PTEN deficiency sensitizes endometrioid endometrial cancer to compound PARP-PI3K inhibition but not PARP inhibition as monotherapy.

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising cancer therapeutics especially for tumors with deficient homologous recombination (HR) repair. However, as HR-deficient tumors represent only a small fraction of endometrial cancers, the therapeutic utility of PARP inhibitors is limited in this disease. Somatic loss of phosphatase and tensin homolog (PTEN), a tumor suppressor that counteracts phosphoinositide 3-kinase (PI3K) activity, is one of the most common genetic aberrations in endometrioid endometrial cancer. While previous works have identified the role of PTEN in DNA double-strand break repair, vulnerabilities of PTEN-deficient endometrioid endometrial cancers to PARP inhibition remain controversial. Here we find that PTEN-deficient endometrioid endometrial cancer cells are not responsive to PARP inhibitor Olaparib alone, but instead show superior sensitivity to compound inhibition with PI3K inhibitor BKM120, as evidenced by reduced clonogenic cell growth and three-dimensional (3D) spheroid disintegration. Mechanistically, PI3K blockade by BKM120 attenuated HR competency with γH2AX accumulation and reduced RAD51 and BRCA1 expression in Ishikawa, AN3CA and Nou-1 cells, but the same combination treatment led to enhanced phosphorylation of DNA-PK, a non-homologous end joining repair protein, in Hec-108 cells. Furthermore, we show that CRISPR/Cas9-mediated PTEN depletion rendered PTEN wild-type Hec-1A endometrioid endometrial cancer cells responsive to combined inhibition of PARP/PI3K, with concomitantly induced DNA damage accumulation and repair defects. The combination of BKM120 and Olaparib cooperated to inhibit tumor growth in a genetic mouse model of Pten-deficient endometrioid endometrial cancer. Together, these results suggest PI3K inhibition may be a plausible approach to expand the utility of PARP inhibitors to endometrioid endometrial cancers in a PTEN-deficient setting.

A small spacecraft for multipoint measurement of ionospheric plasma.

Measurement of ionospheric plasma is often performed by a single in situ device or remotely using cameras and radar. This article describes a small, low-resource, deployed spacecraft used as part of a local, multipoint measurement network. A B-field aligned sounding rocket ejects four of these spin-stabilized spacecraft in a cross pattern. In this application, each spacecraft carries two retarding potential analyzers which are used to determine plasma density, flow, and ion temperature. An inertial measurement unit and a light-emitting diode array are used to determine the position and orientation of the devices after deployment. The design of this spacecraft is first described, and then results from a recent test flight are discussed. This flight demonstrated the successful operation of the deployment mechanism and telemetry systems, provided some preliminary plasma measurements in a simple mid-latitude environment, and revealed several design issues.

PI3K-p110α mediates resistance to HER2-targeted therapy in HER2+, PTEN-deficient breast cancers.

Human epidermal growth factor receptor-2 (HER2) amplification/overexpression (HER2+) frequently co-occurs with PI3K pathway activation in breast tumors. PI3K signaling is most often activated by PIK3CA mutation or PTEN loss, which frequently results in sensitivity to p110α or p110ß inhibitors, respectively. To examine the p110 isoform dependence in HER2+, PTEN-deficient tumors, we generated genetic mouse models of breast tumors driven by concurrent Her2 activation and Pten loss coupled with deletion of p110α or p110ß. Ablation of p110α, but not p110ß, significantly impaired the development of Her2+/Pten-null tumors in mice. We further show that p110α primarily mediates oncogenic signaling in HER2+/PTEN-deficient human cancers while p110ß conditionally mediates PI3K/AKT signaling only upon HER2 inhibition. Combined HER2 and p110α inhibition effectively reduced PI3K/AKT signaling and growth of cancer cells both in vitro and in vivo. Addition of the p110ß inhibitor to dual HER2 and p110α inhibition induced tumor regression in a xenograft model of HER2+/PTEN-deficient human cancers. Together, our data suggest that combined inhibition of HER2 and p110α/ß may serve as a potent and durable therapeutic regimen for the treatment of HER2+, PTEN-deficient breast tumors.

PIK3CA(H1047R)- and Her2-initiated mammary tumors escape PI3K dependency by compensatory activation of MEK-ERK signaling.

Human breast cancers that have HER2 amplification/overexpression frequently carry PIK3CA mutations, and are often associated with a worse prognosis. However, the role of PIK3CA mutations in the initiation and maintenance of these breast cancers remains elusive. In the present study, we generated a compound mouse model that genetically mimics HER2-positive breast cancer with coexisting PIK3CA(H1047R). Induction of PIK3CA(H1047R) expression in mouse mammary glands with constitutive expression of activated Her2/Neu resulted in accelerated mammary tumorigenesis with enhanced metastatic potential. Interestingly, inducible expression of mutant PIK3CA resulted in a robust activation of phosphatidylinositol-3-kinase (PI3K)/AKT signaling but attenuation of Her2/Her3 signaling, and this can be reversed by deinduction of PIK3CA(H1047R) expression. Strikingly, although these Her2(+) PIK3CA(H1047R)-initiated primary mammary tumors are refractory to HER2-targeted therapy, all tumors responded to inactivation of the oncogenic PIK3CA(H1047R), a situation closely mimicking the use of a highly effective inhibitor specifically targeting the mutant PIK3CA/p110a. Notably, these tumors eventually resumed growth, and a fraction of them escaped PI3K dependence by compensatory ERK activation, which can be blocked by combined inhibition of Her2 and MEK. Together, these results suggest that PIK3CA-specific inhibition as a monotherapy followed by combination therapy targeting MAPK and HER2 in a timely manner may be an effective treatment approach against HER2-positive cancers with coexisting PIK3CA-activating mutations.

Imaging free-falling particles for multipoint measurement of plasma fluctuations.

The measurement of plasma fluctuations by insertable probes is sometimes limited by the perturbation of the probe on the plasma, and some non-invasive diagnostics such as photodiode arrays can only measure integrated values. In this paper, we introduce a new approach to plasma fluctuation measurement using small, free-falling particles imaged with a fast camera to provide simultaneous multipoint measurement of visible light emissions surrounding each particle. We find that the fluctuations measured in this manner are in agreement with existing diagnostics, and the particle signals are correlated to those measured on inserted floating potential probes. Signals from multiple particles demonstrate an application of multipoint measurement of the plasma spatial structure and coherence.

Polyoma small T antigen triggers cell death via mitotic catastrophe.

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila.

Cytokines regulate the development and differentiated functions of hematopoietic cells by activating multiple signaling pathways, including the Jak-Stat pathway, the PI3-kinase pathway, and the Ras/Raf pathway. While the Jak-Stat interaction has been extensively studied, the relationship between this pathway and other cytokine-induced signaling pathways is not fully understood. In Drosophila melanogaster, mutations that result in hyperactivity of the Jak kinase Hopscotch (Hop) cause an activation of the larval blood cell encapsulation response, including blood cell aggregation and differentiation of plasmatocytes into apparent lamellocytes. Here, we demonstrate that Hop requires the activity of the Raf pathway to promote the activation response of larval plasmatocytes, and provide evidence to suggest that the Hop and D-Raf proteins physically interact. We also show that basal level activity of the Raf pathway is required for the accumulation of circulating blood cells.

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

Retroviral RNA elements integrate components of post-transcriptional gene expression.

Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency, and may even lack pathogenicity, but all retroviruses require cytoplasmic expression of intron-containing mRNA. In the cytoplasm, the primary viral transcript has two essential roles as mRNA template for protein synthesis and as genomic RNA for packaging into progeny virions. Cellular proteins are used by the virus to modulate synthesis, processing, and translation of the viral RNA. To subvert the normal RNA processing cascade and achieve nuclear export of intron-containing viral RNA, retroviruses utilize structured RNA elements and viral or cellular protein partners. These nuclear interactions determine the cytoplasmic fate of viral RNAs by facilitating RNA stability, nuclear export, translational efficiency, and even assembly of progeny virions. The HIV Rev responsive element (RRE) and Rev protein have been a informative paradigm for dissection of the process of eukaryotic RNA nuclear export. Rev is an adapter protein that bridges RRE-containing RNA and the CRM1 nuclear export receptor, which delivers intron-containing RNA to a nuclear export pathway typically used for 5s rRNA and protein transport. This review summarizes data indicating that Rev/RRE also targets cytoplasmic transcripts to the cytoskeletal polysomes and activates their translational efficiency. The interesting parallel is discussed that genetically simpler retroviruses lack a Rev-like protein and recruit cellular proteins to distinct RNA elements that modulate post-transcriptional gene expression through different export pathways. These pathways include the global mRNA export pathway mediated by Tap, and Tap- and CRM1-independent pathways. The CRM1-independent nuclear export pathway accessed by the spleen necrosis virus post-transcriptional control element is functionally linked to RU5-mediated translational enhancement in the cytoplasm. The simple retroviral post-transcriptional control elements also modulate RNA splicing efficiency, stability, assembly of virions, and subsequent viral egress from the cell. Thus, multiple layers of post-transcriptional control are executed by these retroviral RNA elements, which serve as a compact platform for interaction with nuclear and possibly cytoplasmic protein partners. Further characterization of the cellular partners and their regulation will be an important step to full understanding of nuclear-cytoplasmic connections that hardwire post-transcriptional gene expression in eukaryotic cells.

Induction of p53-independent apoptosis by simian virus 40 small t antigen.

Simian virus 40 small t antigen (st) is required for optimal transformation and replication properties of the virus. We find that in certain cell types, such as the human osteosarcoma cell line U2OS, st is capable of inducing apoptosis, as evidenced by a fragmented nuclear morphology and positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of transfected cells. The cell death can be p53 independent, since it also occurs in p53-deficient H1299 cells. Genetic analysis indicates that two specific mutants affect apoptosis induction. One of these (C103S) has been frequently used as a PP2A binding mutant. The second mutant (TR4) lacks the final four amino acids of st, which have been reported to be unimportant for PP2A binding in vitro. However, TR4 unexpectedly fails to bind PP2A in vivo. Furthermore, a long-term colony assay reveals a potent colony inhibition upon st expression, and the behavior of st mutants in this assay reflects the relative frequency of nuclear fragmentation observed in transfections using the same mutants. Notably, either Bcl-2 coexpression or broad caspase inhibitor treatment could restore normal nuclear morphology. Finally, fluorescence-activated cell sorting analysis suggests a correlation between the ability of st to modulate cell cycle progression and apoptosis. Taken together, these observations underscore that st does not always promote proliferation but may, depending on conditions and cell type, effect a cell death response.

Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling.

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.

Simian virus 40 large tumor antigen modulates the Raf signaling pathway.

The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.

Alterations in the oxidative metabolic profile in vascular smooth muscle from hyperlipidemic and diabetic swine.

High cholesterol, especially LDL cholesterol, has been associated with the development of atherosclerotic plaques in arteries. To investigate the changes in cellular substrate metabolism early in the atherogenic process, Sinclair miniature swine were treated for 12 weeks with either a control diet, a high fat diet, or a high fat diet with the addition of alloxan to induce diabetes. The fractional entry into the TCA cycle of 1,2-(13)C-acetate (5 mM), 1-(13)C-glucose (5 mM), and unlabeled, endogenous lipids was determined in control, hyperlipidemic, and diabetic/hyperlipidemic pigs using 13C-isotopomer analysis of glutamate. The diabetic state of the pigs was validated by plasma glucose measurements made after 10 weeks of alloxan treatment for control (65 +/- 6 mg/dL), hyperlipidemic (63 +/- 5 mg/dL), and diabetic/hyperlipidemic (333 +/- 52 mg/dL) pigs. Plasma glucose values did not correlate with the percentage of glucose entry into the TCA cycle (R2 = 0.0819, n = 10). Alterations in the pattern of substrate oxidation were better correlated with changes in plasma lipids (cholesterol and triglycerides) than with changes in plasma glucose. Plasma total cholesterol and total triglyceride levels significantly correlated with changes in acetate metabolism (R2 = 0.7768 and R2 = 0.4787, respectively) and with changes in glucose metabolism (R2 = 0.6067 and R2 = 0.4506, respectively). We conclude that alterations in lipid profile, especially those that were observed in the diabetic milieu, are associated with early changes in vascular smooth muscle oxidative metabolism. These changes in oxidative metabolism may precede alterations in smooth muscle phenotype and, therefore, may play an important role in the early pathogenesis of atherosclerosis.

Schwann cell proliferative responses to cAMP and Nf1 are mediated by cyclin D1.

In most mammalian cells, the cAMP-dependent protein kinase A pathway promotes growth arrest and cell differentiation. However in Schwann cells, the reverse is true. Elevated levels of cAMP function as the cofactor to a broad range of mitogenic cues in culture and in animals. Previous studies have suggested that cAMP acts at an early point in the Schwann cell mitogenic response, perhaps by stimulating the expression of growth factor receptors. We show here that cAMP acts downstream rather than upstream of growth factor receptor expression. The essential function(s) of cAMP is exerted as Schwann cells progress through the G(1) phase of the cell cycle. Ectopic expression studies using an inducible retroviral vector show that the G(1) phase requirement for cAMP can be alleviated by a single protein, cyclin D1. We show, in addition, that at least one function of the Nf1 tumor suppressor is to antagonize the accumulation of cAMP and the expression of cyclin D1 in Schwann cells. Thus a G(1) phase-specific protein, cyclin D1, accounts for two salient features of Schwann cell growth control: the promitotic response to cAMP and the antimitotic response to the Nf1 tumor suppressor.

How the assembly dynamics of the nematode major sperm protein generate amoeboid cell motility.

Nematode sperm are amoeboid cells that use a major sperm protein (MSP) cytoskeleton in place of a conventional actin cytoskeleton to power their amoeboid motility. In these simple, specialized cells cytoskeletal dynamics is tightly coupled to locomotion. Studies have capitalized on this feature to explore the key structural properties of MSP and to reconstitute motility both in vivo and in vitro. This review discusses how the mechanistic properties shared by the MSP machinery and actin-based motility systems lead to a "push-pull" mechanism for amoeboid cell motility in which cytoskeletal assembly and disassembly at opposite ends of the lamellipodium are associated with independent forces for protrusion of the leading edge and retraction of the cell body.

The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity.

The activities of Src-family non-receptor tyrosine kinases are regulated by structural changes that alter the orientation of key residues within the catalytic domain. In this study, we investigate the effects of activation loop mutations on regulation of the lymphocyte-specific kinase Lck (p56lck). Substitution of 5 - 7 residues amino terminal to the conserved activation loop tyrosine (Y394) increases kinase activity and oncogenic potential regardless of regulatory C-terminal tail phosphorylation levels (Y505), while most mutations in the 13 residues carboxyl to Y394 decrease kinase activity. Phosphorylation of the C-terminal regulatory tail is carried out by the cytosolic tyrosine kinase Csk and we find that mutations upstream or downstream of Y394 or mutation of Y394 do not affect the level of Y505 phosphorylation. In addition, we report that mutations on either side of Y394 affect substrate specificity in vivo. We conclude that the high degree of conservation across the entire activation loop of Src-family kinases is critical for normal regulation of kinase activity and oncogenicity as well as substrate selection. Oncogene (2000) 19, 3961 - 3961.

The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA.

The RU5 region at the 5' RNA terminus of spleen necrosis virus (SNV) has been shown to facilitate expression of human immunodeficiency virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate gag RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the cis-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV gag reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (luc) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the luc transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5'-proximal position for efficient function. Our results indicate that RU5 contains stimulatory sequences that function in a 5'-proximal position to enhance initiation of translation of a nonretroviral reporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5' untranslated region of the retroviral RNA.

A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1.

Sciatic nerve axons in cyclin D1 knockout mice develop normally, become properly ensheathed by Schwann cells, and appear to function normally. However, in the Wallerian degeneration model of nerve injury, the mitotic response of Schwann cells is completely inhibited. The mitotic block is Schwann cell autonomous and developmentally regulated. Rescue analysis (by "knockin" of cyclin E) indicates that D1 protein, rather than regulatory elements of the D1 gene, provides the essential Schwann cell function. Genetic inhibition of the Schwann cell cycle shows that neuronal responses to nerve injury are surprisingly independent of Schwann cell mitotic responses. Even axonal regrowth into the distal zone of a nerve crush injury is not markedly impaired in cyclin D1-/- mice.

Constitutive cellular expression of PI 3-kinase is distinct from transient expression.

The discovery that the PTEN tumor suppressor encodes a phosphoinositide 3-phosphatase has raised interest in the effects of constitutive activation of PI 3-kinase. To gain insight into PI 3-kinase function, we have stably expressed a myristoylated form of the catalytic subunit p110alpha (myr-p110) in cells. The myr-p110 associated with the endogenous p85 regulatory subunit and retained lipid and protein kinase activity. Stable lines expressing myr-p110 had 2- to 4-fold more PI 3-kinase activity than controls. Expression of myr-p110 altered cellular morphology and increased the saturation density in culture. These clones were morphologically transformed but Akt and pp70(s6k) were not constitutively activated in contrast to transient assays and from tumor cell lines deficient in PTEN. In addition, the ability of PDGF to induce activation of Akt and pp70(s6k) was diminished. Therefore, expression of a myristoylated PI 3-kinase in murine fibroblasts induces a morphological transformation of the cells.