Integrin β4 synergizes with E-and P-selectin to regulate tumor growth: Unravelling the molecular mechanisms

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Sandra Hanika
Vera Labitzky
Florian Gebauer
Daniel Wicklein
Hanna Maar
Tobias Gosau
Jennifer Schröder-Schwarz
Stefan Linder
Kristoffer Riecken
Udo Schumacher
Tobias Lange

Abstract

Purpose/Objectives: Metastases are responsible for 90% of cancer-related deaths. Scientific evidence has accumulated showing that adhesion molecules including selectins and integrins play a crucial role in the process of cancer metastasis. In particular, upregulation of integrin β4 (ITGB4) is associated with tumor progression in various tumor entities. Thus, we aimed at detecting a potential crosstalk between selectins and integrins in tumor formation and metastatic spread.


Material/Methods: Knockdown of ITGB4 in PC3 (prostate cancer), PaCa5061 (pancreatic cancer) and SKOV3 (ovarian cancer) cells using shRNA, proliferation and colony forming assays, ELISA, WB, 3D chemotaxis assays with human macrophages, tumor initiation assays in vivo, immunohistochemistry


Results: Knockdown of ITGB4 affected prostatic PC3, pancreatic PaCa5061 and ovarian SKOV3 cancer xenografts already at the primary tumor site. In particular, tumor growth was
strongly inhibited when the ITGB4-knockdown was combined with E-/P-selectin deficiency of mice while selectin-knockout alone had no effect. In this study we wanted to investigate the molecular mechanisms underlying this synergistic growth retardation. Delayed tumor growth was associated with increased apoptosis. Immunostainings further revealed an enhanced immune cell infiltration in ITGB4-knockdown tumors grown in immunodeficient mice. On the contrary, leukocytes were mainly located at the outer tumor periphery of both ITGB4-knockdown and control tumors in E-/P-selectin-knockout mice. The increased leukocyte infiltration in ITGB4-knockdown tumors in control mice seemed to result from a greater chemotactic potential of ITGB4-knockdown cells. CCL2, 5 and 20 were identified as potential chemotactic factors upregulated upon ITGB4-knockdown in PaCa5061 and SKOV3 cells while the chemoattractant playing a role in ITGB4-knockdown PC3 cells remains to be determined.


Conclusion: Taken together, our results suggest that ITGB4-knockdown tumor initiation depends on pro-tumoral signals from attracted tumor-associated leukocytes. These leukocytes appear to depend on endothelial selectins for infiltration of the tumor stroma. Investigation of how leukocytes actually stabilize the survival of ITGB4-knockdown xenografts is the subject of ongoing experiments.

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Section
Section 8: Immunomodulation / Tumor Biology