eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

MAPL loss dysregulates bile and liver metabolism in mice.

Mitochondrial and peroxisomal anchored protein ligase (MAPL) is a dual ubiquitin and small ubiquitin-like modifier (SUMO) ligase with roles in mitochondrial quality control, cell death and inflammation in cultured cells. Here, we show that MAPL function in the organismal context converges on metabolic control, as knockout mice are viable, insulin-sensitive, and protected from diet-induced obesity. MAPL loss leads to liver-specific activation of the integrated stress response, inducing secretion of stress hormone FGF21. MAPL knockout mice develop fully penetrant spontaneous hepatocellular carcinoma. Mechanistically, the peroxisomal bile acid transporter ABCD3 is a primary MAPL interacting partner and SUMOylated in a MAPL-dependent manner. MAPL knockout leads to increased bile acid production coupled with defective regulatory feedback in liver in vivo and in isolated primary hepatocytes, suggesting cell-autonomous function. Together, our findings establish MAPL function as a regulator of bile acid synthesis whose loss leads to the disruption of bile acid feedback mechanisms. The consequences of MAPL loss in liver, along with evidence of tumor suppression through regulation of cell survival pathways, ultimately lead to hepatocellular carcinogenesis.

CD34 Staining as a Useful Tool in Disorders of Collagen Degeneration.

ABSTRACT: The human progenitor-cell antigen CD34 is expressed in dermal dendritic cells and is lost in several disorders affecting dermal collagen. The loss of CD34 immunohistochemical staining has been demonstrated to be helpful in the histologic diagnosis of morphea, lichen sclerosus, and the classic pattern of granuloma annulare. This study characterized CD34 expression in 2 sclerosing disorders affecting the subcutis: lipodermatosclerosis (LDS) and the sclerodermoid form of chronic graft-versus-host disease (ScGVHD). In addition, we applied CD34 staining to the interstitial pattern of granuloma annulare (IGA), which is a diagnostically challenging entity with subtle amounts of dermal collagen degeneration. Fifteen cases of LDS, 6 cases of ScGVHD, and 4 cases of IGA were identified and stained with CD34. All cases of LDS showed loss of CD34 within subcutaneous septa, and 9 cases (60%) also exhibited full-thickness dermal loss of interstitial staining. All 6 cases of ScGVHD showed varying degrees of CD34 loss within the dermis and/or subcutaneous septa. The normal subcutis showed diffuse septal staining with CD34, with a density equal to that seen in the dermis. CD34 staining was lost in areas of dermal inflammation in half of the IGA cases. We conclude that CD34 staining is a useful ancillary test in disease processes affecting the subcutaneous collagen such as LDS and ScGVHD. Its utility also extends to diagnostically challenging disorders of dermal collagen degeneration such as IGA.

Phosphatidylinositol 4-kinase III beta regulates cell shape, migration, and focal adhesion number.

Cell shape is regulated by cell adhesion and cytoskeletal and membrane dynamics. Cell shape, adhesion, and motility have a complex relationship and understanding them is important in understanding developmental patterning and embryogenesis. Here we show that the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß) regulates cell shape, migration, and focal adhesion (FA) number. PI4KIIIß generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol and is highly expressed in a subset of human breast cancers. PI4KIIIß and the PI4P it generates regulate a variety of cellular functions, ranging from control of Golgi structure, fly fertility, and Akt signaling. Here, we show that loss of PI4KIIIß expression decreases cell migration and alters cell shape in NIH3T3 fibroblasts. The changes are accompanied by an increase in the number of FA in cells lacking PI4KIIIß. Furthermore, we find that PI4P-containing vesicles move to the migratory leading edge during migration and that some of these vesicles tether to and fuse with FA. Fusion is associated with FA disassembly. This suggests a novel regulatory role for PI4KIIIß and PI4P in cell adhesion and cell shape maintenance.

Human gene expression variability and its dependence on methylation and aging.

BACKGROUND: Phenotypic variability of human populations is partly the result of gene polymorphism and differential gene expression. As such, understanding the molecular basis for diversity requires identifying genes with both high and low population expression variance and identifying the mechanisms underlying their expression control. Key issues remain unanswered with respect to expression variability in human populations. The role of gene methylation as well as the contribution that age, sex and tissue-specific factors have on expression variability are not well understood. RESULTS: Here we used a novel method that accounts for sampling error to classify human genes based on their expression variability in normal human breast and brain tissues. We find that high expression variability is almost exclusively unimodal, indicating that variance is not the result of segregation into distinct expression states. Genes with high expression variability differ markedly between tissues and we find that genes with high population expression variability are likely to have age-, but not sex-dependent expression. Lastly, we find that methylation likely has a key role in controlling expression variability insofar as genes with low expression variability are likely to be non-methylated. CONCLUSIONS: We conclude that gene expression variability in the human population is likely to be important in tissue development and identity, methylation, and in natural biological aging. The expression variability of a gene is an important functional characteristic of the gene itself and the classification of a gene as one with Hyper-Variability or Hypo-Variability in a human population or in a specific tissue should be useful in the identification of important genes that functionally regulate development or disease.

Mandatory helmet legislation as a policy tool for reducing motorcycle fatalities: Pinpointing the efficacy of universal helmet laws.

This study uses repeated cross-sections of individual level crash data to study the effectiveness of motorcycle helmet legislation. Results suggest that motorcycle helmet laws reduce average individual fatality risks by 20.5%. From a policy standpoint, large states such as Florida and Texas can reduce annual motorcycle fatalities by an average 100 deaths through reinstating universal helmet laws. Valuing these fatality reductions at the U.S. DOT suggested $9.4 million value of a statistical life yields aggregate annual state benefits of approximately $940 million. The effectiveness of helmet legislation can be attributed to the technological efficacy of helmets as well as enhancing behavior in the form of reduced risk taking among motorcyclists. Specifically, motorcyclists who use helmets in order to comply with mandatory helmet laws are 29.8% less likely to receive a traffic citation for risky driving behavior (speeding, alcohol, etc.), travel at a 6 mph lower average speed, and have a 47.4% reduction in the probability of "severely" damaging their motorcycle in a crash.

Outcomes for multilevel surgery for sleep apnea: Obstructive sleep apnea, transoral robotic surgery, and uvulopalatopharyngoplasty.

OBJECTIVES/HYPOTHESIS: This study evaluates the outcomes of multilevel surgery for patients with obstructive sleep apnea (OSA) who underwent transoral robotic surgery (TORS) (i.e., posterior glossectomy and limited lateral pharyngectomy) with uvulopalatopharyngoplasty (UPPP). STUDY DESIGN: Prospective, nonrandomized trial with historical controls. METHODS: All patients underwent pre- and postoperative polysomnography, preoperative magnetic resonance imaging of the neck, preoperative drug-induced sleep endoscopy, surgery, including UPPP if this had not occurred previously, and OSA TORS. Outcomes measures included apnea-hypopnea index (AHI), Epworth Sleepiness Scale (ESS), O2 saturation nadir, and total sleep time spent at < 90% O2 saturation. RESULTS: Patients who had no prior surgery achieved an average AHI reduction from 58.4 to 19.5 (67%, P < .0001), a surgical success rate of 56%, and a surgical response rate of 73%. Patients with prior pharyngeal surgery achieved an AHI reduction from 55.0 to 45 (24%, P = .19), a surgical success rate of 30%, and a surgical response rate of 40%. Total sleep time spent at <90% O2 saturation was improved from 14% to 3.6% (P < .0003) for patients without prior surgery, and 21.1% to 12.5% (P < .17)for those with prior surgery. ESS improved for all patients combined from 12.8 to 5.8 (P < .0001). CONCLUSIONS: Outcomes for the combined approach of OSA TORS and UPPP provide strong evidence in favor of this multilevel approach for the surgical management of OSA. The benefit of the current surgical approach is most significant for previously unoperated patients.

Offsetting or Enhancing Behavior: An Empirical Analysis of Motorcycle Helmet Safety Legislation.

This study uses state-level panel data from a 33-year period to test the hypotheses of offsetting and enhancing behavior with regards to motorcycle helmet legislation. Results presented in this article find no evidence of offsetting behavior and are consistent with the presence of enhancing behavior. State motorcycle helmet laws are estimated to reduce motorcycle crashes by 18.4% to 31.9%. In the absence of any behavioral adaptations among motorcyclists mandatory helmet laws are not expected to have any significant impact on motorcycle crash rates. The estimated motorcycle crash reductions do not appear to be driven by omitted variable bias or nonclassical measurement error in reported crashes. Overall, the results strongly suggest that mandatory helmet laws yield significant changes in motorcycle mobility in the form of reduced risk taking and/or decreased utilization.

Phosphorylation of filamin A by Cdk1 regulates filamin A localization and daughter cell separation.

In cell culture, many adherent mammalian cells undergo substantial actin cytoskeleton rearrangement prior to mitosis as they detach from the extracellular matrix and become spherical. At the end of mitosis, the actin cytoskeleton is required for cytokinesis and the reassembly of interphase structures as cells spread and reattach to substrate. To understand the processes regulating mitotic cytoskeletal remodeling, we studied how mitotic phosphorylation regulates filamin A (FLNa). FLNa is an actin-crosslinking protein that was previously identified as a cyclin-dependent kinase 1 (Cdk1) binding partner and substrate in vitro. Using quantitative label-based mass spectrometry, we find that FLNa serines 1084, 1459 and 1533 are phosphorylated in mitotic HeLa cells and all three sites match the phosphorylation consensus sequence of Cdk1. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine residues and expressed GFP-tagged FLNa(S1084A,S1459A,S1533A) (FLNa-AAA GFP) in a FLNa-deficient human melanoma cell line called M2. M2 cells expressing FLNa-AAA GFP have enhanced FLNa-AAA GFP and actin localization at sites of contact between daughter cells, impaired post-mitotic daughter cell separation and defects in cell migration. Therefore, mitotic phosphorylation of FLNa is important for successful cell division and interphase cell behavior.

The lipid kinase PI4KIIIß is highly expressed in breast tumors and activates Akt in cooperation with Rab11a.

Emerging evidence now implicates phosphatidylinositol 4-kinases (PI4K), enzymes that generate PI(4)P from phosphatidylinositol (PtdIns), in cancer. In this study, we investigate the role of PI4KIIIß, one of four mammalian PI4Ks, in breast cancer. Although PI4KIIIß protein levels are low in normal breast tissue, we find that approximately 20% of primary human breast tumors overexpress it. Expression of PI4KIIIß in breast carcinoma cells leads to increased Akt activation, dependent on increased PI(3,4,5)P3 production. However, a kinase-inactive version of PI4KIIIß also led to increased Akt activation, and no changes in PI(4)P or PI(4,5)P2 lipid abundance were detected in the PI4KIIIß-overexpressing cells. This implies that PI4KIIIß regulates PI(3,4,5)P3 and Akt independent of PI(4)P production. We find that the PI4KIIIß-binding protein, Rab11a, a small GTPase that regulates endosomal recycling, is involved in PI4KIIIß-mediated activation of Akt, as RNAi depletion of Rab11a impairs Akt activation. Furthermore, ectopic PI4KIIIß expression alters cellular Rab11a distribution and enhances recruitment of PI4KIIIß and Rab11a to recycling endosomes. This work suggests that PI4KIIIß affects PI3K/Akt signaling through Rab11a and endosomal trafficking, independent of its lipid kinase activity. Thus, PI4KIIIß likely plays a role in breast oncogenesis and that cooperation between Rab11a and PI4KIIIß represents a novel Akt activation pathway.

FiloDetect: automatic detection of filopodia from fluorescence microscopy images.

BACKGROUND: Filopodia are small cellular projections that help cells to move through and sense their environment. Filopodia play crucial roles in processes such as development and wound-healing. Also, increases in filopodia number or size are characteristic of many invasive cancers and are correlated with increased rates of metastasis in mouse experiments. Thus, one possible route to developing anti-metastatic therapies is to target factors that influence the filopodia system. Filopodia can be detected by eye using confocal fluorescence microscopy, and they can be manually annotated in images to quantify filopodia parameters. Although this approach is accurate, it is slow, tedious and not entirely objective. Manual detection is a significant barrier to the discovery and quantification of new factors that influence the filopodia system. RESULTS: Here, we present FiloDetect, an automated tool for detecting, counting and measuring the length of filopodia in fluorescence microscopy images. The method first segments the cell from the background, using a modified triangle threshold method, and then extracts the filopodia using a series of morphological operations. We verified the accuracy of FiloDetect on Rat2 and B16F1 cell images from three different labs, showing that per-cell filopodia counts and length estimates are highly correlated with the manual annotations. We then used FiloDetect to assess the role of a lipid kinase on filopodia production in breast cancer cells. Experimental results show that PI4KIII ß expression leads to an increase in filopodia number and length, suggesting that PI4KIII ß is involved in driving filopodia production. CONCLUSION: FiloDetect provides accurate and objective quantification of filopodia in microscopy images, and will enable large scale comparative studies to assess the effects of different genetic and chemical perturbations on filopodia production in different cell types, including cancer cell lines.

Transoral robot-assisted lingual tonsillectomy and uvulopalatopharyngoplasty for obstructive sleep apnea.

OBJECTIVES: We assessed the use of transoral robot-assisted lingual tonsillectomy and uvulopalatopharyngoplasty for the surgical management of tongue base obstruction in patients with obstructive sleep apnea. METHODS: In a prospective, nonrandomized trial using historical controls, patients underwent drug-induced sleep endoscopy, transoral robot-assisted lingual tonsillectomy with uvulopalatopharyngoplasty, and preoperative and postoperative polysomnography. RESULTS: Twenty patients have completed the study to date. The rate of surgical success was 45%, and the rate of surgical response was 65%. The mean preoperative apnea-hypopnea index of 55.6 decreased by 56.7%, to a mean postoperative value of 24.1 (p < 0.001), and the minimum arterial oxygen saturation increased from the mean preoperative value of 75.8% to the mean postoperative value of 81.7% (p = 0.013). The mean Epworth Sleepiness Scale score improved from 13.4 to 5.9 (p = 0.003). One patient had postoperative bleeding that required cauterization, resulting in a major complication rate of 4.2%. CONCLUSIONS: Transoral robot-assisted lingual tonsillectomy with uvulopalatopharyngoplasty is a novel technique for the surgical management of obstructive sleep apnea that results in a significant decrease in the apnea-hypopnea index, a significant improvement in minimum arterial oxygen saturation, and a significant improvement in the Epworth Sleepiness Scale score and has an acceptable complication rate.

The ability to induce microtubule acetylation is a general feature of formin proteins.

Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.

The lipid kinase PI4KIIIß and the eEF1A2 oncogene co-operate to disrupt three-dimensional in vitro acinar morphogenesis.

The study of in vitro morphogenesis is fundamental to understanding neoplasia since the dysregulation of morphogenic pathways that create multi-cellular organisms is a common hallmark of oncogenesis. The in vitro culture of human breast epithelial cells on reconstituted basement membranes recapitulate some features of in vivo breast development, including the formation of a three-dimensional structure termed an acinus. Importantly, the capacity to disrupt in vitro acinar morphogenesis is a common property of human breast oncogenes. In this report, we find that phosphatidylinositol 4-kinase IIIß (PI4KIIIß), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIß protein localizes to the basal surface of acini created by human MCF10A cells and ectopic expression of PI4KIIIß induces multi-acinar devlopment. Furthermore, expression of an oncogenic PI4KIIIß activator, eEF1A2 (eukaryotic elongation factor 1 alpha 2), phenocopies the PI4KIIIß multi-acinar phenotype. Ectopic expression of PI4KIIIß or eEF1A2 alters the localization of PI(4)P and PI(4,5)P(2) within acini, indicating the importance of these lipids in acinar development. Our work shows that PI4KIIIß, eEF1A2 and PI(4)P likely play an important role in mammary neoplasia and acinar development.

The good oncogene: When bad genes identify good outcome in cancer.

Some cancer patients live many decades after diagnosis while others are not so fortunate. Understanding why this occurs is a fundamental issue in cancer research. We hypothesize that among the factors controlling favorable outcome are a class of genes that we describe as "good oncogenes". These genes have a paradoxical function in cancer in that they are prognostic markers for favorable survival but have strong transforming and tumour-promoting properties. As such, good oncogenes both promote neoplasia and constrain it. We propose that good oncogenes enhance outcome probability by allowing early tumor detection, sensitizing cancer cells to senescence or by attenuating metastatic progression and tumour self-renewal. We believe that understanding the signaling pathways regulated by good oncogenes provides mechanistic insight into the biochemical basis for long-term survival in cancer.

Robust patterns in the stochastic organization of filopodia.

BACKGROUND: Filopodia are actin-based cellular projections that have a critical role in initiating and sustaining directional migration in vertebrate cells. Filopodia are highly dynamic structures that show a rich diversity in appearance and behavior. While there are several mathematical models of filopodia initiation and growth, testing the capacity of these theoretical models in predicting empirical behavior has been hampered by a surprising shortage of quantitative data related to filopodia. Neither is it clear how quantitatively robust the cellular filopodial network is and how perturbations alter it. RESULTS: We have measured the length and interfilopodial separation distances of several thousand filopodia in the rodent cell line Rat2 and measured these parameters in response to genetic, chemical and physical perturbation. Our work shows that length and separation distance have a lognormal pattern distribution over their entire detection range (0.4 µm to 50 µm). CONCLUSIONS: We find that the lognormal distribution of length and separation is robust and highly resistant to perturbation. We also find that length and separation are independent variables. Most importantly, our empirical data is not entirely in agreement with predictions made based on existing theoretical models and that filopodial size and separation are an order of magnitude larger than what existing models suggest.

The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas.

Ovarian epithelial carcinomas (OECs) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In our study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

Eukaryotic elongation factor 1A2 cooperates with phosphatidylinositol-4 kinase III beta to stimulate production of filopodia through increased phosphatidylinositol-4,5 bisphosphate generation.

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P(2)]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), and we find that production of eEF1A2-dependent PI(4,5)P(2) and generation of filopodia require PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both the production of PI(4,5)P(2) and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIIIbeta, and activated PI4KIIIbeta stimulates production of PI(4,5)P(2) and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P(2) signaling and actin remodeling.

The prognostic significance of elongation factor eEF1A2 in ovarian cancer.

OBJECTIVE: To determine whether eukaryotic elongation factor 1 alpha 2 (eEF1A2), a transforming gene previously shown to be highly expressed in primary human ovarian tumours, is a prognostic marker. METHODS: We have used an antibody specific for eEF1A2 to measure eEF1A2 protein expression in 500 primary ovarian tumours in a tissue microarray. We have also ectopically expressed eEF1A2 in SK-OV-3 cells, a clear cell carcinoma line that does not normally express eEF1A2. RESULTS: We have shown that eEF1A2 has high expression levels in approximately 30% of all primary ovarian tumours. 50% of serous tumours, 30% of endometrioid, 19% of mucinous and 8% of clear cell tumours highly express eEF1A2. Ectopic expression of eEF1A2 in the SK-OV-3 clear cell carcinoma line enhances their in vitro proliferative capacity and ability to form tumour-like spheroids in hanging drop culture. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In serous cancer, eEF1A2 is an independent prognostic marker for survival and high eEF1A2 protein expression was associated with increased probability of 20-year survival. CONCLUSIONS: eEF1A2 is highly expressed in ovarian carcinomas. Its expression enhances cell growth in vitro, and eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian cancer patients with serous tumours.

Mutational analysis of the FXNPXY motif within LDL receptor-related protein 1 (LRP1) reveals the functional importance of the tyrosine residues in cell growth regulation and signal transduction.

LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.