Shuxian Jiang

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.

Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor α

The interaction between 17β‐estradiol and estrogen receptor alpha (ER‐α) plays an important role in breast carcinogenesis and breast cancer treatment. ER‐α is a critical growth regulatory gene in breast cancer and its expression level is tightly linked to the prognosis and treatment outcomes of breast cancer patients. Loss of ER‐α expression in breast epithelial cells is critical for breast cancer progression. The underlying molecular mechanisms for this loss, however, are poorly defined. Histone deacetylases (HDACs) are implicated in the alteration of chromatin assembly and tumorigenesis. We show that histone deacetylase 1 (HDAC1) interacts with ER‐α in vitro and in vivo and suppresses ER‐α transcription activity. The interaction of HDAC1 with ER‐α was mediated by the AF‐2 and DBD domains of ER‐α. We observed an endogenous interaction of HDAC1 with ER‐α in breast cancer cells, which was decreased in the presence of estrogen. Interestingly, overexpression of HDAC1 in stable transfected MCF‐7 clones induced loss of ER‐α and significantly increased cell proliferation and colony formation, as compared to the control MCF‐7 cells, whereas treatment of stable MCF‐7 clones with the HDAC specific inhibitor trichostatin A (TSA) induced re‐expression of ER‐α mRNA and protein. Our findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in association with a reduction in both ER‐α protein expression and transcriptional activity. Thus, HDAC1 may be a potential target for therapeutic intervention in the treatment of a subset of ER‐negative breast cancers.

DIRECT ASSOCIATION OF CSK HOMOLOGOUS KINASE (CHK) WITH THE DIPHOSPHORYLATED SITE TYR568/570 OF THE ACTIVATED C-KIT IN MEGAKARYOCYTES

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2CHK to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2CHK-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2CHK. These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2CHK-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2CHK to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2CHK binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2CHK. This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

HIV-1 Tat-Mediated Apoptosis in Human Brain Microvascular Endothelial Cells

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-l-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-l-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.

Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.

The Csk Homologous Kinase Associates with TrkA Receptors and Is Involved in Neurite Outgrowth of PC12 Cells

Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.

HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells.

The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells (BMECs). The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia despite the lack of productive HIV infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. In this study we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in vitro on primary human BMECs (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites compared with control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat Abs or with the specific inhibitor SU-1498, which interferes with vascular endothelial growth factor receptor type 2 (Flk-1/KDR) phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and was found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant interfering mutant form, FAK-related nonkinase, significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time-dependent manner. Tat also impaired BBB permeability, as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs via activation of FAK, leading to cytoskeletal reorganization and permeability changes.

Expression and Function of Cannabinoid Receptors CB1 and CB2 and Their Cognate Cannabinoid Ligands in Murine Embryonic Stem Cells

Background Characterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES) cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation. Methods and Findings The cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are members of the G-protein coupled receptor (GPCR) family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively) induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Δ9-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists. Conclusions This work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell survival and hematopoietic differentiation.

Angiopoietin-2 mediates blood–brain barrier impairment and colonization of triple-negative breast cancer cells in brain

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple‐negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood–brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO‐1 and claudin‐5 tight junction (TJ) protein structures. Angiopoietin‐2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang‐2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang‐2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang‐2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang‐2 may serve as potential therapeutics for brain metastasis. Copyright

Genetic alterations of the NRP/B gene are associated with human brain tumors

Nearly all brain tumors develop following the progressive accumulation of genetic alterations of oncogenes and tumor suppressor genes (such as p53 and retinoblastoma protein). Furthermore, aberrations in the nuclear matrix often contribute to genomic instabilities and the development of cancer. We have previously shown that nuclear-restricted protein/brain (NRP/B), a member of the BTB/Kelch repeat family, is a nuclear matrix protein normally expressed in neurons but not in astrocytes, and that it is an early and specific marker of neurons during the development of the central nervous system. Here, we show aberrant expression of NRP/B in human brain tissues. NRP/B is expressed in the cytoplasm of human brain tumor cells (glioblastoma, GBM) arising from astrocytes. NRP/B mutations (13 mutations in the Kelch domains, two in the intervening sequence (IVS) domain and two in the BTB domain) were detected in brain tumor cell lines (A-172, CCF-STTG1, SK-N-SH and U87-MG) and in primary human malignant GBM tissues (eight samples). More importantly, we found that NRP/B mutants, but not wild-type (wt) NRP/B, increased the activation of ERK and consequently promoted cell proliferation, attenuated caspase activation and suppressed the cellular apoptosis induced by the stressful stimulus cisplatin (10 μM). These events were observed to occur via a p53-mediated pathway. In addition, while wt NRP/B was associated with actin, mutations in the Kelch domains of NRP/B led to its reduced binding affinity to actin. Thus, alterations and gene mutations within the NRP/B gene may contribute to brain tumorigenesis by promoting cell proliferation, suppressing apoptosis and by affecting nuclear cytoskeleton dynamics.