No description available.

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.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

No description available.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

Eukaryotic Elongation Factor 1A (eEF1A) plays a key role in protein synthesis by delivering aminoacylated tRNAs to the A site of the ribosome. In higher vertebrates, two isoforms of eEF1A exist called eEF1A1 and eEF1A2, with eEF1A2 being expressed in adult brain, heart and skeletal muscle. Since 2012, several different de novo heterozygous missense mutations in EEF1A2 have been identified in humans and these cause epilepsy, intellectual disability and autism. Before considering treatment options, it is vital to determine whether these mutations cause loss or gain of protein function. I performed a battery of behavioural tests using two mouse lines with heterozygous loss of function mutations in eEF1A2. The aim was to determine whether there were any behavioural phenotypes consistent with intellectual disability and/or autism. Using heterozygous wasted mice (Eef1a2+/wst), I analysed the effects of aging on behaviour and found that Eef1a2+/wst mice showed reduced marble burying activity and reduced movement in the open field test with age. In a test of social behaviour, Eef1a2+/wst mice showed a significantly reduced preference for social novelty at all ages tested. The second heterozygous null line, Del22.ex3, was generated on a pure C57BL/6J genetic background. This new line was made in order to reduce the level of variation observed in data from the wasted line, which was on a mixed genetic background. The genetic background was shown to have an influence on behaviour as the results differed between this line and the wasted line. Del22.ex3 Eef1a2+/- mice showed significantly reduced engagement in repetitive behaviours compared with wild-type littermates and normal preference for social novelty. Using CRISPR/Cas9, a mouse line with the D252H missense mutation was generated and I repeated my behavioural testing on heterozygotes from this line. I found no behavioural abnormalities in this line suggesting a mouse-human difference in the ability to tolerate eEF1A2 missense mutations. Previous attempts to make a line with the G70S missense mutation were unsuccessful but as a product of this experiment, it was found that mice expressing G70S eEF1A2 had a comparable phenotype to and died at the same age as complete knockouts. This suggested that the G70S protein is non-functional and cannot compensate for loss of wild-type eEF1A2. These experiments have improved our understanding of the phenotypic effects of Eef1a2 mutations in mice and have shown, for the first time, that mutations in Eef1a2 affect mouse behaviour.

In mammals, there are two isoforms of eukaryotic translation elongation factor 1A (eEF1A) called eEF1A1 and eEF1A2. They share 98% similarity at the amino acid level, and the main function of both is to facilitate the elongation process in protein translation. However, they have very different expression patterns. While eEF1A1 is universally expressed, eEF1A2 is strictly expressed in muscle, brain and heart. The over-expression of eEF1A2 has been found in cancers, such as ovarian and breast cancer. The factors influencing the different expression patterns of the two isoforms and the mechanisms by which eEF1A2 can act as an oncogene are not clear, therefore, the main aim of this study was to further investigate these two areas. The first aim was to find out whether the resveratrol induced down-regulation of eEF1A2 was mediated by miR-663. Western blotting in MCF7 cells showed that the level of endogenous eEF1A2 was decreased after resveratrol treatment while eEF1A1 remained stable. In contrast, NIH-3T3 stable cell lines which stably express the eEF1A2 coding sequence (CDS) only did not show this down-regulation, suggesting that the untranslated regions (UTRs) might play a role in this regulation. I then showed that miR-663 has ability to down-regulate a reporter linked to the UTRs of eEF1A2. The same reporter gene harbouring UTRs in which the binding sites of miR-663 had been deleted also showed down-regulation after resveratrol treatment, suggesting that the UTRs of EEF1A2 are key to the down-regulation of eEF1A2 by resveratrol but that miR-663 does not mediate this decrease. The second project aimed to address why eEF1A2 is an oncogene but eEF1A1 is not. The 3D structure of human eEF1A1 and eEF1A2 shows that the most of the highly conserved amino acids differences between the two isoforms are Ser and Thr residues, which are potential sites for phosphorylation. I mutated these three sites in eEF1A2 expression constructs to the equivalent amino acid from eEF1A1. Firstly, by transient transfection, all the mutant eEF1A2 were shown expressed and the sub-cellular locations of eEF1A2 remain unchanged after site-directed mutagenesis. Then, stable cell lines were generated. Anchorage independent growth (soft agar) and focus formation assays showed that the stable cell lines harbouring wild type eEF1A2 were significantly more transformed that those expressing the eEF1A2 mutants. However, there was no apparent difference in global protein synthesis between these cell lines. The results suggest that the potential phosphorylated sites in eEF1A2 play an important role in its oncogenicity and that this oncogenicity is not related to the canonical function of eEF1A2.

Eukaryotic Translation Elongation Factor 1 Alpha (EEF1A) exists as two forms with different tissue specificities and encoded by separate loci: eEF1A1 on 6q13 and eEF1A2 on 20q13.3. eEF1A1 is ubiquitously expressed whereas eEF1A2 expression is normally limited to the heart, brain and skeletal muscles. eEF1A proteins are GTP-binding proteins that recruit an amino-acylated tRNA to the ribosome during the elongation phase of protein translation. eEF1A2 mRNA and protein are highly expressed in 50–60% of primary human breast tumors and metastases but not in normal breast epithelium. Since it is also overexpressed in 30% of primary human ovarian tumors, transforms rodent fibroblasts and increases their tumorigenicity in nude mice, eEF1A2 is considered to be a potential human oncogene. The mechanism of eEF1A2 expression is yet to be determined. Studies showed no gene mutation and no correlation between locus amplification or methylation and gene expression. Phosphate Tyrosine Kinase-6 (PTK6) is also located on 20q13.3. It is 48kb upstream of EEF1A2. PTK6 is a non-receptor tyrosine-kinase that is normally expressed in epithelial linings, prostate, skin and oral epithelium but it is not detected in the normal human mammary epithelium. PTK6 has been found to be expressed in many breast cancer cell lines and in approximately 60% of primary human breast tumors but it has not been detected in normal human breast tissue nor in fibroadenomas. Like other tyrosine kinases, PTK6 phosphorylates and activates downstream substrates that would be predicted to lead to increased transcriptional activity and therefore mediates proliferation of breast cancer cells. PTK6 is considered a prognostic marker of metastasis-free survival in breast cancer independent of the classical markers of tumor size, lymph node involvement and HER2 status. The aim of this project was to characterize for the first time the genomic region containing the two mentioned breast cancer oncogenes and understand their various roleswhether they act in tandem or independently in breast tumorigenesis. Immunohistochemistry was performed on tissue microarrays from 300 breast cancer patients to detect the expression levels of eEF1A2 and PTK6. Tumors that showed a high co-expression were analyzed for the genes’ copy number. An increased copy number of PTK6 was detected but not of eEF1A2 nor of adjacent genes on the 20q13.3 amplicon. To understand the effect of EEF1A2 expression on other genes, microarray analysis was performed on NIH-3T3 cells stably transfected with EEF1A2. Many upregulated genes were associated with different types of cancers. This was further confirmed by real-time PCR. However, when the NIH-3T3 cells were transiently transfected with EEF1A2, the genes that were upregulated in the microarray study showed no change in expression. In conclusion, EEF1A2 and PTK6 act independently and each acts through a different mechanism in breast tumorigenesis.