Abstract 2144: RasGRP1 induces autophagy in primary human epidermal keratinocytes resembling fail-safe mechanisms triggered by oncogenic Ras

Abstract

Abstract RasGRP1 is a guanyl nucleotide exchange factor for Ras that is abundantly expressed in lymphocytes, regulating their maturation and activation. Its aberrant expression has been associated to transformation in various cell types, although the role in human cancer remains largely unexplored. Our lab has previously demonstrated expression of RasGRP1 in epidermal keratinocytes, followed by studies that showed a critical role in skin carcinogenesis in mouse models of cutaneous squamous cell carcinoma through its ability to increase the dosage of active Ras. To explore whether RasGRP1 could contribute to transformation of human keratinocytes, we utilized primary epidermal keratinocytes (HEK) isolated from neonatal foreskin. In a model of tissue reconstruct, or 3-D culture, HEK manipulated to overexpress RasGRP1 (RasGRP1+) failed at generating a stratified epithelium. In fact, the RasGRP1 + cells underwent changes resembling autophagy and cell death in culture, with vacuolated cytoplasm as well as rounded bodies. Autophagy was confirmed at the molecular level by measuring increases in the LC3-II species by Western blot and the formation of a LC3 punctate pattern by immunofluorescence. Surprisingly, there was no caspase-3 activation in the RasGRP1+ HEK despite the morphological signs of apoptosis, although we noticed an increase in the expression of the BH3-only protein NOXA as early as 48 hours post RasGRP1 overexpression. Since oncogenic signals are known to induce fail-safe mechanisms that restrict cell expansion, we presumed that the changes observed in the HEK cell overexpressing RasGRP1 were due to Ras hyperactivation (oncogenic)-induced cell death. As predicted, levels of active Ras-GTP were 3 times higher in the RasGRP1+ cells compared to the wild type HEK, with a preponderance of the H-Ras isoform being activated. In line with these observations, enforced expression of an oncogenic version of the H-Ras isoform (H-RasQ61L) also resulted in cytoplasmatic vacuolization of HEK and increases in LC3-II levels. In contrast to the RasGRP1+ cells, H-RasQ61L caused activation of caspase-3 while increases in NOXA were delayed compared to those caused by RasGRP1. While RasGRP1 and oncogenic H-RasQ61L differ in some aspect of their downstream molecular pathways, probably as a result of the magnitude of Ras activation, the present findings reveal a novel RasGRP1-Ras axis that can lead to oncogenic signals in human keratinocytes. This warrants further investigation into the potential participation of RasGRP1 in keratinocyte transformation, particularly contributing to cutaneous squamous cell carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2144. doi:1538-7445.AM2012-2144