Abstract 3836: A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer.

Abstract

Background: Pancreatic Ductal Adenocarcinoma (PDA) ranks fourth among cancer-related deaths in the United States. The poor prognosis of PDA is due to resistance to current therapies. Contributing to this resistance is the tumor architecture comprising dense desmoplastic stroma, extracellular matrix (ECM), tumor cells and tumor hypoxia. We hypothesized that destabilizing the compact tumor architecture by weakening the interactions between tumor cells alone or tumor cells along with the ECM may result in more effective therapies for PDA. Methods and Results: Using the pancreatic cell line Panc1, we developed a three dimensional (3D) spheroid model system, that exhibits oxygen gradients and hypoxia that mimics the tumor microenvironment. We used the pancreatic cancer cell line PANC1-HRE that stably expresses HRE-luciferase and detects HIF-1α activation, and a platform comprising a non-matrix nano-structured scaffold, to generate compact spheroids with hypoxic inner cores and HIF-1α activation. A high throughput siRNA screen using the kinome siRNA library and our 3D spheroid modal system identified kinases whose silencing reduced both the level of hypoxia and activity of HIF-1α. The positive control, siRNA against HIF-1α, reduced activity of HIF-1α with no effect on the levels of hypoxia within the spheroids. We identified the kinases Toll-like receptor 4 (TLR4) and its downstream target spleen tyrosine kinase (SYK) as our lead candidates following target validation. Image analysis of spheroids transfected with siRNA against both TLR4 and SYK resulted in the loss of compactness of the spheroids. In addition, these spheroids also demonstrated a significant reduction in the levels of hypoxia determined by the hypoxia probe Lox-1. These findings suggest that the alteration of the spheroid architecture resulted in the reduction in hypoxia. Previous studies have shown that SYK is a direct target of TLR4. Therefore to determine the mechanism through which TLR4 dependent signaling regulates the integrity of the spheroidal architecture we are generating stable cell lines in Panc1 of shRNA targeting TLR4 under an inducible system. We will use our inducible shRNA targeting TLR4 and the two 3D model systems, the hanging drop system (analogous to a primary tumor) and the nanoculture plate system (analogous to the progression of a metastatic loci) to further confirm our findings. Conclusion: TLR4 mediated signaling regulates the integrity of spheroid architecture. Inhibiting this pathway destabilized the tumor architecture altering the hypoxic state of the spheroid. Our long-term goals are to use known pharmacological inhibitors of TLR4 to study the mechanisms by which this kinase regulates tumor architecture, determine the pre-clinical significance of targeting TLR4 utilizing autochtonous mouse models for PDA and develop TLR4-targeted therapy for PDA. Citation Format: Geoffrey A. Bartholomeusz, Alex Campos, Geoffrey Grandjean, Garth Powis. A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2013-3836