The Role of Toll Like Receptor 9 (TLR9) in Breast Cancer

The role of Toll Like Receptor 9 (TLR9) in breast cancer

Uzma Hasan (CIRI Lyon) & Nathalie Bendriss-Vermare (CRCL, Lyon)

1 Immunity and Cell death

FACTS on TLR9 TLR9 dsDNA

Highly expressed on immune cells Cellular (pDC and B cells human) transformation Weakly expressed on epithelial cells (skin and cervix human)

Immune system Activated by dsDNA

Breast cancer1 Expressed in the ER - shifts to endosome

1R3 exhibition Melanie Planche and Floriane Desseigne www. R³ air / aire / ère TLR9 pivotal in immune responses and cell cycle control

Oncogenic stress 5, 6, 7, 8 TLR9

Immune response 1, 2, 3 ,4 Type I IFN by pDC

1 Kadowaki, N. et al. J. Exp. Med 2001 5 Proliferation, Colorectal cancer, Si Ming Man et al., Cell 2015 2 Krug A et al., Eur J Immunol 2001 6 Senescence, Gluck et al., Nature Cell Biology 2017 3 Lund J et al., J Exp Med 2003 7 Proliferation, Pacini et al., J Virology 2015 8 4 Lande et al, Nature 2007 p16 up regulation, Parroche et al., Oncogenesis 20163

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Question Content Role of TLR9 in Breast Cancer? may be subject to Methods copyright. 5 WPs TLR9 tumor Team 1 UH KCL Objective 1. TLR9 expression Cohort (Kings College London)

Objective 2. Mechanism BC human in vitro and ex vivo models

Objective 3. Mechanism BC murine in vivo models

TLR9 pDC TEAM 2 NVB TEAM 3 OT

Detection of miRNA-210 expression in triple- negative breast cancer (TNBC) by in situ hybridization (ISH). A-D: miR-210 expression is detected in tumor cells. C-F. Strong miR-210 expression is detected in the tumor microenvironment. (Scale bar: 50 µm.) Abbreviations: Tu, tumor; TME, tumor microenvironment.

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Content may be subject tTLR9o in BC tumour cells copyright.

TLR9 tumor Team 1 UH KCL

TLR9 pDC TEAM 2 NVB TEAM 3 OT

Source publication -TLR9 staining on FFPE tissue by Gregory Weitsmann, scoring by Trupti Pai Guys and KCL on breast and BC tissue. Stats by Ommran Allatif CIRI

Objective 1. TLR9 expression RESULTS

Cohort (Kings College London) Normal tissue score =1

x20 zoom

DCIS /Invasive tissue =0

-TLR9 staining on FFPE tissue by Gregory Weitsmann, scoring by Trupti Pai Guys and KCL on breast and BC tissue. Stats by Ommran Allatif CIRI

EXPRESSION TLR9?

30 sections 30 sections 220 TMA -TLR9 staining on FFPE tissue by Gregory Weitsmann, scoring by Trupti Pai Guys and KCL on breast and BC tissue. Stats by Ommran Allatif CIRI

TLR9 expression is blocked in several virus-induced cancers

HPV161 2 3 6 4 5 HCV HPV38 HBV MCPyV EBV 1. Hasan UA et al.. J Exp Med 2013 2. Fischer J et al. Gut, 2017 3. Pacini L et al. J Virol, 2015b TLR9 4. Shahzad N et al.. J Virol, 2013 promoter NF-κB (B) NF-κB (C) CRE C/EBPβ NF-κB (D) 5. Fathallah I et al. J Immunol, 2010 6. Tout et al. JI 2018 -3327 bp -1237 -1024 -679 -620 -403 TSS

Ex vivo In vitro

6 RESULTS HMEL_breast

5,5 Immortalized cell lines

(n=8) 5 Breast cancer cell lines (n=59) 4,5

4 TLR9 expression (Log2) expressionTLR9

3,5

3 http://www.broadinstitute.org/ccle/home http://brainarray.mbni.med.umich.edu/Brainarray/Database/ T2.1 HMEC: Does TLR9 is regulated during senescence ?

Objective 2. Mechanism + BC human in vitro models T2.1 HMECT2.1: HMDoes EHMECC hT ETLRT RR9:as Does is regul TLR9ate ids rdurinegulateg sdene durinscencg seene ? scence ?

TLR9 TLR7

+ +

HMEC hTERT RasHM EC hTERT Ras

• Proliferation: colony assay Breast cancer cell line T2.1 - T2.2 • SASP (Senescence-Associated Secretory Phenotype )

Breast cancerBrea celstl canlinecer cell line • Tumour suppressor T2.1 - T2.2T 2.1 - T2.2

8 T2.1 HMEC: Does TLR9 is regulated during senescence ? RESULTS:Re Proliferationsults : Cell gisro blockedwth by TLR9 in breast cancerModel :cells

TLR9 TLR7MDA B361 cells (same results for MDA231) +

HMEC hTERT Ras

Results : Cell growth Breast cancer cell line Model : N=4 T2.1 -MDA T2.2B361 cells (same results for MDA231) TLR9

N=4

TLR9 reduce colony formation in BC cell lines

TLR9 reduce colony formation in BC cell lines T2.1 HMEC: Does TLR9 is regulated during senescence ? Sénescence cellulaire : Detection de Results Results cytokines

RESULTS: TLR9 induces tumour suppressorsCell senescence: and SASP Cyto kine detection Expression of tumour suppressors+ Results

DNA damage, hypoxia, cellular stress, oncogene, radiation, other factors ...HM EC hTERT Ras Results: SASP factors are upregulated when TLR9 is - +. DOX expressed Confirm results SASP factors A Confirm results p53 CXCL-1 CXCL-1 MA characteristic of 5% CO2 à MAp53 INK4a p16 senescence: 37°C p16 IL-6, IL-8 + dox CXCL-1 TLR93 + j ours TLR7 + IL-6 IL-8 IL-6 IL-8 actin (Chien Y et al., Confirm results 2011) MA Cytokines p21CIP CDK dependent VE VE GF GF Lignée Lignée Statut Statut cellulair Statut Statut PR Statut Triple négatif cellulair ER Statut PR HER2 Triple négatif e ER HER2 e 100 MDA Breast cancer cell line MDA - - - OUI

MB 231 - - - OUI 80

6 6

MB 231 -

paper - L

Nanostring: L

60 T2.1 - T2.2 m

Sebastien Viel m

cell N

Stop cell N 40 R

Lignée senescence R A Statut Statut cycle A cellulair Statut PR Triple négatif 20 ER HER2 e 0 MDA - - - OUI TLR7+ TLR9+ MB 231

b-gal

10 TLR7 x3 TLR9 x3 CONCLUSIONS and next steps

Add top regulators for tlr9 increased Tlr9 down regulated

TLR9 tumour cells

IL-6 • Senescence

• Slow down in cell cycle GOTERM _BP_DIR ECT cell proliferation negative IL-8 GOTERM regulation of _BP_DIR ECT cell division TLR7 x3 TLR9 x3

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Content may be subject toTLR9 immunity in BC copyright.

TLR9 tumor Team 1 UH KCL

TLR9 pDC TEAM 2 NVB TEAM 3 OT

Source publication pDC are specialized in antiviral responses via type I IFN production

Steady state Activated Resting pDC pDC Microbial stimulation

T T T T L L IFN-I L L R R R R 7 9 7 9 MyD88 early Interferon- IRF7 Stimulated Genes late NFkB MAPK pDC TNF

13 pDC at the center of an immunosuppressive microenvironment in breast and ovarian cancers

Elimination Premalignant Immuno- lesion surveillance

Tumor Advanced Equilibrium Immuno- Inhibitory and non-IFN-I cells oncogenesis selection inducing endogenous activating stimuli

Any role for pDC and type I IFNs? neutrophil ? Elimination Tumor cells Premalignant Immuno- lesion surveillance IFN-α pDC

IFN-α producing TApDC

Tumor Advanced Equilibrium Immuno- Inhibitory and non-IFN-I cells oncogenesis selection inducing endogenous activating stimuli

IFN-α pDC Escape Treg Tumor Immuno- expansion growth subversion Altered TApDC Th2 differentiation Labidi-Galy et al, Can Res 2011 Sisirak et al, Can Res 2012 Faget, Can Res 2012 Sisirak et al, Int J Cancer 2013 Ghirelli et al, Can Res 2015 Do pDC play a dual role in breast cancer immunity ? What is the role for TLR9 ?

Any role for pDC and type I IFNs? neutrophil ? Elimination Tumor cells Premalignant Immuno- lesion surveillance IFN-α pDC

IFN-α producing TApDC

Tumor Advanced Equilibrium Immuno- TLR9 ? Inhibitory and non-IFN-I cells oncogenesis selection inducing endogenous activating stimuli

IFN-α pDC Escape Treg Tumor Immuno- expansion growth subversion Altered TApDC Th2 differentiation Labidi-Galy et al, Can Res 2011 Sisirak et al, Can Res 2012 Faget, Can Res 2012 Sisirak et al, Int J Cancer 2013 Ghirelli et al, Can Res 2015 Objective 1. TLR9 is strongly expressed in the TLR9 expression Cohort (Kings College London and CLB, Lyon) microenvironment of human breast tumors Tonsil (positive control) Normal mammary tissue TLR9 TLR9

o c

s 2

x20 x20 +

9 1

R L Peritumoral tissue Invasive tumor T 0

e e r TLR9 x20 TLR9 u u o s s s m i is u t t t l l a a e r v m i r o s o m a n u v it in r e p

x20

17 Vey, Mussard et al, in preparation Do pDC play a dual role in breast cancer immunity ? What is the role for TLR9 ?

Elimination Premalignant Immuno- lesion surveillance

Tumor Advanced Equilibrium Immuno- TLR9 ? Inhibitory and non-IFN-I cells oncogenesis selection inducing endogenous activating stimuli

IFN-α pDC Escape Treg Tumor Immuno- expansion growth subversion Altered TApDC Th2 differentiation Labidi-Galy et al, Can Res 2011 Sisirak et al, Can Res 2012 Faget, Can Res 2012 Sisirak et al, Int J Cancer 2013 Ghirelli et al, Can Res 2015 Objective 2. BAD-LAMP controls TLR9 trafficking and Mechanism BC human in vitro and ex vivo models signaling in human pDC

BAD-LAMP pDC

Unstim. CpG-A 24h

Single cells from Villani et al. 2017 Combes et al. Nature Comm. 2017

BAD-LAMP & TLR9 NS 1h CpGA 24h CpGA BAD-LAMP BAD-LAMP downmodulation overexpression

IFN TNF IFN TNF

19 Combes et al, Nature Communications 2017 Objective 2. BAD-LAMP expression is enhanced in Mechanism breast tumor pDCs and its downregulation BC human in vitro and ex vivo models is prevented by inhibitory tumor supernatants

A Blood patients’ pDC Breast Tumor pDC

20 Combes et al, Nature Communications 2017 Do pDC play a dual role in breast cancer immunity ? What is the role for TLR9 ?

Any role for pDC and type I IFNs? neutrophil ? Elimination Tumor cells Premalignant Immuno- lesion surveillance IFN-α pDC

IFN-α producing TApDC

Advanced Equilibrium Immuno- TLR9 ? oncogenesis selection

Escape Tumor Immuno- growth subversion Objective 2. Type 1 IFN pathway contributes to breast Mechanism tumor immunosurveillance in mice BC human in vitro and ex vivo models A B C

22 Vey, Mussard et al, in preparation Objective 2. Evidence for the activation of type I IFN Mechanism pathway in human breast tumors BC human in vitro and ex vivo models

Invasive tumor Invasive tumor CD15/MxA/BDCA2 CD15/MxA/TLR9

IFN-a MxA

23 Vey, Mussard et al, in preparation Objective 2. Endogenous TLR agonists are present in Mechanism breast tumors and are able to activate or BC human in vitro and ex vivo models potentiate pDC activation in vitro

LL37 HMGB1

Breast tumor Breast normal Breast tumor Breast normal 24 Conclusion and next steps

• Further characterize the intratumor endogenous TLR ligands - Mitochondrial DNA vs Genomic DNA - Demonstrate the role of TLR

• Further characterize immune infiltrate and immune pathways (TLR9, IFN) dominating in early breast cancers versus invasive breast cancers in patients

• Demonstrate the role of pDC and neutrophils in breast tumor immunosurveillance in vivo by depleting experiments

• Establish a new model of spontaneous mammary tumor model in B6 mice (BRCA122-24, p53null - Drost RM et al, Jonkers Lab. Br J Cancer 2009)

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Content Joint conclusion may be subject to TLR9 tumorco pyright. TLR9 TME Team 1 UH TLR9 in BC TEAM 2 NVB KCL TEAM 3 OT

Strong Expression expression in is lost tumor pDC tumour cells

Activation Slows down blocked by proliferation BAD-LAMP

Induces Endogenous senesence ligands are present in BC

Source publication

Christophe Caux Philippe Pierre Tony Ng Nelly Vey Evelina Gatti Greg Weitsmann Maire Marotel Julie Mussard Alexis Combes Trupti Pai Michelle Ainouze Aurélien Voissière Cheryl Gillet Guillaume Roblot Natalie Woodman Kyohei Yamanaka CLB Salome Amouyal Omran Allatif Clinicians and patients Biological ressources center Platforms

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Confirmed Invited Speakers Thematic sessions

KEYNOTE LECTURES 1.Innate Immunity Pr. Harald Zur Hausen, Germany 2.Immuno-metabolism Pr. Ruslan Medzhitov, USA 3.Immunotherapy and clinics 4.Microbiota Ido Amit, Israel Eran Elinav, Israel Dan Littman, USA 5.Genetics and Epigenetics Esteban Ballestar, Spain Nelson Gekara, Sweden Mala Maini, UK Antonio Bertoletti, Singapore Thomas Gajewski, USA Frederica M-Berg, UK 6.Oncopathogens and immune responses Menna Clatworthy, UK Ping-Chih Ho, Switzerland Anne Puel, France Julie Dechanet-Merville, France Jonathan Kagan, USA John Wherry, USA Sandra Diebold, UK Mansun Law, USA 28 To register go to: www.irci2020.insight-outside.fr