Abstract 4943: Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma
Authors: Hayashi Masamichi , Reis Leonardo O. , Baras Alexander , Maldonado Leonel , Guida Eliza , Izumchenko Evgeny , Brait Mariana , Bivalacqua Trinity , Netto George J. , Koch Wayne , Sidransky David , Hoque Mohammad O. 
Published: August 2015

GULP1 functions as a cytoplasmic adaptor protein in the engulfment of apoptotic corpses and cells. It was also reported to be an adapter protein of LRP1 which is known to be a key regulator of TGF-β signaling. TGF- β signaling pathway is known to contribute to urothelial carcinoma (UC) development by increasing invasiveness and metastasis, and inducing epithelial to mescenchymal (EMT) transition. By an integrated genetic approach, we recently identified GULP1 as a potential tumor suppressor gene and epigenetically silenced in ovarian cancer. In the present study we evaluated the promoter methylation of GULP1 in urothelial carcinoma (UC) and functionally characterize GULP1 in urothelial carcinogenesis.

We first determined GULP1 protein expression by immunohistochemistry on a customized muscle-invasive UC (MIUC) tissue-microarray. The mechanism of gene silencing was probed using quantitative methylation-specific PCR (QMSP). The association between GULP1 expression and promoter methylation was analyzed by using a subset of primary UC and UC cell lines. Effects of forced overexpression and siRNA-mediated GULP1-knockdown were measured by cell proliferation and colony forming assays. Western blot analysis was performed for several key molecules to determine the effect of GULP1 modulation in the respective pathways.

Complete loss of GULP1 expression was observed in 89 out of 104 MIUC (85.6%). In primary tumor tissues and cell lines, promoter methylation of GULP1 were inversely correlated with GULP1 expression and reactivation of GULP1 was observed by 5-aza-dC treatment of UC cell lines. Additionally QMSP assay of GULP1 was performed for 98 primary UC tissues of different cell types and 21 normal urothelial samples. Our initial analysis by using an empiric cut off value revealed GULP1 promoter methylation was significantly associated with UC (P = 0.007, Fisher's exact test). To determine the feasibility of GULP1 QMSP assay for non-invasive detection of UC using urine DNA, we tested urine from 58 UC patients and 46 healthy people. As expected, 33 out of 58(56.9%) UC cases were methylation positive while very low level of methylation were detected in 4 out of 46 urine control samples (8.7%) (P&lt;0.001, Fisher's exact test). Effect of in vitro modulation of GULP1 was determined by MTT assay, BrdU assay and colony formation assay. The results consistently revealed that GULP1 had anti-proliferative activities. In order to detect the potential pathway related to GULP1, RNA from T24 and shGULP1 transfected T24 cells were applied to Cancer pathway finder array (Qiagen). Heme oxygenase 1 (HMOX1) solely showed over 3 fold increase in T24shGULP1 cells. Other associated molecules altered due to GULP1 knock-down include TGF-β RI, p-Smad3C, p15 and LRP1.

In conclusion, GULP1 is a candidate tumor suppressor in UC carcinogenesis and it may have a potential for non-invasive UC detection and targeting therapy.

Citation Format: Masamichi Hayashi, Leonardo O. Reis, Alexander Baras, Leonel Maldonado, Eliza Guida, Evgeny Izumchenko, Mariana Brait, Trinity Bivalacqua, George J. Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2015-4943