FOXP3-miR-146-NFkB axis and its role in breast cancer • FOXP3-regulated micRNA200 family as biomarks in Breast cancer

  10. MiR-146a/b contributes to FOXP3-induced tumor suppression and apoptosis in breast cancer cells * * 25 30 miR-146a miR-146b * 25 * 20 GFP GFP 20 15 %RNU6B %RNU6B GFP-FOXP3 GFP-FOXP3 15 FOXP3-Tet-off MCF7 cells 10 10 B 5 5 A 160 * 0 0 140 Cell number (1x104) 0h 24h 48h 0h 24h 48h 0h 24h 48h 0h 24h 48h 120 -Dox * -Dox 100 * 80 * FOXP3- FOXP3+ 60 FOXP3+miR-146a -i 40 FOXP3+miR-146b-i FOXP3+miR-146a/b-is 20 0 0 1 3 5 7 Days (-Dox) FOXP3+ FOXP3- C 100 80 Apoptosis (%) 60 7-AAD FOXP3+miR-146b-i FOXP3+miR-146a/b-is FOXP3+miR-146a-i * 40 * * 20 0 FOXP3 - + + + + miR-146a-i - - + - + miR-146b-i - - - + + Annexin V

  11. FOXP3 (+) miR-146a-i FOXP3 (+) miR-146b-i FOXP3 (+) miR-146a&b-is FOXP3 inhibits cell migration independent miR-146 in breast cancer cells FOXP3 (-) FOXP3 (+) 0h Scratch assay 24h Transwell migration 160 Cell number 120 * * * * 80 40 0 FOXP3 - + + + + miR-146a-i - - + - + miR-146b-i - - - + +

  12. FOXP3-miR-146-NFkB axis in breast cancer cells FOXP3-Tet-off MCF7 cells at 3d FOXP3 * * * * 4000 3000 FOXP3 - + + + + 2000 1000 miR-146a-i - - + - + 0 25 IRAK1 2.5 miR-146b-i - - - + + 20 2.0 * 1.5 * * 15 1.0 FOXP3 0.5 10 0.0 * * 5 BCL2L1 IRAK1 TRAF6 * * 3.0 0 * % GAPDH TRAF6 2.0 % GAPDH * * 0.3 1.0 0.0 p65 0.2 N-p65 0.1 TRAF1 * Lamin 0.0 RELA(P65) 3 1.2 Actin 0.8 2 FOXP3- 0.4 FOXP3+ 0.0 FOXP3+miR-146a-i FOXP3 - + + + + 1 TRAF2 - - + - + miR-146a-i FOXP3+miR-146b-i * - - - + + miR-146b-i FOXP3+miR-146a+b-is 0 Days (-Dox) 0 1 2 3 4 5 6 7

  13. FOXP3-miR-146-NFkB axis in mouse breast in vivo p = 7.50 x 10-5 p = 5.63 x 10-4 Cre cKO Foxp3 flox/flox miR-146 (% snoRNA202) Foxp3 breast conditional knockout (cKO) MMTV-Cre b c a 12wks (n = 10) 20wks (n = 10) MMTV-Cre cKO Bcl2l1 4.0 8.0 Foxp3 Irak1 cKO/Cre= 4.74 p = 0.0028 3.0 cKO/Cre = 4.04 p = 0.0044 6.0 % Hprt % Hprt 2.0 4.0 1.0 2.0 0.0 0.0 Irak1 14 Traf6 1.5 1.5 12 Traf1 100 µm 100 µm cKO/Cre = 2.26 p = 0.0138 cKO/Cre = 3.89 p = 0.0014 10 1.0 1.0 % Hprt Nongestational Gestational % Hprt 8 0.5 Traf6 0.5 6 4 0.0 Micro-dissected breast epithelial cells 0.0 100 µm cKO/Cre = 0.97 p = 0.9367 100 µm p65 2 Traf2 10.0 25 cKO/Cre = 5.27 p = 0.0022 0 8.0 20 20-week-old 6.0 15 % Hprt % Hprt 4.0 10 5 2.0 0 0.0 cKO (n = 5) MMTV-Cre (n = 5) cKO (n = 5) MMTV-Cre (n = 5)

  14. FOXP3-miR-146-NFkB axis in human breast cancer FOXP3+ FOXP3- 50 µm 50 µm 16 miR-146a miR-146b IRAK1 TRAF6 16 14 12 p = 0.0302 12 p = 0.0195 10 %RNU6B %GAPDH 8 8 6 p = 0.0084 p = 0.0041 4 4 2 0 0 FOXP3+ FOXP3- FOXP3+ FOXP3- FOXP3+ FOXP3- FOXP3+ FOXP3- (n = 8) (n = 12) (n = 8) (n = 12) (n = 8) (n = 12) (n = 8) (n = 12) (micro-dissected cancer cells)

  15. FOXP3 directly targets the miR-146a promoter in MCF7 cells A Forkhead domain DNA binding core motif 1.0 kb miR-146a 5q34 Real-time PCR fragment TSS -10.0 kb DQ658414 Exon 1 DQ658414 Exon 2 16.0kb -1.0kb -7.5kb -4.5kb -0.35kb 11.0kb B 25 20 15 Relative Units (%) 10 5 D Forkhead domain DNA binding core motif 0 FOXP3 protein Nuclear lyses C NF-kB binding element Relative luciferase activity Protein or lyses - + + + + - + + + TSS Biotin-probe + + + + + + + + + 200 bp DQ658414 Intron 1 WT-comp - - - - + - - + - 10 20 30 40 50 60 70 80 -1.0 kb 0 Mut-comp - - + - - - - - + DL-comp - - - + - - - - - 0d-Foxp3 pGL2 WT 0d-IgG 2d-Foxp3 FOXP3 FOXP3 pGL2 Mut A 2d-IgG pGL2 Mut B Vector NS Mut A+B pGL2 dual-luciferase reporter assay FOXP3 NS Gel shift assay FP FP

  16. FOXP3 inhibits cell growth and metastasis in breast cancer cells 1 × 104 FOXP3(-)/(+) MDA-MB231 iv. injection NSG (NOD SCID gamma) Sacrifice for histologic examination Age (wks) 0 8 14 Lung Liver FOXP3(+)/(-)=0.53 p = 0.0386 FOXP3(+)/(-)=0.32 p = 0.0283 50 Tumor nodules Tumor nodules 30 40 30 20 20 10 10 0 0 FOXP3(+)/(-)=0.51 p = 0.0355 FOXP3(+)/(-)=0.32 p = 0.0115 Tumor burden % Tumor burden % 30 25 20 20 15 10 10 5 0 0 FOXP3- FOXP3+ FOXP3- FOXP3+ (n = 10) (n = 10) (n = 10) (n = 10) FOXP3- FOXP3+ FOXP3+ FOXP3-

  17. Summary II: FOXP3-miRNA-146 and NF-κB feedback loop in cancer cells EGFR/HER2 FOXP3 miR-146 PI3K P P • miR-146a/b can target two NF-kB inducers IRAK1 and TRAF6 to inhibit NF-kB activity. • NF-kB can also induce miR-146 transcription, suggesting miR-146 and NF-kB feedback loop in cancer cells. • FOXP3 directly interacted with NF-kB p65 to regulate an miR-146–NF-kB negative feedback loop in normal epithelial and tumor cells. • The FOXP3-miR146-NFkB axis plays an important role in FOXP3-mediated tumor suppression, and may provide a new therapeutic approach for FOXP3 deficient cancers. P IRAK1/ TRAF6 AKT IRAK1 NF-κB TRAF6 NF-kB NF-κB BCL2L1/ TRAF1/ TRAF2 miR-146 FOXP3 Apoptosis miR-146 Tumor growth

  18. Outlines • FOXP3 is an X-linked tumor suppressor gene in breast cancer • FOXP3-miR-146-NFkB axis and its role in breast cancer • FOXP3-regulated micRNA200 family as biomarks in Breast cancer

  19. A Tet off MCF7 cells FOXP3 induces miR-200c and miR-141 in breast cancer cells GFP FOXP3/GFP -Dox 0 24 48 0 24 48 hours FOXP3 B Hours 0h miR-200 cluster 1 (Chr 1) miR-200 cluster 2 (Chr 12) ACTIN 24h Relative expression 4.0 miR-200b miR-200a miR-429 miR-200c miR-141 * 48h * * 3.0 2.0 1.0 0.0 FOXP3 (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) T47D C BT474 MDM-MB-468 * * 6 * 8 1.6 5 * * 6 1.2 4 % RNU6B % RNU6B % RNU6B 3 4 0.8 * 2 2 0.4 1 0 0 0 Vector FOXP3 Vector FOXP3 Vector FOXP3 Vector FOXP3 Vector FOXP3 Vector FOXP3 Vector FOXP3-V5 Vector FOXP3-V5 Vector FOXP3-V5 miR-200c miR-141 miR-200c miR-141 miR-200c miR-141 V5 V5 V5 ACTIN ACTIN ACTIN D TCGA data CD25(IL2RA)-high CD25(IL2RA)-low Relative expression * * * miR-200b miR-200b miR-200a miR-200a miR-429 miR-429 miR-200c miR-200c miR-141 miR-141 * * * * 542 Samples n=135 n=135 n=135 n=135 n=135 n=135 n=135 n=135 n=135 n=135 n=136 n=136 n=136 n=136 n=136 n=136 n=136 n=136 n=136 n=136 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3 FOXP3

  20. Changes in miR-200 s in tumor cells and plasma during breast cancer progression A Benign breast Breast Cancer Foxp3sf/WT lung meta Foxp3WT/WT Foxp3sf/WT Foxp3sf/WT tumor 5 4 10 3 C 8 2 1 6 0 4 Lung metastasis, n= 5 2 Plasma Breast tumor, n=5 No tumor, n=5 0 B miR-200 cluster 1 (Chr 1) miR-200 cluster 2 (Chr 12) 20 miR-200c Relative expression 16 WT, Foxp3wt/wt, n= 5 * sf/wt, Foxp3sf/wt, n=5 12 * Tu, Foxp3sf/wt with tumor, n=5 * % snoRNA202 * 8 4 * * 0 Months 13 15 17 19 21 23 WT sf/wt Tu WT sf/wt Tu WT sf/wt Tu WT sf/wt Tu WT sf/wt Tu miR-200b miR-200a miR-429 miR-200c miR-141 miR-141 * * Months 13 15 17 19 21 23 Foxp3sf/+ female mice laser-capture microdissection Vertical arrows: The time points of breast tumor development Red arrows and blue arrows for metastatic and non-metastatic mice

  21. Plasma levels of miR-200c and miR-141 are elevated in patients with metastatic breast cancer A Plasma B Plasma (a lower adjusted Ct value correlated with a higher level) miR-141 miR-200c 38 45 36 * 40 * 34 * 35 32 aCt value aCt value 30 30 28 25 26 141cpCt 20 24 200cpCt D C n=50 n=50 n=25 n=50 n=50 n=25 Metastatic versus Control Localized versus Control 22 n=44 n=30 n=21 n=6 n=33 Control Control Localized Localized Metastatic Metastatic DCIS Breast cancer Family Benign Control Sensitivity Sensitivity AUC AUC miR-200c 0.557 miR-200c 0.790 miR-141 0.582 miR-141 0.862 Specificity Specificity DCIS: ductal carcinoma in situ

  22. Plasma levels of miR-200c and miR-141 are elevated in patients with metastatic breast cancer. Blood cells E miR-141 miR-200c 5 4 % RNU6B 3 2 n=10 n=10 n=10 n=10 n=10 n=10 1 Control Control Localized Localized Metastatic Metastatic F Plasma miR-200c miR-141 miR-200c miR-141 miR-200c miR-141 40 35 aCt value 30 25 20 n=29 n=46 n=29 n=46 n=34 n=41 n=34 n=41 n=47 n=28 n=47 n=28 (-) (-) (-) (-) (-) (-) (+) (+) (+) (+) (+) (+) ER PR HER2

  23. Levels of tumor-derived exosomal miR-200c and miR-141 in breast cancer cells FOXP3/GFP Tet off MCF7 cells -Dox 0 1 2 3 5 days FOXP3 ACTIN A miR-200c miR-141 Culture cells * * 8.0 20 * * * 15 6.0 * * * % RNU6B % RNU6B 10 4.0 5 2.0 0 0.0 2 3 5 0 1 Days 2 3 5 0 1 B miR-200c miR-141 Culture medium * * 10 * 30 * 8 * * 6 20 % cel-mir-39 % cel-mir-39 * 4 10 2 0 0 Days 2 3 5 0 1 2 3 5 0 1 miR-200c miR-141 C Exosomes * 4.0 4.0 * * * 3.0 3.0 % cel-mir-39 % cel-mir-39 2.0 2.0 1.0 1.0 0.0 0.0 Days 2 5 0 Days 2 5 0

  24. Levels of miR-200c and miR-141 in Foxp3sf/+ breast cancer mice Blood cells miR-200 cluster 2 (Chr 12) 14 12 10 8 % snoRNA202 6 4 2 0 4 3 Exosomes % cel-mir-39 2 * * 1 * 0 * No n=5 No n=5 Tu n=5 Tu n=5 Meta n=5 Meta n=5 No n=5 No n=5 Tu n=5 Tu n=5 Meta n=5 Meta n=5 Peripheral blood cells miR-200c miR-200c miR-141 miR-141

  25. FOXP3 induces the transcriptional activity of miR-200c and miR-141 in breast cancer cells through KAT2B B Forkhead domain DNA binding core motif RYMAAYA (R=A,G; M=A,C; Y=C,T)) FOXP3 (-) FOXP3 (+) pre-miR-200c-141 cluster miR-200c 1 2 3 4 5 1 2 3 4 5 miR-141 Real-time PCR fragment 12p13.31 0.5 kb TSS -5.0 kb +2.0 kb A -2.4kb -1.3kb +1.2kb -4.5kb -4.2kb +0.23kb +0.7kb D C FOXP3/GFP Tet off MCF7 cells 0 24 48 h FOXP3 FOXP3/GFP Tet off MCF7 cells GFP Tet off MCF7 cells 0 3 KAT2B 0h 24h 48h 25 20 0h 24h 48h * * 20 16 PITX2 0.7 15 12 0.6 ACTIN % GAPDH % GAPDH * * 0.5 * 10 8 0.4 * * Relative Units (%) * 5 4 * 0.3 0.2 0 0 0.1 0.0 0h-Foxp3 0h-IgG 48h-Foxp3 48h-IgG

  26. FOXP3 induces the transcriptional activity of miR-200c and miR-141 in breast cancer cells through KAT2B FOXP3/GFP Tet off MCF7 cells miR-200c miR-141 Relative expression * * * * * * * * * * * * * Scr siR1 siR2 siR1 siR2 Scr siR1 siR2 siR1 siR2 KAT2B PITX2 KAT2B PITX2 4.0 0h 24h 3.0 48h 2.0 1.0 E F 0.0 Scr siR1 siR2 Scr siR1 siR2 KAT2B PITX2 ACTIN ACTIN

  27. Summary • FOXP3 is an X-linked tumor suppressor gene in breast cancer. • FOXP3 as a transcriptional factor regulates two micRNA families, miR-146s and miR-200s. • Plasma miR-200c and 141 are potential biomarkers for early detection of breast cancer metastasis. • MiR-200c and 141 appear to be released from breast cancer cells, through exosomes, into the circulation.

  28. Acknowledgments University of Alabama at Birmingham Department of Genetics Liu’s lab Kenney Zhang Wei Zhang Lisa Wang Tracy Zeng Kevin Gao Courtney M. Dugas Mateus S V Mota Binh Vu Sadhvi Batra Department of Medicine Erica Stringer-Reasor Sejong Bae Dongquan Chen Department of Pathology Shi Wei Selvarangan Ponnazhagan Stanford University Stanley L. Qi Dehua Liu University of Michigan Dr. Pan Zheng Mercer University Wei-Hsiung Yang Funding support: DOD-PC140308 NCI-CA179282 UAB-Comprehensive Cancer Center NCTN –LAPS

  29. Thank You !!!

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