Login to Access Video or Poster Abstract: MP98-13
Sources of Funding: Wilmot Foundation Cancer Research Fellowship

Introduction

_x000D_ Previously we identified the long non-coding RNA Hox antisense intergenic transcript (HOTAIR) enriched in urothelial bladder cancer (UBC) cell lines, extracellular vesicles (ECVs), patient tumors and urinary ECVs. Importantly, HOTAIR affects the expression of genes involved in epithelial-to-mesenchyme transition (EMT). Critically, we found reduced HOTAIR expression correlates with decreased in vitro migration and invasion. Many of the genes affected by HOTAIR expression are in the canonical Wnt-pathway. HOTAIR is a known to facilitate EMT through the canonical Wnt-pathway in other tumors. Determining the importance of the Wnt-pathway in UBC may open up new treatment options. Here we show that HOTAIR is necessary for Wnt-responsiveness and its expression increases during Wnt-pathway activation. EMT is also regulated through intercellular communication mediated by ECVs. Given HOTAIR regulates thousands of genes, we hypothesized that ECVs from HOTAIR knockdown cells would have limited ability to facilitate EMT. In fact, HOTAIR knockdown cells produce fewer exosomes with altered protein cargo and do not facilitate migration or invasion, suggesting that targeting of HOTAIR therapeutically would affect EMT through both the Wnt-pathway and ECVs functionality. _x000D_ _x000D_ Objective: _x000D_ To evaluate the role of HOTAIR in WNT-mediated and EVC-mediated EMT_x000D_

Methods

_x000D_ UBCs treated with LiCl or rWNT and gene expression was analyzed by qRTPCR, western blot and immunohistochemistry. We used scratch and 3D spheroid invasion assays to measure in vitro EMT in rWNT treated or untreated UBC cells. shRNA or siRNA against HOTAIR were used and WNT target and antagonist gene expression was measured by qRT-PCR. Migration and invasion were measured using scratch wound assay and 3D spheroid assay. TCF7L2 binding sites were identified in the promoter region of HOTAIR by sequencing. siRNA against TCF7L2 or beta-catenin reduced HOTAIR expression. ECVs isolatedd by ultracentrifugation and sucrose gradient were analyzed using the Nanosight. ECVs protein analysis was performed with LC MS/MS mass spectrometry and western blot. EVC-mediated migration and invasion was evaluated by wound and 3D invasion assay. _x000D_

Results

_x000D_ TCGA data reveals WNT pathway genes are affected in human UBC. LiCl or rWNT treated UBCs have increased EMT related gene expression. rWnt facilitates UBC in vitro migration and invasion in a HOTAIR-dependent fashion. Reduced HOTAIR expression correlates with decreased WNT-target and increased WNT-antagonist gene expression. Importantly, HOTAIR is a target of canonical WNT signaling. Reduced HOTAIR expression affects UBC EVC number, content and in vitro migration and invasion._x000D_

Conclusions

_x000D_ These data support a role for the canonical WNT-pathway in UBC in a manner dependent on HOTAIR expression. Therefore, therapeutic targeting of the WNT-pathway may affect UBC tumor progression through reduced HOTAIR expression. Importantly, loss of HOTAIR affects the expression of hundreds of genes that results in reduced ECVs number, content, and ability to affect in vitro migration and invasion. _x000D_

Funding

Wilmot Foundation Cancer Research Fellowship