Angiopoietin-4 increases permeability of blood vessels and promotes lymphatic dilation.

The angiopoietin (Ang) ligands are potential therapeutic targets for lymphatic related diseases, which include lymphedema and cancer. Ang-1 and Ang-2 functions are established, but those of Ang-4 are poorly understood. We used intravital fluorescence microscopy to characterize Ang-4 actions on T241 murine fibrosarcoma-associated vessels in mice. The diameters of lymphatic vessels draining Ang-4- or VEGF-C (positive control)-expressing tumors increased to 123 and 135 µm, respectively, and parental, mock-transduced (negative controls) and tumors expressing Ang-1 or Ang-2 remained at baseline (∼60 µm). Ang-4 decreased human dermal lymphatic endothelial cell (LEC) monolayer permeability by 27% while increasing human dermal blood endothelial cell (BEC) monolayer permeability by 200%. In vivo, Ang-4 stimulated a 4.5-fold increase in tumor-associated blood vessel permeability compared with control when measured using intravital quantitative multiphoton microscopy. Ang-4 activated receptor signaling in both LECs and BECs, evidenced by tyrosine kinase with Ig and endothelial growth factor homology domains-2 (TIE2) receptor, protein kinase B, and Erk1,2 phosphorylation detectable by immunoblotting. These data suggest that Ang-4 actions are mediated through cell-type-specific networks and that lymphatic vessel dilation occurs secondarily to increased vascular leakage. Ang-4 also promoted survival of LECs. Thus, blocking Ang-4 may prune the draining lymphatic vasculature and decrease interstitial fluid pressure (IFP) by reducing vascular permeability.

Vascular endothelial growth factor-C enhances radiosensitivity of lymphatic endothelial cells.

Radiation therapy after lymph node dissection increases the risk of developing painful and incurable lymphedema in breast cancer patients. Lymphedema occurs when lymphatic vessels become unable to maintain proper fluid balance. The sensitivity of lymphatic endothelial cells (LECs) to ionizing radiation has not been reported to date. Here, the radiosensitivity of LECs in vitro has been determined using clonogenic survival assays. The ability of various growth factors to alter LEC radiosensitivity was also examined. Vascular endothelial growth factor (VEGF)-C enhanced radiosensitivity when LECs were treated prior to radiation. VEGF-C-treated LECs exhibited higher levels of entry into the cell cycle at the time of radiation, with a greater number of cells in the S and G2/M phases. These LECs showed higher levels of γH2A.X-an indicator of DNA damage-after radiation. VEGF-C did not increase cell death as a result of radiation. Instead, it increased the relative number of quiescent LECs. These data suggest that abundant VEGF-C or lymphangiogenesis may predispose patients to radiation-induced lymphedema by impairing lymphatic vessel repair through induction of LEC quiescence.

Androgen receptor phosphorylation and activity are regulated by an association with protein phosphatase 1.

Androgen receptor (AR) is phosphorylated at multiple sites in response to ligand binding, but the functional consequences and mechanisms regulating AR phosphorylation remain to be established. We observed initially that okadaic acid, an inhibitor of the major PPP family serine/threonine phosphatases PP2A and protein phosphatase 1 (PP1), had cell type-dependent effects on AR expression. More specific inhibitors of PP2A (fostriecin) and PP1 (tautomycin and siRNA against the PP1alpha catalytic subunit) demonstrated that PP1 and protein phosphatase 2A had opposite effects on AR protein and transcriptional activity. PP1 inhibition enhanced proteasome-mediated AR degradation, while PP1alpha overexpression increased AR expression and markedly enhanced AR transcriptional activity. Coprecipitation experiments demonstrated an AR-PP1 interaction, while immunofluorescence and nuclear-cytoplasmic fractionation showed androgen-stimulated nuclear translocation of both AR and PP1 in prostate cancer cells. Studies with phosphospecific AR antibodies showed that PP1 inhibition dramatically increased phosphorylation of Ser-650, a site in the AR hinge region shown to mediate nuclear export. Significantly, PP1 inhibition caused a marked decrease in nuclear localization of the wild-type AR, but did not alter total or nuclear levels of a S650A mutant AR. These findings reveal a critical role of PP1 in regulating AR protein stability and nuclear localization through dephosphorylation of Ser-650. Moreover, AR may function as a PP1 regulatory subunit and mediate PP1 recruitment to chromatin, where it can modulate transcription and splicing.

Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor.

The tumor antigens simian virus 40 small t antigen (ST) and polyomavirus small and medium T antigens mediate cell transformation in part by binding to the structural A subunit of protein phosphatase 2A (PP2A). The replacement of B subunits by tumor antigens inhibits PP2A activity and prolongs phosphorylation-dependent signaling. Here we show that ST mediates PP2A A/C heterodimer transfer onto the ligand-activated androgen receptor (AR). Transfer by ST is strictly dependent on the agonist-activated conformation of AR, occurs within minutes of the addition of androgen to cells, and can occur in either the cytoplasm or the nucleus. The binding of ST changes the conformation of the A subunit, and ST rapidly dissociates from the complex upon PP2A A/C heterodimer binding to AR. PP2A is transferred onto the carboxyl-terminal half of AR, and the phosphatase activity is directed to five phosphoserines in the amino-terminal activation function region 1, with a corresponding reduction in transactivation. Thus, ST functions as a transfer factor to specify PP2A targeting in the cell and modulates the transcriptional activity of AR.

Subcellular localization modulates activation function 1 domain phosphorylation in the androgen receptor.

Although the steady-state distribution of the androgen receptor (AR) is predominantly nuclear in androgen-treated cells, androgen-bound AR shuttles between the nucleus and the cytoplasm. In the present study we have addressed how nucleocytoplasmic shuttling contributes to the regulation of AR. Nuclear transport signal fusions were used to force AR localization to the nucleus or cytoplasm of prostate cancer cells, and the effect of localization on shuttling, transcription, androgen binding, and phosphorylation was determined. Fusing the simian virus 40 nuclear localization signal or c-Abl nuclear export signal to AR resulted in androgen-independent localization to the nucleus or cytoplasm, respectively. AR forced to the nucleus was transcriptionally active on prostate-specific antigen and mouse mammary tumor virus promoters driving reporter genes. AR forced to the cytoplasm was largely inactive on the prostate-specific antigen promoter, but, surprisingly, AR was active on the mouse mammary tumor virus promoter and on two endogenous genes examined. Thus, highly transient nuclear localization of AR is sufficient to activate transcription. Androgen dissociation rates and the dissociation constant (K(D)) of AR for androgen were similar whether AR was localized to the cytoplasm or the nucleus, suggesting the ligand-binding cycle of AR is not strictly linked to its compartmentalization. Using phosphosite antibodies, we found that compartmentalization influences the phosphorylation state of AR. We show there is a bias for androgen-dependent phosphorylation of Ser81, Ser256, and Ser308 in the nucleus and androgen-independent phosphorylation of Ser94 in the cytoplasm. We propose that one function of nucleocytoplasmic shuttling is to integrate the signaling environment in the cytoplasm with AR activity in the nucleus.

Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization.

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

Preventing the Androgen Receptor N/C Interaction Delays Disease Onset in a Mouse Model of SBMA.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor (AR) and is associated with misfolding and aggregation of the mutant AR. We investigated the role of an interdomain interaction between the amino (N)-terminal FxxLF motif and carboxyl (C)-terminal AF-2 domain in a mouse model of SBMA. Male transgenic mice expressing polyQ-expanded AR with a mutation in the FxxLF motif (F23A) to prevent the N/C interaction displayed substantially improved motor function compared with N/C-intact AR-expressing mice and showed reduced pathological features of SBMA. Serine 16 phosphorylation was substantially enhanced by the F23A mutation; moreover, the protective effect of AR F23A was dependent on this phosphorylation. These results reveal an important role for the N/C interaction on disease onset in mice and suggest that targeting AR conformation could be a therapeutic strategy for patients with SBMA.

Lymphatic vessels in health and disease.

The lymphatic vasculature plays vital roles in tissue fluid balance, immune defense, metabolism, and cancer metastasis. In adults, lymphatic vessel formation and remodeling occur primarily during inflammation, development of the corpus luteum, wound healing, and tumor growth. Unlike the blood circulation, where unidirectional flow is sustained by the pumping actions of the heart, pumping actions intrinsic to the lymphatic vessels themselves are important drivers of lymphatic flow. This review summarizes critical components that control lymphatic physiology.

Androgen induces a switch from cytoplasmic retention to nuclear import of the androgen receptor.

The androgen receptor (AR) has critical functions as a transcription factor in both normal and cancer cells, but the specific mechanisms that regulate its nuclear localization are not well defined. We found that an AR mutation commonly reported in prostate cancer generates an androgen-independent gain of function for nuclear import. The substitution, Thr877Ala, is within the ligand-binding domain, but the nuclear import gain of function is mediated by the bipartite nuclear localization signal (NLS) spanning the DNA-binding domain (DBD) and hinge region. Bipartite NLS activity depends on the structure provided by the DBD, and protein interactions with the bipartite NLS are repressed by the hinge region. The bipartite NLS is recognized by importin 7, a nuclear import receptor for several proteins. Importin 7 binding to AR, however, inhibits import by shielding the bipartite NLS. Androgen binding relieves the inhibition by inducing a switch that promotes exchange of importin 7 for karyopherin alpha import receptors. Importin 7 contributes to the regulation of AR import by restraining import until androgen is detected in the cytoplasm.

Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression.

BACKGROUND: The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. METHODS: AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. RESULTS: AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. CONCLUSIONS: Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.