IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer.

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.

Nuclear Insulin-Like Growth Factor Binding Protein-3 As a Biomarker in Triple-Negative Breast Cancer Xenograft Tumors: Effect of Targeted Therapy and Comparison With Chemotherapy.

Triple-negative breast cancer (TNBC) typically has a worse outcome than other breast cancer subtypes, in part owing to a lack of approved therapeutic targets or prognostic markers. We have previously described an oncogenic pathway in basal-like TNBC cells, initiated by insulin-like growth factor binding protein-3 (IGFBP-3), in which the epidermal growth factor receptor (EGFR) is transactivated by sphingosine-1-phosphate (S1P) resulting from sphingosine kinase (SphK)-1 activation. Oncogenic IGFBP-3 signaling can be targeted by combination treatment with the S1P receptor modulator and SphK inhibitor, fingolimod, and the EGFR kinase inhibitor, gefitinib (F + G). However, the interaction of this treatment with chemotherapy has not been documented. Since we observed nuclear localization of IGFBP-3 in some TNBC tumors, this study aimed to evaluate the prognostic significance of nuclear IGFBP-3 in pre-clinical models of basal-like TNBC treated with F + G and doxorubicin. Orthotopic xenograft tumors were grown in nude mice from the human basal-like TNBC cell lines MDA-MB-468 and HCC1806, and were treated with gefitinib, 25 mg/Kg, plus fingolimod, 5 mg/Kg, 3-times weekly. In some studies, doxorubicin was also administered once weekly for 6 weeks. Tumor tissue proteins were quantitated by immunohistochemistry (IHC). Interaction between doxorubicin and F + G was also studied in proliferation assays in vitro. In both tumor models, tissue staining for IGFBP-3 was predominantly nuclear. Combination of F + G significantly enhanced mouse survival, decreased nuclear IGFBP-3 and Ki67 staining, and increased apoptosis (cleaved caspase-3) staining. Kaplan-Meier survival analysis showed that a high tumor IGFBP-3 IHC score (>median), like a high Ki67 score, was significantly associated with shorter survival time, whereas a high apoptosis score was associated with prolonged survival. Studied in vitro in both cell lines, low-dose doxorubicin that had little effect alone, strongly enhanced the cytostatic effect of low-dose F + G combination. However, in both in vivo models, doxorubicin at maximum-tolerated dose neither inhibited tumor growth when administered alone, nor enhanced the significant inhibitory effect of F + G. We conclude that doxorubicin may not add benefit to the inhibitory effect of F + G unless its dose-limiting toxicity can be overcome. Nuclear IGFBP-3 appears to have potential as a prognostic marker in TNBC and could be evaluated for clinical utility.

Inhibition of basal-like breast cancer growth by FTY720 in combination with epidermal growth factor receptor kinase blockade.

BACKGROUND: New molecular targets are needed for women with triple-negative breast cancer (TNBC). This pre-clinical study investigated the combination of the EGFR inhibitor gefitinib with the sphingosine kinase (SphK) inhibitor FTY720 (Fingolimod), aiming to block tumorigenic signaling downstream of IGFBP-3, which is abundantly expressed in basal-like TNBC. METHODS: In studies of breast cancer cell growth in culture, proliferation was monitored by IncuCyte live-cell imaging, and protein abundance was determined by western blotting. In vivo studies of mammary tumor growth used two models: orthotopic xenograft tumors derived from three basal-like TNBC cell lines, grown in immune-deficient mice, and syngeneic murine 4T1 tumors grown in immune-competent mice. Protein abundance in tumor tissue was assessed by immunohistochemistry. RESULTS: Quantitated by live-cell imaging, the inhibitor combination showed synergistic cytostatic activity in basal-like cell lines across several TNBC molecular subtypes, the synergy being decreased by IGFBP-3 downregulation. Suppression of the tumorigenic mediator CD44 by gefitinib was potentiated by FTY720, consistent with CD44 involvement in the targeted pathway. In MDA-MB-468 and HCC1806 orthotopic TNBC xenograft tumors in nude mice, the drug combination inhibited tumor growth and prolonged mouse survival, although this effect was not significant for the gefitinib-resistant cell line HCC70. Combination treatment of murine 4T1 TNBC tumors in syngeneic BALB/c mice was more effective in immune-competent than immune-deficient (nude) mice, and a relative loss of tumor CD3 (T-cell) immunoreactivity caused by FTY720 treatment alone was alleviated by the drug combination, suggesting that, even at an FTY720 dose causing relative lymphopenia, the combination is still effective in an immune-competent setting. Immunohistochemistry of xenograft tumors showed significant enhancement of caspase-3 cleavage and suppression of Ki67 and phospho-EGFR by the drug combination, but SphK1 downregulation occurred only in MDA-MB-468 tumors, so is unlikely to be integral to treatment efficacy. CONCLUSIONS: Our data indicate that targeting IGFBP-3-dependent signaling pathways through gefitinib-FTY720 co-therapy may be effective in many basal-like breast cancers, and suggest tissue IGFBP-3 and CD44 measurement as potential biomarkers of treatment efficacy.

Involvement of p53 in insulin-like growth factor binding protein-3 regulation in the breast cancer cell response to DNA damage.

Chemotherapy drugs that induce apoptosis by causing DNA double-strand breaks, upregulate the tumor suppressor p53. This study investigated the regulation of the growth-regulatory protein insulin-like growth factor binding protein-3 (IGFBP-3), a p53 target, by DNA-damaging agents in breast cancer cells. IGFBP-3 was upregulated 1.4- to 13-fold in response to doxorubicin and etoposide in MCF-10A, Hs578T, MCF-7 and T47D cells, which express low to moderate basal levels of IGFBP-3. In contrast, IGFBP-3 was strongly downregulated by these agents in cells with high basal levels of IGFBP-3 (MDA-MB-231, MDA-MB-436 and MDA-MB-468). In MDA-MB-468 cells containing the R273H p53 mutation, reported to display gain-of-function properties, chemotherapy-induced suppression of IGFBP-3 was not reversed by the p53 reactivating drug, PRIMA-1, or by p53 silencing, suggesting that the decrease in IGFBP-3 following DNA damage is not a mutant p53 gain-of-function response. SiRNA-mediated downregulation of endogenous IGFBP-3 modestly attenuated doxorubicin-induced apoptosis in MDA-MB-468 and Hs578T cells. IGFBP-3 downregulation in some breast cancer cell lines in response to DNA-damaging chemotherapy may have clinical implications because suppression of IGFBP-3 may modulate the apoptotic response. These observations provide further evidence that endogenous IGFBP-3 plays a role in breast cancer cell responsiveness to DNA damaging therapy.

Targeting Insulin-Like Growth Factor Binding Protein-3 Signaling in Triple-Negative Breast Cancer.

Insulin-like growth factor binding protein-3 (IGFBP-3) is a key regulatory molecule of the IGF axis and can function in a tissue-specific way as both a tumor suppressor and promoter. Triple-negative breast cancer (TNBC) has high tumor expression of IGFBP-3 associated with markers of poor prognosis and, although accounting for 15-20% of all breast cancers, is responsible for disproportionate rates of morbidity and mortality. Because they lack estrogen and progesterone receptors and overexpression of HER2, TNBC are resistant to treatments that target these molecules, making the development of new therapies an important goal. In addition to frequent high expression of IGFBP-3, these tumors also express EGFR highly, but targeting EGFR signaling alone in TNBC has been of little success. Identification of a functional growth-stimulatory interaction between EGFR and IGFBP-3 signaling prompted investigation into cotargeting these pathways as a novel therapy for TNBC. This involves inhibition of both EGFR kinase activity and a mediator of IGFBP-3's stimulatory bioactivity, sphingosine kinase-1 (SphK1), and has shown promise in a preclinical setting. Functional interaction between EGFR and IGFBP-3 may also promote chemoresistance in TNBC, and delineating the mechanisms involved may identify additional targets for development of therapies in cancers that express both IGFBP-3 and EGFR.

The first crop plant genetically engineered to release an insect pheromone for defence.

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-ß-farnesene (Eßf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eßf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

Repression of breast cancer cell growth by proteasome inhibitors in vitro: impact of mitogen-activated protein kinase phosphatase 1.

Mitogen activated protein kinase phosphatase-1 (MKP-1) has emerged as an important protein mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies, especially proteasome inhibitors. In this in vitro study, we utilized the breast cancer epithelial cell lines MCF-7 and MDA-MB-231, in comparison to MCF-10A control cells, to examine the impact of MKP-1 on breast cancer cell growth and repression by proteasome inhibitors. We confirm that proteasome inhibitors MG-132 and bortezomib induce MKP-1 protein upregulation and we show that one of the ways in which bortezomib increases MKP-1 in breast cancer cells, in addition to inhibition of ubiquitin-proteasome system, is via upregulation of MKP-1 mRNA expression in p38 MAPK-mediated manner. Notably, these effects are specific to cancer cells, as bortezomib activated p38 MAPK and induced MKP-1 in MCF-7 and MDA-MB-231 breast cancer cells, but not in control cells (MCF-10A). We took a dual approach toward targeting MKP-1 to show that bortezomib-induced effects are enhanced. Firstly, treatment with the non-specific MKP-1 inhibitor triptolide reduces breast cancer cell growth and augments proteasome inhibitor-induced effects. Secondly, specific knock-down of MKP-1 with siRNA significantly repressed cell viability by reduced cyclin D1 expression, and enhanced repression of cancer cell growth by proteasome inhibitors. Taken together, these results indicate that removing the unwanted (MKP-1-inducing) effects of bortezomib significantly improves the efficacy of proteasome inhibition in breast cancer cells. Thus, future development of drugs targeting MKP-1 offer promise of combination therapies with reduced toxicity and enhanced cell death in breast cancer.

Involvement of the insulin-like growth factor binding proteins in the cancer cell response to DNA damage.

The complex mechanisms that cells have evolved to meet the challenge of constant exposure to DNA-damaging stimuli, also serve to protect cancer cells from the cytotoxic effects of chemo- and radiotherapy. IGFBPs appear to be involved, directly or indirectly, in some of these protective mechanisms. Activation of p53 is an early response to genotoxic stress, and all six human IGFBP genes have predicted p53 response elements in their promoter and/or intronic regions, at least some of which are functional. IGFBP3 has been extensively characterized as a p53-inducible gene, but in some cases it is suppressed by mutant p53 forms. DNA double-strand breaks (DSBs), induced by radiotherapy and some chemotherapies, potentially lead to apoptotic cell death, senescence, or repair and recovery. DSB damage can be repaired by homologous recombination or non-homologous end-joining (NHEJ), depending on the cell cycle stage, availability of key repair proteins, and other factors. The epidermal growth factor receptor (EGFR) has been implicated in the NHEJ pathway, and EGFR inhibition may inhibit repair, promoting apoptosis and thus improving sensitivity to chemotherapy or radiotherapy. Both IGFBP-3 and IGFBP-6 interact with components of the NHEJ pathway, and IGFBP-3 can facilitate this process through direct interaction with both EGFR and the catalytic subunit of DNA-PK. Cell fate after DNA damage may in part be regulated by the balance between the sphingolipids ceramide and sphingosine-1-phosphate, and IGFBPs can influence the production of both lipids. A better understanding of the involvement of IGFBPs in the DNA damage response in cancer cells may lead to improved methods of sensitizing cancers to DNA-damaging therapies.

Inhibition of insulin-like growth factor-binding protein-3 signaling through sphingosine kinase-1 sensitizes triple-negative breast cancer cells to EGF receptor blockade.

The type I EGF receptor (EGFR or ErbB1) and insulin-like growth factor-binding protein-3 (IGFBP-3) are highly expressed in triple-negative breast cancer (TNBC), a particularly aggressive disease that cannot be treated with conventional therapies targeting the estrogen or progesterone receptors (ER and PR), or HER2. We have shown previously in normal breast epithelial cells that IGFBP-3 potentiates growth-stimulatory signaling transduced by EGFR, and this is mediated by the sphingosine kinase-1 (SphK1)/sphingosine 1-phosphate (S1P) system. In this study, we investigated whether cotargeting the EGFR and SphK1/S1P pathways in TNBC cells results in greater growth inhibition compared with blocking either alone, and might therefore have novel therapeutic potential in TNBC. In four TNBC cell lines, exogenous IGFBP-3 enhanced ligand-stimulated EGFR activation, associated with increased SphK1 localization to the plasma membrane. The effect of exogenous IGFBP-3 on EGFR activation was blocked by pharmacologic inhibition or siRNA-mediated silencing of SphK1, and silencing of endogenous IGFBP-3 also suppressed EGF-stimulated EGFR activation. Real-time analysis of cell proliferation revealed a combined effect of EGFR inhibition by gefitinib and SphK1 inhibition using SKi-II. Growth of MDA-MB-468 xenograft tumors in mice was significantly inhibited by SKi-II and gefitinib when used in combination, but not as single agents. We conclude that IGFBP-3 promotes growth of TNBC cells by increasing EGFR signaling, that this is mediated by SphK1, and that combined inhibition of EGFR and SphK1 has potential as an anticancer therapy in TNBC in which EGFR and IGFBP-3 expression is high.

Responses of herbivore and predatory mites to tomato plants exposed to jasmonic acid seed treatment.

Jasmonic acid (JA) signalling can influence plant defense and the production of plant volatiles that mediate interactions with insects. Here, we tested whether a JA seed treatment could alter direct and indirect defenses. First, oviposition levels of herbivorous mites, Tetranychus urticae, on JA seed-treated and control tomato plants were compared. They were not significantly different on tomato cv. 'Moneymaker', however, there was a significant reduction in oviposition on treated plants in additional experiments with cv. 'Carousel'. Second, responses of predatory mites, Phytoseiulus persimilis, were assessed in a Y-tube olfactometer. Volatiles from JA seed-treated tomato cv. 'Moneymaker' plants were significantly more attractive than volatiles from control plants. Volatiles collected from plants were analysed by GC/MS, and samples from JA seed-treated plants contained more methyl salicylate and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) than samples from control plants. Our results indicate that JA seed treatment can make tomato plants more attractive to predatory mites, but that direct effects on herbivorous mites are variable and cultivar dependent.

Silencing the mannose 6-phosphate/IGF-II receptor differentially affects tumorigenic properties of normal breast epithelial cells.

Although loss of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) in breast cancer is believed to play a role in tumorigenesis, it has not been demonstrated that M6P/IGF-IIR loss is sufficient to confer a malignant phenotype in an untransformed cell. We investigated the impact of M6P/IGF-IIR silencing using phenotypically normal (MCF-10A) and oncogenically transformed (MCF-10T, the c-Ha-ras transformed derivative of MCF-10A) human breast epithelial cell lines as model systems. In both cell lines, silencing of M6P/IGF-IIR increased cell proliferation and motility, with the effects being more pronounced in MCF-10A cells. Although anchorage-independent growth was increased by M6P/IGF-IIR silencing in MCF-10T cells, MCF-10A cells did not acquire the ability to grow in soft agar. Conversely, reduced M6P/IGF-IIR expression increased the invasive potential of MCF-10A cells, but did not enhance the already high rate of invasion of MCF-10T cells. M6P/IGF-IIR silencing had no effect on basal or IGF-II-stimulated IGF-I receptor (IGF-IR) or AKT phosphorylation in either cell line, but both were abrogated by IGF-IR kinase inhibition, which also reduced the stimulatory effect of M6P/IGF-IIR silencing on proliferation under basal and IGF-II-stimulated conditions in both cell lines. However, cell motility was neither stimulated by IGF-II nor reduced by IGF-IR inhibition, suggesting that potentiation of specific tumorigenic features in response to M6P/IGF-IIR silencing involves IGF-II- dependent and -independent mechanisms. Collectively, these data suggest that M6P/IGF-IIR silencing alone is insufficient to confer a tumorigenic phenotype, but can enhance tumorigenicity in an already transformed cell.

Activation of defence in sweet pepper, Capsicum annum, by cis-jasmone, and its impact on aphid and aphid parasitoid behaviour.

BACKGROUND: Two important pests of the sweet pepper, Capsicum annuum, are the peach potato aphid, Myzus persicae, and the glasshouse potato aphid, Aulacorthum solani. Current aphid control measures include the use of biological control agents, i.e., parasitic wasps, but with varying levels of success. One option to increase parasitoid efficiency is to activate plant defence. Therefore, sweet pepper plants were treated with the naturally occurring plant defence activator cis-jasmone, and its impact upon the behaviour and development of aphids and aphid parasitoids was investigated. RESULTS: Growth rate studies revealed that the intrinsic rate of population increase of A. solani and M. persicae on sweet pepper plants treated with cis-jasmone (cJSP) was not affected compared with untreated plants (UnSP), but the positive behavioural response of alate M. persicae towards the volatile organic compounds (VOCs) from UnSP was eliminated by cis-jasmone treatment 48 h previously (cJSP48). In addition, the aphid parasitoid Aphidius ervi preferred VOCs from cJSP48 compared with UnSP, and a significant increase in foraging time was also observed on cJSP. Analysis of VOCs collected from cJSP48 revealed differences compared with UnSP. CONCLUSION: There is evidence that treatment with cis-jasmone has the potential to improve protection of sweet pepper against insect pests.

Signalling pathways of insulin-like growth factors (IGFs) and IGF binding protein-3.

Although the insulin-like growth factor (IGF) system is essential for normal growth and development, its dysregulation has been implicated in a range of pathological states. The peptide growth factors IGF-I and IGF-II exert their effects by binding to cell-surface heterotetrameric tyrosine kinase receptors and activating multiple intracellular signalling cascades, leading to changes in the expression of proteins essential for cell proliferation, survival and differentiation. The IGF system comprises multiple ligands, receptors and high-affinity IGF binding proteins (IGFBPs), with added complexity arising from crosstalk between its receptors and other key growth-regulatory pathways such as those activated by steroid hormones, integrins and other receptor tyrosine kinases. The IGFBPs are also increasingly recognised for their intrinsic growth-regulatory activity, and the ability of IGFBP-3 to modulate signalling pathways of nuclear hormone and growth factor receptors, as well as novel receptors, is believed to play a role both in normal physiology and in disease.

Involvement of insulin-like growth factor-binding protein-3 in the effects of histone deacetylase inhibitor MS-275 in hepatoma cells.

Insulin-like growth factor-binding protein-3 (IGFBP-3) expression is frequently suppressed in liver cancers and can be reactivated by histone deacetylase (HDAC) inhibition. This study examined the role of IGFBP-3 in mediating the effects of the HDAC inhibitor MS-275 in liver cancer cells and identified IGFBP-3-dependent proteins that regulate proliferation and migration. In HepG2 cells, MS-275 inhibited DNA synthesis, cell cycle activity, and cell viability concomitantly with increased binding of acetylated histone H3 to IGFBP-3 promoter sequences and induction of IGFBP-3 expression. IGFBP-3 down-regulation by siRNA significantly reversed the inhibition of cell viability and DNA synthesis by MS-275, indicating an intermediary role for IGFBP-3. Induction of the cyclin-dependent kinase inhibitor p21 by MS-275 was attenuated by IGFBP-3 down-regulation, providing an explanation for IGFBP-3-dependent effects of MS-275 on cell cycle activity. In contrast, MS-275 stimulated HepG2 cell migration, an effect also inhibited by IGFBP-3 down-regulation. Among genes whose induction by MS-275 was attenuated by IGFBP-3 down-regulation, LYVE1 and THBS2 (thrombospondin-2) were identified as mediators of IGFBP-3-dependent effects of MS-275. Silencing of either protein had no effect on the inhibition of HepG2 viability by MS-275 but reversed its stimulatory effect on cell migration. We conclude that among genes up-regulated by MS-275, IGFBP-3 is a key mediator of effects on hepatoma cell growth and migration, involving IGFBP-3-dependent proteins p21 (proliferation) and LYVE1 and THBS2 (migration). The enhanced cell motility that accompanies reactivation of IGFBP-3 expression in liver cancer by HDAC inhibition suggests the possibility of increased metastatic spread despite inhibited cell proliferation.

Development of semiochemical attractants for monitoring bean seed beetle, Bruchus rufimanus.

BACKGROUND: Bruchus rufimanus is a serious pest of field beans. The objective here was to develop a semiochemical-baited trapping system to facilitate monitoring of the pest. RESULTS: Volatile compounds that were electrophysiologically active with the antennae of B. rufimanus females were identified from headspace samples of Vicia faba flowers and from male B. rufimanus. Selected headspace samples and synthetic compounds were tested in olfactometer bioassays. The semiochemicals were then formulated in lures for traps and evaluated in a field trapping experiment. Cone traps baited with a three-component blend of floral volatiles, releasing (R)-linalool (17.7 mg day(-1)), cinnamyl alcohol (0.4 mg day(-1)) and cinnamaldehyde (0.77 mg day(-1)), caught significantly more of both sexes of B. rufimanus than unbaited control traps. A male volatile, 1-undecene, was EAG active with female antennae. It was attractive to females in an olfactometer, indicating that it is a sex pheromone. However, in the field it only enhanced trap catches if it was released together with the floral volatiles. CONCLUSION: The blends of semiochemicals identified were shown to be attractive in cone traps under field conditions. The prototype trapping system developed could be used as a monitoring tool to determine infestation levels of B. rufimanus in bean fields.

Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity.

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

Rapamycin treatment for a child with germline PTEN mutation.

BACKGROUND: A 9-month-old boy with Proteus syndrome and a de novo germline mutation in the tumor suppressor PTEN was referred to a specialist centre for management. Over the first years of life, the patient developed life-threatening respiratory dysfunction and malnutrition because of progressive growth of hamartomas affecting the chest, mediastinum, abdomen and pelvis. INVESTIGATIONS: Physical examination, CT scans of the mediastinum, pelvis and abdomen, measurement of serum insulin-like growth factor binding protein-2, and investigation of the effect of the PTEN mutation on phosphatidylinositol 3-kinase/mammalian target of rapamycin signaling in an in vitro cell model. DIAGNOSIS: PTEN hamartoma tumor syndrome, specifically Proteus syndrome. MANAGEMENT: Oral rapamycin.

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum.

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.

Expression of insulin-like growth factor binding protein-2 by MCF-7 breast cancer cells is regulated through the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway.

IGF binding protein-2 (IGFBP-2) has been implicated in the development and spread of a number of tumor types, and its abrogation in experimental models of cancer is associated with decreased tumor growth. This suggests that targeted inhibition of IGFBP-2 expression in some cancers may have therapeutic benefit. In this study, we investigated signaling pathways involved in extracellular IGFBP-2 expression in an IGF- and estrogen-responsive breast cancer cell line, MCF-7. IGFBP-2 was present at approximately 150 ng per 10(6) cells in serum-free MCF-7-conditioned medium and constituted the predominant IGFBP. Inhibition of the phosphatidylinositol 3-kinase signaling pathway using LY294002, or the downstream signaling intermediate mammalian target of rapamycin using rapamycin, markedly reduced IGFBP-2 in conditioned medium to approximately 25% of untreated levels (P < 0.001); there was no effect of inhibition of p38 MAPK, and an inhibitor of p44/42 MAPK activation, PD98059, caused only a slight reduction in extracellular IGFBP-2. IGFBP-2 levels were increased 25-30% by estradiol, whereas IGF-I (100 ng/ml) increased IGFBP-2 levels 2-fold (P < 0.001) by a type 1 IGF receptor (IGFR1)-dependent mechanism. Estradiol enhanced the effect of IGF-I on IGFBP-2 levels, and this was associated with increased phosphorylation of IGFR1. Basal, IGF-, or estradiol-stimulated IGFBP-2 was abrogated by LY294002 and rapamycin and an inhibitor of IGFR1 tyrosine kinase activity, AG1024. Modulation of intracellular hypoxia-inducible factor-1alpha had no effect on IGFBP-2 expression. These findings indicate that IGFBP-2 is regulated predominantly through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway, the target of a number of anticancer agents currently in clinical trial and use.

Development of a pheromone trap monitoring system for orange wheat blossom midge, Sitodiplosis mosellana, in the UK.

Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.