Phosphorylation of dedicator of cytokinesis 1 (Dock180) at tyrosine residue Y722 by Src family kinases mediates EGFRvIII-driven glioblastoma tumorigenesis.

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence.

MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 pm melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of beta-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.

Angiopoietin-2 stimulates breast cancer metastasis through the alpha(5)beta(1) integrin-mediated pathway.

Acquisition of a metastatic phenotype by breast cancer cells includes alternations of multigenic programs that permit tumor cells to metastasize to distant organs. Here, we report that angiopoietin-2 (Ang2), a known growth factor, is capable of promoting breast cancer cell invasion leading to metastasis. Analysis of 185 primary human breast cancer specimens that include 97 tumors showing lymph node and/or distant metastasis reveals a significant correlation between the expression of Ang2 and E-cadherin, Snail, metastatic potential, tumor grade, and lymph-vascular invasion during breast cancer progression. Using a xenograft model, we show that overexpression of Ang2 in poorly metastatic MCF-7 breast cancer cells suppresses expression of E-cadherin and induces Snail expression and phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) promoting metastasis to the lymph nodes and lung. In cell culture, Ang2 promotes cell migration and invasion in Tie2-deficient breast cancer cells through the alpha(5)beta(1) integrin/integrin-linked kinase (ILK)/Akt, GSK-3beta/Snail/E-cadherin signaling pathway. Inhibition of ILK and the alpha(5)beta(1) integrin abrogates Ang2 modulation of Akt, GSK-3beta, Snail, and E-cadherin and Ang2-stimulated breast cancer cell migration and invasion. Together, these results underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also by promoting metastasis, and provide a mechanism by which breast cancer cells acquire an enhanced invasive phenotype contributing to metastasis.

ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion.

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.

Estrogen deprivation and inhibition of breast cancer growth in vivo through activation of the orphan nuclear receptor liver X receptor.

Estrogen plays an important role in normal physiology. It is also a risk factor for breast cancer, and antiestrogen therapies have been shown to be effective in the treatment and prevention of breast cancers. The liver is important for estrogen metabolism, and a compromised liver function has been linked to hyperestrogenism in patients. In this report, we showed that the liver X receptor (LXR) controls estrogen homeostasis by regulating the basal and inducible hepatic expression of estrogen sulfotransferase (Est, or Sult1e1), an enzyme critical for metabolic estrogen deactivation. Genetic or pharmacological activation of LXR resulted in Est induction, which in turn inhibited estrogen-dependent uterine epithelial cell proliferation and gene expression, as well as breast cancer growth in a nude mouse model of tumorigenicity. We further established that Est is a transcriptional target of LXR, and deletion of the Est gene in mice abolished the LXR effect on estrogen deprivation. Interestingly, Est regulation by LXR appeared to be liver specific, further underscoring the role of liver in estrogen metabolism. Activation of LXR failed to induce other major estrogen-metabolizing enzymes, suggesting that the LXR effect on estrogen metabolism is Est specific. In summary, our results have revealed a novel mechanism controlling estrogen homeostasis in vivo and may have implications for drug development in the treatment of breast cancer and other estrogen-related cancerous endocrine disorders.

Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 expression through the alphavbeta1 integrin and focal adhesion kinase signaling pathway.

Accumulating evidence reveals a significant correlation between angiopoietin 2 (Ang2) expression and tumor invasion and metastasis in various human cancers, but the major focus of recent studies has been on the angiogenic effects of Ang2. We recently reported that Ang2-stimulated glioma cell invasion results from the up-regulation and activation of matrix metalloprotease 2 (MMP-2) in tumor cells. In this study, we identify a novel mechanism by which Ang2 stimulates MMP-2 expression leading to glioma cell invasion. We show that Ang2 interacts with alpha(v)beta(1) integrin in Tie2-deficient human glioma cells, activating focal adhesion kinase (FAK), p130(Cas), extracellular signal-regulated protein kinase (ERK) 1/2, and c-jun NH(2)-terminal kinase (JNK) and substantially enhancing MMP-2 expression and secretion. The Ang2/alpha(v)beta(1) integrin signaling pathway was attenuated by functional inhibition of beta(1) and alpha(v) integrins, FAK, p130(Cas), ERK1/2, and JNK. Furthermore, expression of a negative regulator of FAK, FAK-related nonkinase, by U87MG/Ang2-expressing glioma xenografts suppressed Ang2-induced MMP-2 expression and glioma cell infiltration in the murine brain. These data establish a functional link between Ang2 interaction with alpha(v)beta(1) integrin and glioma cell invasion through the FAK/p130(Cas)/ERK1/2 and JNK-mediated signaling pathway.

Estradiol and nicotine exposure enhances A549 bronchioloalveolar carcinoma xenograft growth in mice through the stimulation of angiogenesis.

Angiogenesis is required for lung cancer growth, which is mediated by various growth factors such as vascular endothelial growth factor (VEGF). Increases in VEGF and angiogenesis have been correlated with poor prognosis and survival in patients with lung cancer. In addition, recent reports show that estradiol and nicotine play important roles in lung tumor initiation and progression. In this report, we demonstrate that estradiol and nicotine exposure enhances the growth of A549 bronchioloalveolar carcinoma xenografts in mice through the stimulation of cell proliferation, VEGF secretion and angiogenesis. We detect a four-fold increase in microvascular density in tumors from mice exposed to estradiol and nicotine compared to control tumors resulting in an increase in tumor growth. Intriguingly, the effects on angiogenesis and tumor growth by the combination of agents were additive when compared to either agent alone. Furthermore, estradiol promotes VEGF secretion from various non-small cell lung carcinoma (NSCLC) cells and this effect is augmented by nicotine in a tumor xenograft model. These results indicate that aside from their roles in promoting cell proliferation, estradiol and nicotine appear to have additive effects on the induction of angiogenesis through the stimulation of VEGF secretion during NSCLC progression.

Melatonin receptors and their regulation: biochemical and structural mechanisms.

There is growing evidence demonstrating the complexity of melatonin's role in modulating a diverse number of physiological processes. This complexity could be attributed to the fact that melatonin receptors belong to two distinct classes of proteins, that is, the G-protein coupled receptor superfamily (MT(1), MT(2)) and the quinone reductase enzyme family (MT(3)) which makes them unique at the molecular level. Also, within the G-protein coupled receptor family of proteins, the MT(1) and MT(2) receptors can couple to multiple and distinct signal transduction cascades whose activation can lead to unique cellular responses. Also, throughout the 24-hour cycle, the receptors' sensitivity to specific cues fluctuates and this sensitivity can be modulated in a homologous fashion, that is, by melatonin itself, and in a heterologous manner, that is, by other cues including the photoperiod or estrogen. This sensitivity of response may reflect changes in melatonin receptor density that also occurs throughout the 24-hour light/dark cycle but out of phase with circulating melatonin levels. The mechanisms that underlie the changes in melatonin receptor density and function are still not well-understood, but data is beginning to show that transcriptional events and G-protein uncoupling may be involved. Even though this area of research is still in its infancy, great strides are being made everyday in elucidating the mechanisms that underlie melatonin receptor function and regulation. The focus of this review is to highlight some of these discoveries in an attempt to reveal the uniqueness of the melatonin receptor family while at the same time provide thought-provoking ideas to further advance this area of research. Thus, a brief overview of each of the mammalian melatonin receptor subtypes and the signal transduction cascades to which they couple will be discussed with a greater emphasis placed on the mechanisms underlying their regulation and the domains within the receptors essential for proper signaling.

ADP-ribosylation factor 6 regulates glioma cell invasion through the IQ-domain GTPase-activating protein 1-Rac1-mediated pathway.

A common pathobiological feature of malignant gliomas is the insidious infiltration of single tumor cells into the brain parenchyma, rendering these deadly tumors virtually incurable with available therapies. In this study, we report that ADP-ribosylation factor 6 (ARF6), a Ras superfamily small GTPase, is abundantly expressed in invasive human glioma cells. Cellular depletion of ARF6 by small interfering RNA decreased Rac1 activation, impaired HGF-stimulated and serum-stimulated glioma cell migration in vitro, and markedly decreased the invasive capacity of invasive glioma in the brain. Furthermore, ectopic expression of ARF6 in glioma cells promoted cell migration via the activation of Rac1. Upon stimulation of glioma cells with HGF, we show that IQ-domain GTPase-activating protein 1 (IQGAP1) is recruited and overlaps with ARF6 at the leading edge of migrating cells. However, cellular depletion of ARF6 abrogated this recruitment of IQGAP1 and attenuated the formation of surface protrusions. ARF6 forms complexes with Rac1 and IQGAP1 in glioma cells upon HGF stimulation, and knockdown of IQGAP1 significantly inhibits ARF6-induced Rac1 activation and cell migration. Taken together, these data suggest that ARF6-mediated Rac1 activation is essential for glioma cell invasion via a signaling pathway that requires IQGAP1.

Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity.

Acquisition of insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Slit2, a chemorepulsive factor, controls cell migration of neuronal and glial cells during development and inhibits chemotaxic migration of various types of cells in vitro. However, the role of Slit2 in vitro remains controversial, and the biological significance of Slit2 expression in cancer cell invasion in vivo has not yet been determined. In the present study, we characterized the effects of Slit2 expression on the migration and invasion of invasive glioma cells in vitro and in vivo. By reverse transcriptase polymerase chain reaction (PCR) analyses, Slit2 was found to be expressed at lower levels in primary glioma specimens and invasive glioma cells compared with normal human brain cells and astrocytes. Ectopic expression of Slit2 or treatment with recombinant Slit2 on glioma cells attenuates cell migration and invasion through inhibition of Cdc42 activity in vitro. Cellular depletion of Robo1, a cognate receptor for Slit2, prevented Slit2 inhibition of Cdc42 activity and glioma cell migration. In vivo, expression of Slit2 by invasive SNB19 glioma cells markedly inhibited glioma cell infiltration into the brain of mice. Moreover, impediment of glioma cell invasion by Slit2 did not affect the expression of N-cadherin and beta-catenin in glioma cells. These results provide the first evidence demonstrating that Slit2-Robo1 inhibits glioma invasion through attenuating Cdc42 activity in vitro and in the brain. Understanding the mechanisms of Slit2-Robo1 inhibition of glioma cell invasion will foster new treatments for malignant gliomas.

Glucocorticoids antagonize estrogens by glucocorticoid receptor-mediated activation of estrogen sulfotransferase.

Glucocorticoids and estrogens are two classes of steroid hormones that have essential but distinct physiologic functions. Estrogens also represent a risk factor for breast cancer. It has been suggested that glucocorticoids can attenuate estrogen responses, but the mechanism by which glucocorticoids inhibit estrogenic activity is unknown. In this study, we show that activation of glucocorticoid receptor (GR) by dexamethasone (DEX) induced the expression and activity of estrogen sulfotransferase (SULT1E1 or EST), an enzyme important for the metabolic deactivation of estrogens, because sulfonated estrogens fail to activate the estrogen receptor. Treatment with DEX lowered circulating estrogens, compromised uterine estrogen responses, and inhibited estrogen-dependent breast cancer growth in vitro and in a xenograft model. We further showed that the mouse and human SULT1E1 genes are transcriptional targets of GR and deletion of Sult1e1/Est in mice abolished the DEX effect on estrogen responses. These findings have revealed a novel nuclear receptor-mediated and metabolism-based mechanism of estrogen deprivation, which may have implications in therapeutic development for breast cancers. Because glucocorticoids and estrogens are widely prescribed drugs, our results also urge caution in avoiding glucocorticoid-estrogen interactions in patients.

Modulation of melatonin receptors and G-protein function by microtubules.

Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin-induced outgrowths in MT1-expressing CHO cells, also prevents MT1 receptor desensitization by affecting G(alpha)-GTP exchange on G-proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin-induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin-treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G-proteins. Depolymerization of microtubules during melatonin-induced desensitization, attenuated forskolin-induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of G(i alpha) protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into G(i) proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/G(q) coupling nor did it affect melatonin-induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin-induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on G(i) proteins and impact on downstream signaling cascades.

Up-regulation of angiopoietin-2, matrix metalloprotease-2, membrane type 1 metalloprotease, and laminin 5 gamma 2 correlates with the invasiveness of human glioma.

Diffuse infiltration of malignant human glioma cells into surrounding brain structures occurs through the activation of multigenic programs. We recently showed that angiopoietin-2 (Ang2) induces glioma invasion through the activation of matrix metalloprotease-2 (MMP-2). Here, we report that up-regulation of Ang2, MMP-2, membrane type 1-MMP (MT1-MMP), and laminin 5 gamma 2 (LN 5 gamma 2) in tumor cells correlates with glioma invasion. Analyses of 57 clinical human glioma biopsies of World Health Organization grade I to IV tumors displaying a distinct invasive edge and 39 glioma specimens that only contain the central region of the tumor showed that Ang2, MMP-2, MT1-MMP, and LN 5 gamma 2 were co-overexpressed in invasive areas but not in the central regions of the glioma tissues. Statistical analyses revealed a significant link between the preferential expression of these molecules and invasiveness. Protein analyses of microdissected primary glioma tissue showed up-regulation and activation of MT1-MMP and LN 5 gamma 2 at the invasive edge of the tumors, supporting this observation. Concordantly, in human U87MG glioma xenografts engineered to express Ang2, increased expression of MT1-MMP and LN 5 gamma 2, along with MMP-2 up-regulation, in actively invading glioma cells was also evident. In cell culture, stimulation of glioma cells by overexpressing Ang2 or exposure to exogenous Ang2 promoted the expression and activation of MMP-2, MT1-MMP, and LN 5 gamma 2. These results suggest that up-regulation of Ang2, MMP-2, MT1-MMP, and LN 5 gamma 2 is associated with the invasiveness displayed by human gliomas and that induction of these molecules by Ang2 may be essential for glioma invasion.