Tissue-specific variants of translation elongation factor eEF1A and their role in cancer
Eukaryotic translation elongation factor eEF1A exists in two closely related variant forms, eEF1A1 and eEF1A2, that are encoded by separate loci. The former is the second most abundant protein in the cell and is almost ubiquitously expressed but eEF1A2 expression is more limited and its presence was defined predominantly in neurons and muscle cells. Both perform equally well in translation elongation and are responsible for delivering aminoacylated tRNA to the A site of the ribosome in a GTP-dependent manner. Translation factor eEF1A2 was identified as an oncogene due to inappropriate expression being observed in the high proportion of ovarian, breast, lung, colon and pancreatic tumours. Additionally, its forced expression in rodent fibroblasts resulted in soft agar colony formation along with tumours when overexpressing cells were injected into nude mice. The mechanism by which eEF1A2 contributes to oncogenesis remains unclear. Gene amplification is not solely responsible for eEF1A2 upregulation and neither activating mutations nor methylation status changes are seen in tumours. Interestingly, no connection of eEF1A1 with any malignancy has been made. It is proposed that the oncogenic properties of eEF1A2 might be associated with its conventional role in translation or perhaps with non-canonical functions that differ from those of the eEF1A1 variant. The main objectives of this PhD project were to elucidate the differential functions of both variants of eEF1A in cancer and to investigate other possible mechanism of eEF1A2 upregulation. In order to compare the contribution of overexpressed eEF1A variants to cellular transformation, stable cell lines were generated in NIH-3T3 mouse fibroblasts and tested in a panel of in vitro transformation assays. Mammalian expression plasmids used for transfection contained each eEF1A variant coding sequence with or without its own 5‟UTR and each variant with the 5‟UTR from the other eEF1A form. Transient transfections with the same mammalian expression plasmids were performed to observe that incorporation of exogenous eEF1A1 and eEF1A2 resulted in a decrease of the endogenous eEF1A1 expression at the mRNA and protein level. The dynamic interplay between exogenous and endogenous variants occurred within the first 48 hours post transfection but Eef1a1 returned to the levels seen in controls as soon as the expression of any of the exogenous eEF1A forms started to decline. In contrast, in almost all tested stable cell lines, the levels of endogenous eEF1A1 remained unchanged, at both the mRNA and protein level. NIH-3T3 lines constitutively expressing eEF1A forms were subsequently subjected to various in vitro transformation assays. Stable cell lines of eEF1A1 coding sequence origin formed colonies and foci but with lower efficiency when compared to the eEF1A2 coding sequence variant. It was also shown that anchorage independent growth and foci formation were affected by incorporating either the eEF1A1 or eEF1A2 5‟UTR in front of either eEF1A1 or eEF1A2 coding sequence. There was no apparent increase in migration and invasion of the cell lines stably expressing eEF1A. No significant association between protein synthesis rate or increased overall eEF1A level and transformed phenotype in all tested stable cell lines was observed. Expression of eEF1A1 or eEF1A2 was also determined immunohistologically in panels of different tumour arrays. Moderate to high expression of eEF1A2 protein was observed in 43% of colorectal cancers analysed. The level of eEF1A2 expression appeared to be inversely correlated (P = 0.024) with metastasis in lymph nodes in one of the tested colorectal tumour arrays. Moreover, no substantial upregulation of eEF1A2 at the protein level was confirmed in hepatocellular carcinoma and malignant melanoma arrays. In contrast, eEF1A1 protein expression was mostly weak or absent in these malignancies.