Abstract 4052: Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress.
Authors: You Min , Chen Shu-Mei , Wu Chunjing , Li Ying-Ying , Feun Lynn , Wangpaithitr Medhi , Dinh Vy , Savaraj Niramol 
Published: August 2014

Abstract
We have previously shown that primary brain tumor from patients and cell lines possess type I alpha 1 procollagen. Procollagen type I, III, and IV have also been reported to be expressed in certain glioma subtypes. Although the function of these procollagens is not known, they are most likely involved in invasion and signaling. We have further investigated whether manipulation of the secretion of these procollagens can be used to kill glioma cells. We have studied the expression of procollagen type I alpha 1 and2 (Pro-col1A1 & A2), procollagen type III alpha 1 (Pro-col3A1) and procollagen type IV alpha 1&2 (Proc-col4A1 & A2) in 5 cell lines (Glioma-1, U-373, U-118, U-87, and A-172) using real time RT-PCR (qPCR) and western blot when antibody was available. Our data showed that A-172 expressed all 5 types of procollagen, while U-87, U-118 and Glioma-1 expressed primary Pro-colA1 and A2, and U-373 expressed only pro-col1A2 and pro-col4A1 and A2. Hsp47, which is a major chaperone in the folding of procollagen type I, is not expressed in U-373. This cell line also possessed very high levels of GRP-78 which is indicative of increased ER stress. We also investigated the presence of collagen receptor Endo 180 and DDR1 (discodin domain receptor 1) in these cell lines. Endo 180 was present in all 5 cell lines, but to a much lesser extent in U-373. The DDR1 expression by qPCR was the highest in A-172 followed by U-118 (&gt;5 arbitrary units) while U-373, U-87 and Glioma-1 have less than 3 arbitrary units. It is known that procollagen needs to be folded at ER and then secreted out of the cells, where it will be processed to form collagen. Collagen can then bind DDR1, a receptor tyrosine kinase which plays a role in attachment, migration, proliferation and survival. In this regard, we have detected pro-col1A1 and A2 in the media which verified that pro-col1A1 and A2 are secreted outside the cells. We hypothesize that inability to secrete procollagen will result in both ER stress and inability for signaling. To accomplish this, we have studied the cytotoxic effect of brefeldin (BFA) which is known to block the transport of secretory protein from ER/Golgi network to the cell surface in these five cell lines. All five cell lines are very sensitive to brefeldin with IC50 of 3.0 ng/ml for U-118, A-172 and U-87. In U-373 and Glioma-1 the IC50 was 5.0 ng/ml. Interestingly, cancer stem cells isolated from U-87 did not possess pro-co1A1, but DDR1 mRNA was present and was 3 times higher than in the non stem cells. They also have different metabolic profiling than their parental cells, such as they do not express argininosuccinate synthetase, a key enzyme to synthesize arginine which made them sensitive to arginine deprivation treatment. Whether these differences exist in vivo is not known. Overall, our data indicate that the expression of procollagen and its secretory pathway can be used as a new paradigm to treat primary brain tumor through ER stress-mediated cell death.
Citation Format: Min You, Shu-Mei Chen, Chunjing Wu, Ying-Ying Li, Lynn Feun, Medhi Wangpaithitr, Vy Dinh, Niramol Savaraj. Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress. [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 4052. doi:10.1158/1538-7445.AM2013-4052