The role of RasGRP1 in Ras-induced human epidermal neoplasia
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2014-12
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University of Hawaii at Manoa
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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 laboratory 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 human epidermal keratinocytes isolated from neonatal foreskin (HKn). In a model of tissue reconstruct, or 3-D culture, HKn manipulated to overexpress RasGRP1 (RasGRP1+) failed at generating a stratified epithelium. In fact, the RasGRP1+ cells underwent changes, including growth arrest and autophagy in culture, which have been described as failsafe mechanisms in response to oncogenic stress. While there was no activation of apoptosis in RasGRP1+, we noticed an increase in expression of the BH3-only protein NOXA as early as 48 hours post RasGRP1 transduction. We presumed that the changes observed in RasGRP1+ were due to Ras hyperactivation (oncogenic)-induced cell failsafe mechanisms. As predicted, levels of active, RasGTP were approximately 5 times higher in the RasGRP1+ cells compared to the wild-type HKn, with a preponderance of the H-Ras isoform being activated. Similarly, enforced expression of an oncogenic version of the H-Ras isoform (HRasQ61L) also resulted in growth arrest and autophagy. In contrast to the RasGRP1+ cells, HRasQ61L caused activation of caspase-3 while increases in NOXA were delayed compared to those caused by RasGRP1. Furthermore, enforced RasGRP1 expression appeared to induce transformation in skin cells with p53 suppression, demonstrating the oncogenic potential of this protein. While RasGRP1 and oncogenic HRasQ61L 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.
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RasGRP1
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering.
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