Abstract 1265: LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target

Abstract

Abstract A combination of poor vasculature perfusion, hypoxia, and increased flux of carbons through fermentative glycolysis lead to extracellular acidosis in solid tumors; with extracellular pH values as low as 6.5. The proton concentration increases within the lumen due to diffusion limitations and increased production of acid from hypoxic-glycolytic cells, causing the interior of the lumen to become highly acidic. The glycolytic phenotype can become “hardwired” as Warburg proposed, leading to the continued generation of metabolic acids even in well-oxygenated conditions. This acidified habitat is constant and imparts a Darwinian selection pressure that favors cells that have adapted mechanisms to be resistant to acid mediated apoptosis, and this likely increases with increasing cancer stage. In order to understand tumor acidity, we investigated protein profiles of acid-adapted and non-adapted breast cancer cells (MCF-7) using SILAC-proteomics followed by LC-MRM confirmation. A large number of lysosomal proteins were observed to be upregulated in acid-adapted cells and, among these was the lysosome associated membrane protein 2 (LAMP2) confirmed by MRM. This protein is of particular interest because it is heavily glycosylated and functions to protect lysosomal membrane from acid proteolysis. Using independent in vitro as well as in vivo approaches such as intravital fluorescent microscopy of dorsal window chamber mouse xenografts, we observed that LAMP2 was associated with acid-adaptation. In vivo, treatment of animals with bicarbonate to reduce tumor acidity led to reductions in LAMP2. Further, mRNA-expression profiling (microarray) of patient tumor samples and IHC of Tissue Microarray (TMA) of breast cancer patient tissue samples, revealed a high correlation of LAMP2 expression with progression and metastasis of breast tumors. We also applied LAMP2 staining on the whole breast tumor samples of different stages and observed that LAMP2 was associated with the peri-luminal regions of DCIS, as well as regions of local invasion, which are expected to be acidic. Finally, we observed both in tumors by IHC and subsequently in acid adapted cells by ICC that, while LAMP2 was localized in lysosomes and late endosomes under normal pH conditions, it was redistributed to the plasma membrane under acidic pH conditions. To further investigate this cell membrane localization we carried out western blot on membrane extract of acid adapted cells. Based on these observations, we propose a novel cell defense mechanism wherein cells chronically exposed to an acidic environment translocate LAMP2 to their surface, which protects the plasmalemma via an unstirred layer effect. To prove the role of LAMP2 on acid adaptation we did shRNA and siRNA knockdown followed by in vitro and in vivo acid exposure of the cells. We further propose that this pathway may provide a novel therapeutic target against breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Mehdi Damaghi, Robert Sprung, Narges Tafreshi, Veronica Estrella, John Koomen, David Morse, Robert Gillies. LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target. [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 1265. doi:10.1158/1538-7445.AM2015-1265