Page 7 - CUA2019 Abstracts - Oncology-Bladder

2019 CUA Abstracts

UP-2.6 on migration was assessed in a wound healing assay with and without
Endocrine disruptors and bladder carcinoma: Is bladder cancer PAR1/2 agonism, and in UC cells that had deleted PAR1/2 expression (using
progression impacted by bisphenols? CRISPR), or overexpression (using lentiviral transfection). Invasion was tested
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Ève Pellerin , Stéphane Chabaud , Anthony Kerever , Andrew Z. Leong , with and without PAR1/2 agonism or expression using a transwell assay.
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Frédéric Pouliot , Martin Pelletier , Stéphane J. Bolduc 1 Results: UC cell lines express functional PAR1/2 and secrete proteases and
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1 Surgery, CHU de Québec-Université Laval Research Centre, Québec protease inhibitors that affect signaling. Neither cell line cleave PAR1, nor
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City, QC, Canada; Infectious and Immune Disease, CHU de Québec- 2 via a paracrine mechanism, but demonstrated robust autocrine cleavage
Université Laval Research Centre, Québec City, QC, Canada of PAR1. Activating PAR1/2 and/or increasing expression-induced migration
CRSNG/NSERC and invasion by both cell lines, deleting expression inhibited this behaviour.
Introduction: Bisphenols (BP) are endocrine disruptors used in the pro- Conclusions: Functional PAR1 and 2 are expressed by UC cells.
duction of a broad range of commonly used products. BP-A is found in Increasing expression levels and agonism of these receptors increases
>90% of urine and blood samples, at concentrations susceptible to induce invasion and migration.
substantial effects. BP-A-free alternatives, such as BP-S, may also disrupt References
hormonal pathways. The exposure to BP is linked to cancer progres- 1. Mihara K, Ramachandran R, Saifeddine M, et al. Thrombin-mediated
sion and metastasis, especially in hormono-sensitive cancers. Even if the direct activation of proteinase-activated receptor-2: Another target
bladder is not a hormono-sensitive tissue, the activation of androgen and for thrombin signaling. Mol Pharm 2016;89:606-14. https://doi.
estrogen receptors plays a role in the initiation and progression of bladder org/10.1124/mol.115.102723
cancer (BCa). A hallmark of tumour progression is the alteration of the 2. Ramachandran R, Mihara K, Chung H, et al. Neutrophil elastase
metabolic profile, known as the Warburg effect: a metabolic switch from acts as a biased agonist for proteinase activated receptor 2 (PAR2).
mitochondrial respiration to glycolysis. Due to the chronic exposition of J Biol Chem 2011;286:24638-48. https://doi.org/10.1074/jbc.
the bladder to BP and their metabolites in urine, we hypothesize that the M110.201988
metabolic switch induced by BP in BCa cells is linked to its progression. 3. Mihara K, Ramachandran R, Renaux B, et al. Neutrophil elasease
Methods: Primary normal urothelial cell (UC), non-invasive (RT4) and and proteinase-3 trigger G protein-biased signaling through protein-
invasive (T24) BCa cell lines were used for this study. Evaluation of bio- ase-activated receptor-1 (PAR1). J Biol Chem 2013;288:32979-90.
energetics in real-time of cell culture was established after 24, 48, or https://doi.org/10.1074/jbc.M113.483123
72 hours BP-A or BP-S treatment at various concentrations (vehicle as
a control) using an XF extracellular flux analyzer. Progression of cancer UP-2.8
cell lines after 72 hours BP-A/S treatment was evaluated by measuring The anti-tumour effect of PD-1 blockade is reduced when
cell migration, proliferation, and MMP activities. combined with TIGIT blockade in the MB49 murine bladder
Results: After 24 hours of BP exposure, energy metabolism was dramati- cancer model
cally reduced in BCa cells, followed by an increase in glycolysis at 72 Alain Bergeron , Fanny Gaignier , Yves Fradet 1,2
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hours, indicating a metabolic switch mimicking the Warburg effect. After 1 Axe Oncologie, Centre de Recherche du CHU de Québec-Université
only a 72-hour BP exposition, proliferation of UC was increased to lev- Laval, Québec City, QC, Canada; Department of Surgery, Université
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els of BCa cell lines. At the same time, RT4 cell migration was slightly Laval, Québec City, QC, Canada
enhanced. MMP activities remained roughly unaffected. Introduction: TIGIT is an emerging immune checkpoint (IC) with a prom-
Conclusions: This increased proliferation of UC after BP exposure could ising potential for tumour immunotherapy. However, the role of TIGIT in
mimic a hyperplasic phenotype leading to potential errors in DNA replica- the immunosuppression of the anti-tumour immune response in compari-
tion and mutation accumulation favouring tumour initiation. son to PD-1 is poorly characterized. Expression of TIGIT, PD-1, and CD3
mRNAs is highly correlated in human bladder tumours, suggesting that
UP-2.7 T-cells infiltrating bladder tumours frequently co-express these ICs. The
Urothelial carcinoma cells produce proteinases that can regulate objective of this study was to assess the anti-tumour effect of the inhibition
proteinase-activated receptors (PARs) 1 and 2, which can induce of PD-1, TIGIT, or both in MB49 murine bladder tumours.
migration and invasion in these cells Methods: MB49 tumour cells were injected subcutaneously in C57BL/6
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Stacy de Lima , Koichiro Mihara , Antoine DuFour , Morley D. Hollenberg , mice. Tumour growth was measured twice a week until tumour volume
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M. Eric Hyndman reached 2 cm³ and mice sacrificed. Tumours were dissociated and ana-
1 Inflammation Research Network, Snyder Institute, Physiology and lyzed by multicolour flow cytometry. Expression of PD-1, TIGIT, and their
Pharmacology Department, University of Calgary, Calgary, AB, Canada; ligands were characterized on immune cells. In vivo blockade was real-
2 McCaig Institute for Bone and Joint Health, Physiology and Pharmacology ized by 4 i.p. injections of antagonistic anti-PD-1 and anti-TIGIT anti-
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Department, University of Calgary, Calgary, AB, Canada; Southern Alberta bodies two days apart, starting three days after tumour cell implantation.
Institute of Urology (SAIU), Calgary, AB, Canada Tumour growth was followed as described above.
Introduction: An unmet challenge for the management of urothelial Results: Most T-lymphocytes expressed TIGIT, as 69% of CD8+ and 48%
carcinoma (UC) is distinguishing indolent from aggressive disease. We of CD4+ T-cells were TIGIT+PD-1+. No CD8+ but 18% of CD4+ cells
hypothesize that microenvironment proteases generated both by tumour were TIGIT+PD-1-. Regarding the expression of their ligands, 56% of
and parenchymal cells play a critical role in determining disease aggres- CD11b+ and 39% of CD103+ dendritic cells were PD-L1+CD155+. In
siveness by dictating cell invasion and metastasis through oncogenic vivo inhibition of TIGIT slightly reduced tumour growth, whereas PD-1
signaling via proteinase-activated receptors (PAR) and explored this by blockade resulted in the cure of 40% of mice. Dual TIGIT and PD-1
evaluating PAR function and protease-secreting properties of UC cell lines. inhibition induced a reduction in tumour growth that was less important
Methods: UC-derived cell lines used included: T24 & HTB9. qPCR vali- than the one observed with PD-1 blockade alone, as no mice were cured
dated PAR1/2 expression. PAR1/2 function was tested in a calcium assay. by the combined treatment.
Proteomic analysis identified cell-secreted protease and inhibitors in UC Conclusions: TIGIT and PD-1, as well as their ligands, are frequently co-
supernatants (SN) and activity was confirmed with enzyme family-selec- expressed on immune cells infiltrating MB49 tumours. TIGIT blockade
tive chromogenic substrates. Two methods were used for assessment of improved the survival of mice but much less than PD-1 blockade. When
UC-secreted proteases that cleave PAR1/2: 1) An N-luciferase tag fused to combined, TIGIT inhibition reduced the anti-tumour effect of PD-1 inhi-
PAR1/2 in a non-UC cell line is released upon cleavage to determine para- bition. Further analysis is needed to understand the interaction between
crine activity; 2) an N-terminal mCherry/RFP;C-terminal eYFP construct PD-1 and TIGIT pathways.
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was transfected into UC cells to visualize receptor status and autocrine
cleavage (intact receptor=yellow; cleaved receptor=green). PAR1/2 effect
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S94 CUAJ • June 2019 • Volume 13, Issue 6(Suppl5)