NOVEL ROLE FOR WNT1 IN LUNG CANCER ESCAPE FROM IMMUNE SURVEILLANCE MECHANISMS. PRELIMINARY RESULTS.

DIMITRA KERDIDANI, VASO KARAVANA, SOFIA MAGKOUTA, AGGELIKI MELIGOVA, CHARIS ROUSSOS, SPYROS ZAKYNTHINOS, IOANNIS KALOMENIDIS, MARIA TSOUMAKIDOU.

CENTRE FOR APPLIED BIOMEDICAL RESEARCH AND TRAINING “MARIANTHE SIMOS”, FIRST DEPARTMENT OF INTENSIVE CARE MEDICINE, EVAGGELISMOS HOSPITAL

LUNG CANCER BURDEN

Lung cancer comprises 14% of cancer diagnoses/30% of cancer deaths.

There are more deaths due to lung cancer than breast, prostate, and colon cancers combined.

1.61 million lung cancer diagnoses in 2012 / 1.38 million deaths.

For all stages combined, the five-year survival rate is 16% for NSCLC and 6% for SCLC.

Jemal A, et al. CA Cancer J

Clinic 2010.

THE CANCER IMMUNOEDITING

HYPOTHESIS

Cancer cells use immune escape mechanisms to evade immunosurveillance.

Such immunosuppressive strategies can be: preexisting arise through outgrowth of escape mutants take place during tumor-sculpting actions by the

immune system

Vesely MD, et al. Annu Rev Immunol; 2011.

THE CANCER IMMUNOEDITING HYPOTHESIS

Vesely MD, et al. Annu Rev

Immunol; 2011.

LUNG CANCER IMMUNE ESCAPE MECHANISMS

Fas ligation induces lung cancer cells to produce PGE2, which attracts Myeloid-Derived Suppressor Cells (Zhang Y, et al. J Immunol 2009).

Lung cancer cell-derived TGF-β and PGE2 drive the generation of IL-10, arginase I-producing regulatory DCs. (Qiuyan Liu, et al. J Immunol ;2009).

Lung cancer-derived galectin-1 endows dendritic cells with immunosuppressive properties (Kuo P, et al. J Immunol; 2011).

URGENT NEED TO IDENTIFY ALTERNATIVE PATHWAYS

THE WNT SIGNALING PATHWAY

Sen M, et al. J Immunol; 2008.

KNOWN FUNCTIONS OF THE CANONICAL WNT PATHWAY IN

CANCER

Canonical Wnt signalling pathway facilitates cancer progression by regulating:

tumor growth cell senescence cell death cell differentiation metastasis

Polakis P, et al. EMBO; 2012.

Alteration of Wnt pathway components in non–small cell

lung cancer

Increased expression

Decreased expression or inhibition of function

Wnt-1Wnt-2Wnt-3Wnt-5aWnt-11

β-CateninFrizzled-8

Dishevelled-1Dishevelled-2Dishevelled-3

PorcupineTCF-4

Pygopus 2

Wnt-7aAXIN

sFRP-1sFRP-2sFRP-4sFRP-5WIF-1Dkk-1Dkk-3

RUNX3APC

CDX2DACT2EMX2

WNT1 AND LUNG CANCER PATIENT OUTCOMES

Wnt1 overexpression associated with tumor proliferation and a poor prognosis in non-small cell lung cancer patients. Oncol Rep. 2008;19(1):203–209.

Aberrant Wnt1/beta-catenin expression is an independent poor prognostic marker of non-small cell lung cancer after surgery. J Thorac Oncol. 2011;6(4):716–724.

Wnt1 overexpression promotes tumor progression in non-small cell lung cancer. Eur J Cancer. 2008;44(17):2680–2688.

Wnt1 pathway activation predicts increased risk of tumor recurrence in patients with stage I nonsmall cell lung cancer. Ann Surg. 2013;257(3):548–554.

Canonical and noncanonical Wnt proteins program dendritic cell responses for tolerance. J Immunol. 2013;190(12):6126-34.

Activation of beta-catenin in dendritic cells regulates immunity versus tolerance in the intestine. Science. 2010;329(5993):849-53.

β-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10. Proc Natl Acad Sci U S A. 2015;112(9):2823-8.

NOVEL ROLES OF THE CANONICAL WNT PATHWAY IN THE INDUCTION OF

IMMUNE TOLERANCE

The Wnt/β-catenin pathway attenuates experimental allergic airway disease. J Immunol. 2014;193(2):485-95.

TLR2-dependent activation of β-catenin pathway in dendritic cells induces regulatory responses and attenuates autoimmune inflammation. Manoharan I, Hong Y, Suryawanshi A, Angus-Hill ML, Sun Z, Mellor AL, Munn DH, Manicassamy S. J Immunol. 2014;193(8):4203-13.

Canonical Wnt signaling in dendritic cells regulates Th1/Th17 responses and suppresses autoimmune neuroinflammation. J Immunol; 2015 Feb 20. [Epub ahead of print]

THE CANONICAL WNT PATHWAY IN

IMMUNOLOGICAL DISEASES

β-catenin-mediates tumor-induced immunosupression by inhibiting cross-priming of CD8+ T ells. Journal of Leukocyte Biology. 2014;95:179-190.

β-Catenin promotes regulatory T-cell responses in tumors by inducing vitamin A metabolism in dendritic cells. Cancer Res. 2015;75(4):656-65.

WHICH FACTOR(S) IN TUMORS ACTIVATE THE CANONICAL Wnt PATHWAY IN DENDRITIC CELLS IS CURRENTLY NOT KNOWN.

NOVEL IMMUNE FUNCTIONS OF THE CANONICAL WNT

PATHWAY IN CANCER

Lung cancer cell-derived Wnt1 acts paracrine to suppress anti-tumor T cell responses and is a critical mechanism of lung cancer escape from immune surveillance.

HYPOTHESIS

HYPOTHESISWNT1-INDUCED SUPPRESSION IN THE

TUMOR MICROENVIRONMENT

Wnt1

Constitutive Wnt1 expression was assessed in Lewis lung carcinoma (LLC) cells by Immunoblot.

Wnt1-deficient LLC cells were obtained by stably transfecting cells with Wnt1-silencing shRNA.

Control (vector-transfected) and Wnt1-silenced LLC cells (LLC and shLLC) were engrafted in the left lung lobe of syngeneic C57BL/6 mice.

T cell responses were assessed on day 10.

METHODS

PICTURE OF MICE

IMMUNOBLOT AND RT-PCR

scramble shwnt10

20

40

60

80

100

120

RT-PCR

wnt1

WESTERN BLOT

Scr

am

bl

e shw

nt1

wnt1

β-actin

gated on total live cells (sytox-green –ve)

Scrambletransfected

ShWnt1 transfected

CD3+ T CELL NUMBERS (% total lung cells)

S CD39bcom.FCS

FSC

FL4

0 1024 2048 3072 409610

0

101

102

103

104

18.5%

W CD39bcom.FCS

FSC

FL4

0 1024 2048 3072 409610

0

101

102

103

104

4.86%

CD

3

FSC

CD

39

CD73

gated on CD45+CD3+CD4+ T cellsgated on CD45+CD3+CD8+ T cells

Scrambletransfected

ShWnt1 transfected

W CD39_014.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

7.93% 1.50%

87.25% 3.32%

W CD39_014.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

4.11% 0.66%

92.47% 2.76%

S CD39.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

4.78% 1.15%

88.99% 5.08%

S CD39.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

2.63% 0.47%

94.94% 1.97%

ECTONUCLEOTIDASE EXPRESSION (% lung T cells)

IFN

-γ

IL-10

Scrambletransfected

ShWnt1 transfected

EXPRESSION OF IL-10 and IFN-γ (% lung T cells)

gated on CD45+CD3+CD4+ T cellsgated on CD45+CD3+CD8+ T cells

T5 D6.1.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

8.79% 1.20%

71.96% 18.05%

T5 D6.1.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

2.76% 0.42%

91.89% 4.93%

T5 E8.1.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

1.73% 0.33%

72.24% 25.70%

T5 E8.1.FCS compensated

FL7

FL2

100

101

102

103

104

100

101

102

103

104

0.18% 0.07%

78.42% 21.33%

CONCLUSION

Silencing Wnt1

increases the numbers of T cells that infiltrate lung tumors in vivo

lowers expression of the ectonucleotidase CD39 and of the immunosuppressive cytokine IL-10 by intratumoral T cells

up-regulates IFNγ expression by intratumoral T cells

• Targeting Wnt1 may act simultaneously on both cancer and immune cells at key regulatory points in the cancer-immune network.

• Targeting this shared signaling pathways in combination with conventional chemotherapy may be a promising strategy for cancer treatment.

• Sustained targeting of Wnt1 after initial surgery, drug therapy and/or radiotherapy might enhance the ability of the immune system to achieve sterilizing immunity.

IMPLICATIONS

THANK YOU FOR YOUR ATTENTION!