Oncogenic activation of EEF1A2 expression: a journey from a putative to an established oncogene.

Protein synthesis via translation is a central process involving several essential proteins called translation factors. Although traditionally described as cellular "housekeepers," multiple studies have now supported that protein initiation and elongation factors regulate cell growth, apoptosis, and tumorigenesis. One such translation factor is eukaryotic elongation factor 1 alpha 2 (EEF1A2), a member of the eukaryotic elongation factor family, which has a canonical role in the delivery of aminoacyl-tRNA to the A-site of the ribosome in a guanosine 5'-triphosphate (GTP)-dependent manner. EEF1A2 differs from its closely related isoform, EEF1A1, in tissue distribution. While EEF1A1 is present ubiquitously, EEF1A2 replaces it in specialized tissues. The reason why certain specialized tissues need to essentially switch EEF1A1 expression altogether with EEF1A2 remains to be answered. Abnormal "switch on" of the EEF1A2 gene in normal tissues is witnessed and is seen as a cause of oncogenic transformation in a wide variety of solid tumors. This review presents the journey of finding increased expression of EEF1A2 in multiple cancers, establishing molecular mechanism, and exploring it as a target for cancer therapy. More precisely, we have compiled studies in seven types of cancers that have reported EEF1A2 overexpression. We have discussed the effect of aberrant EEF1A2 expression on the oncogenic properties of cells, signaling pathways, and interacting partners of EEF1A2. More importantly, in the last part, we have discussed the unique potential of EEF1A2 as a therapeutic target. This review article gives an up-to-date account of EEF1A2 as an oncogene and can draw the attention of the scientific community, attracting more research.

EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia.

BACKGROUND: The eukaryotic elongation factor, EEF1A2, has been identified as an oncogene in various solid tumors. Here, we have identified a novel function of EEF1A2 in angiogenesis. METHODS: Chick chorioallantoic membrane, tubulogenesis, aortic ring, Matrigel plug, and skin wound healing assays established EEF1A2's role in angiogenesis. RESULT: Higher EEF1A2 levels in breast cancer cells enhanced cell growth, movement, blood vessel function, and tubule formation in HUVECs, as confirmed by ex-ovo and in-vivo tests. The overexpression of EEF1A2 could be counteracted by Plitidepsin. Under normoxic conditions, EEF1A2 triggered HIF1A expression via ERK-Myc and mTOR signaling in TNBC and ER/PR positive cells. Hypoxia induced the expression of EEF1A2, leading to a positive feedback loop between EEF1A2 and HIF1A. Luciferase assay and EMSA confirmed HIF1A binding on the EEF1A2 promoter, which induced its transcription. RT-PCR and polysome profiling validated that EEF1A2 affected VEGF transcription and translation positively. This led to increased VEGF release from breast cancer cells, activating ERK and PI3K-AKT signaling in endothelial cells. Breast cancer tissues with elevated EEF1A2 showed higher microvessel density. CONCLUSION: EEF1A2 exhibits angiogenic potential in both normoxic and hypoxic conditions, underscoring its dual role in promoting EMT and angiogenesis, rendering it a promising target for cancer therapy.

Reduced IQGAP2 expression promotes EMT and inhibits apoptosis by modulating the MEK-ERK and p38 signaling in breast cancer irrespective of ER status.

IQGAP2, a member of the IQGAP family, functions as a tumor suppressor in most of the cancers. Unlike IQGAP1 and IQGAP3, which function as oncogenes in breast cancer, the role of IQGAP2 is still unexplored. Here we report a reduced expression of IQGAP2, which was associated with lymph node positivity, lymphovascular invasion, and higher age in breast cancer patients. We found an inverse correlation of IQGAP2 expression levels with oncogenic properties of breast cancer cell lines in estrogen receptor (ER) independent manner. IQGAP2 expression enhanced apoptosis via reactive oxygen species (ROS)-P38-p53 pathway and reduced epithelial-mesenchymal transition (EMT) in a MEK-ERK-dependent manner. IQGAP2-IQGAP1 ratio correlated negatively with phospho-ERK levels in breast cancer patients. Pull-down assay showed interaction of IQGAP1 and IQGAP2. IQGAP2 overexpression rescued, IQGAP1-mediated ERK activation, suggesting the possibility of IQGAP1 sequestration by IQGAP2. IQGAP2 depletion, in a tumor xenograft model, increased tumor volume, tumor weight, and phospho-ERK expression. Overall, our findings suggest that IQGAP2 is negatively associated with proliferative and metastatic abilities of breast cancer cells. Suppression of IQGAP1-mediated ERK activation is a possible route via which IQGAP2 restricts oncogenic properties of breast cancer cells. Our study highlights the candidature of IQGAP2 as a potent target for therapeutic intervention.

The 2'-caged-tethered-siRNA shows light-dependent temporal controlled RNAi activity for GFP gene into HEK293T cells.

Small interfering RNA (siRNA) exhibits gene-specific RNAi activity by the formation of RISC complex with mRNA of gene. The structural modification of siRNA at appropriate positions affects the structure of RISC complex and then RNAi activity. The modified siRNA are mostly prepared from the incorporation of sugar ring modified, and nucleobase modified RNA nucleotides. It is learned that the introduction of the sterically hindered nucleoside at the specific position of siRNA, severely affects siRNA-RISC complex formation. This report describes the syntheses of bulkier siRNA from 2'-caged-tethered-siRNAs, containing bulkier photolabile protecting group (o-nitrobenzyl) at 2'-position of ribose nucleoside. Importantly, these 2'-caged-siRNAs exhibit the light-dependent RNA interference (RNAi) activity into HEK293T cells for the GFP gene expression. The 2'-caged-siRNAs are synthesized by caging the sense and antisense strand of siRNA. The biochemical evaluations of these caged-siRNAs show that antisense-strand caged-siRNAs decrease RNAi activity temporarily in dark while enhancing RNAi activity, almost like control, after exposure withUV- light. However, 2'-caged sense strand siRNA increase RNAi activity temporarily while decreasing RNAi activity after exposure with light. These caged-siRNAs are also stable in the serum (fetal bovine serum) as like native siRNA. Hence these results strongly support that 2'-caged-tethered-siRNAs are promising analogues to control RNAi activity by UV-light.

EEF1A2 triggers stronger ERK mediated metastatic program in ER negative breast cancer cells than in ER positive cells.

AIMS: Ever since EEF1A2's identification as a putative oncogene in breast cancer, it has stimulated curiosity due to its contrasting role in predicting the prognostic values in breast cancer patients. Contradicting reports suggest it to be playing a pro-survival as well as a negative role in the survival of patients. This prompted us to find the association of this protein with molecular subtypes in breast cancer and its effect on EMT in representative cell lines. MAIN METHODS: Data-mining was carried out to ascertain the correlation of EEF1A2 with molecular subtypes in breast cancer patients. Scratch wound healing and transwell invasion assays were carried out to assess its role in migration and invasion. Western blot, qRT-PCR, and ELISA were carried out to determine key signalling pathways, cytokines, and EMT factors responsible for the observed phenotype. KEY FINDINGS: EEF1A2 was associated with ER receptor positivity in breast cancer and was involved in its transcriptional regulation. It induced a robust metastatic program in MDA-MB-231 (a triple-negative cell line), and induced significant changes in its invasive and migratory properties via activation of the ERK pathway. This was not the case in MCF7 which is an ER-positive cell line. SIGNIFICANCE: We highlight the specific tendency of EEF1A2 to enhance invasive properties of cell lines in particular molecular subtype only. This sheds light on its selective role in regulating oncogenic processes in breast cancer and could explain its contradicting association with good survival, despite being an oncogene in a certain cohort of breast cancer patients.

Expression pattern of EEF1A2 in brain tumors: Histological analysis and functional role as a promoter of EMT.

AIMS: Glioblastomas are highly aggressive brain tumors with a very poor survival rate. EEF1A2, the proto-oncogenic isoform of the EEF1A translation factor family, has been found to be overexpressed and promoting tumorigenesis in multiple cancers. Interestingly, recent studies reported reduced expression of this protein in brain tumors, drawing our attention to find the functional role and mechanism of this protein in brain tumor progression. MAIN METHODS: Using representative cell line as models, the role of EEF1A2 in cell proliferation, migration and invasion were assessed using MTS assay, scratch wound-healing assay, transwell migration and invasion assay, respectively. Activation of key signaling pathways was assessed using western blots and real-time PCR. Finally, using immunohistochemistry we checked the protein levels of EEF1A2 in CNS tumors. KEY FINDINGS: EEF1A2 was found to increase the proliferative, migratory and invasive properties of cell lines of both glial and neuronal origin. PI3K activation directly correlated with EEF1A2 levels. Protein levels of key EMT markers viz. Twist, Snail, and Slug were increased upon ectopic EEF1A2 expression. Furthermore, EEF1A2 was found to affect the expression levels of key inflammatory cytokines, growth factors and matrix metalloproteases. IHC analysis showed that EEF1A2 is upregulated in tumor tissues compared to normal tissue. SIGNIFICANCE: EEF1A2 acts as an oncogene in both neuronal and glial cells and triggers an EMT program via PI3K pathway. However, it shows enhanced expression in neuronal cells of the brain than the glial cells, which could explain the previously reported anomaly.

Reduced FRG1 expression promotes prostate cancer progression and affects prostate cancer cell migration and invasion.

BACKGROUND: Prostate cancer is the most common form of cancer in males and accounts for high cancer related deaths. Therapeutic advancement in prostate cancer has not been able to reduce the mortality burden of prostate cancer, which warrants further research. FRG1 which affects angiogenesis and cell migration in Xenopus, can be a potential player in tumorigenesis. In this study, we investigated the role of FRG1 in prostate cancer progression. METHODS: Immunohistochemistry was performed to determine FRG1 expression in patient samples. FRG1 expression perturbation was done to investigate the effect of FRG1 on cell proliferation, migration and invasion, in DU145, PC3 and LNCaP cells. To understand the mechanism, we checked expression of various cytokines and MMPs by q-RT PCR, signaling molecules by western blot, in FRG1 perturbation sets. Results were validated by use of pharmacological inhibitor and activator and, western blot. RESULTS: In prostate cancer tissue, FRG1 levels were significantly reduced, compared to the uninvolved counterpart. FRG1 expression showed variable effect on PC3 and DU145 cell proliferation. FRG1 levels consistently affected cell migration and invasion, in both DU145 and PC3 cells. Ectopic expression of FRG1 led to significant reduction in cell migration and invasion in both DU145 and PC3 cells, reverse trends were observed with FRG1 knockdown. In androgen receptor positive cell line LNCaP, FRG1 doesn't affect any of the cell properties. FRG1 knockdown led to significantly enhanced expression of GM-CSF, MMP1, PDGFA and CXCL1, in PC3 cells and, in DU145, it led to higher expression of GM-CSF, MMP1 and PLGF. Interestingly, FRG1 knockdown in both the cell lines led to activation of p38 MAPK. Pharmacological activation of p38 MAPK led to increase in the expression of GM-CSF and PLGF in DU145 whereas in PC3 it led to enhanced expression of GM-CSF, MMP1 and CXCL1. On the other hand, inhibition of p38 MAPK led to reduction in the expression of above mentioned cytokines. CONCLUSION: FRG1 expression is reduced in prostate adenocarcinoma tissue. FRG1 expression affects migration and invasion in AR negative prostate cancer cells through known MMPs and cytokines, which may be mediated primarily via p38 MAPK activation.

The expression profile and prognostic significance of eukaryotic translation elongation factors in different cancers.

Eukaryotic translation factors, especially initiation factors have garnered much attention with regards to their role in the onset and progression of different cancers. However, the expression levels and prognostic significance of translation elongation factors remain poorly explored in different cancers. In this study, we have investigated the mRNA transcript levels of seven translation elongation factors in different cancer types using Oncomine and TCGA databases. Furthermore, we have identified the prognostic significance of these factors using Kaplan-Meier Plotter and SurvExpress databases. We observed altered expression levels of all the elongation factors in different cancers. Higher expression of EEF1A2, EEF1B2, EEF1G, EEF1D, EEF1E1 and EEF2 was observed in most of the cancer types, whereas reverse trend was observed for EEF1A1. Overexpression of many factors predicted poor prognosis in breast (EEF1D, EEF1E1, EEF2) and lung cancer (EEF1A2, EEF1B2, EEF1G, EEF1E1). However, we didn't see any common correlation of expression levels of elongation factors with survival outcomes across cancer types. Cancer subtype stratification showed association of survival outcomes and expression levels of elongation factors in specific sub-types of breast, lung and gastric cancer. Most interestingly, we observed a reciprocal relationship between the expression levels of the two EEF1A isoforms viz. EEF1A1 and EEF1A2, in most of the cancer types. Our results suggest that translation elongation factors can have a role in tumorigenesis and affect survival in cancer specific manner. Elongation factors have potential to serve as biomarkers and therapeutic drug targets, yet further study is required. Reciprocal relationship of differential expression between EEF1A isoforms observed in multiple cancer types indicates opposing roles in cancer and needs further investigation.

Reduced expression of IQGAP2 and higher expression of IQGAP3 correlates with poor prognosis in cancers.

IQGAPs is a family of proteins which comprises three members, in humans. The expression pattern and role of IQGAP1 has been well established in many cancers, whereas those of IQGAP2 and IQGAP3, have mostly remained unexplored. We used available large datasets, to explore the pan-cancer status of these two genes in-silico. Here we have analysed their mRNA expression and correlation with survivability in eight different cancers, including lung, breast, gastric, brain, colorectal, prostate, liver and kidney cancers and, their subtypes. The mRNA expression of IQGAP2 and IQGAP3 in individual cancers were analysed in two different publicly available databases viz. Oncomine and TCGA. The prognostic value of these genes in lung, breast and gastric cancer was analysed using Kaplan-Meier Plotter database, whereas for brain, colorectal, liver, prostate and kidney cancers, SurvExpress database was used. These results were validated by immunohistochemistry in cancer tissues (stomach, prostate, brain, colorectal). Moreover, we did IQGAP2 and IQGAP3 genomic alteration and, promoter methylation analysis using cBioportal and Wanderer web tool, respectively. Most of the cancer types (lung, breast, prostate, brain, gastric, liver, kidney and colorectal) showed increased IQGAP3 mRNA expression. In contrast, the IQGAP2 transcript levels were reduced across different cancers viz. lung, breast, gastric, liver, kidney and colorectal cancer. IQGAP2 expression correlated positively with survivability, on the contrary, IQGAP3 expression levels correlated inversely with survivability, in most of the cancers. Collectively, enhanced IQGAP3 and reduced IQGAP2 levels were frequently observed in multiple cancers with the former predicting poor survivability and the later opposite. Methylation pattern was significantly altered in most of the cancer types. We found copy no. variation and mutations in specific cancers, for IQGAP2 and IQGAP3. Our in-vivo (IHC) data confirmed the in-silico findings completely. Hence, IQGAP2 and IQGAP3 have potential to be used as prognostic markers or therapeutic targets in specific cancers.

In vitro and in vivo antitumor effects of Peanut agglutinin through induction of apoptotic and autophagic cell death.

In this study we unravel the mechanism underlying the antitumorigenic effects of Peanut agglutinin (PNA) isolated from Arachis hypogea in Dalton's lymphoma (DL) bearing mice and elucidated the mechanism in vitro in HeLa cells. In vivo PNA administration at 1 and 2 mg/kg body weight reduced DL proliferation with increase in autophagic and apoptotic characteristics. In vitro data showed that PNA at 0.1-100 µg/ml dose exhibit selective antiproliferative activity on various cancer cell lines without displaying cytotoxic effect on normal cells. However, heat denatured PNA failed to show any antiproliferative activity. Moreover, PNA was found to induce autophagic and apoptotic cell death in HeLa cells. Exponential increase in reactive oxygen species (ROS) was proved to be the master signal for promoting PNA induced cell death in HeLa cells. Interestingly, when HeLa cells were pre-exposed with N-acetylcysteine (NAC) and followed to PNA treatment, there was sharp decline in autophagy, apoptosis and a concomitant abrogation of antiproliferative potential. PNA at lower doses was also seen to inflict senescence. Hence, this common culinary item derived molecule whose discovery dates back to late 1970s was for the first time evaluated mechanistically in vivo and in vitro as a novel naturally occurring therapeutic agent against cancer.