Candece L. Gladson

Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse.

Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.

TGF-β1 up-regulates paxillin protein expression in malignant astrocytoma cells: requirement for a fibronectin substrate

Cytokines can influence the interactions between members of the integrin cell adhesion receptor family and the extracellular matrix thereby potentially affecting cell function and promoting cell adhesion, growth and migration of malignant astrocytoma tumor cells. As malignant astrocytoma cells synthesize TGF-β1 in vivo, we analysed the effects of TGF-β1 on signaling events associated with integrin receptor ligation, focusing on the effects on paxillin, a phosphorylated adaptor protein, that acts as a scaffold for signaling molecules recruited to focal adhesions. TGF-β1-stimulation of primary astrocytes and serum-starved U-251MG malignant astrocytoma cells attached to fibronectin induced a substantial increase in the levels of paxillin protein (fivefold increase at 2.0u2009ng/ml) in a dose- and time-dependent manner compared to the levels observed on plating onto fibronectin in the absence of stimulation. In the astrocytoma cells, this resulted in an increase in the pool of tyrosine-phosphorylated paxillin, although it did not appear to alter the extent of phosphorylation of the paxillin molecules. In contrast, in primary astrocytes the protein levels were upregulated in the absence of a parallel increase in phosphorylation. The TGF-β1-stimulated increase in paxillin levels required ligation of the fibronectin receptor, as it was not induced when the cells were plated onto vitronectin, collagen or laminin. The increase in the pool of paxillin on TGF-β1 stimulation of the fibronectin-plated astrocytoma cells was associated with an increase in translation, but was not associated with an increase in the steady-state levels of paxillin mRNA. Stimulation with TGF-β1 on a fibronectin substrate increased subsequent attachment and spreading of U-251MG cells onto fibronectin and, to a lesser extent, vitronectin, but not collagen. Our results indicate that physiologic levels of TGF-β1 stimulate the expression of paxillin protein at the level of translation through a process that requires engagement of the fibronectin receptor, and promotes attachment and spreading of malignant astrocytoma cells on fibronectin.

Endothelial Expression of TNF Receptor-1 Generates a Proapoptotic Signal Inhibited by Integrin α6β1 in Glioblastoma

Activation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma, we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (EC) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma (GBM) tumors compared with ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα-induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cellular FLICE inhibitory protein (cFLIP) and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting prodeath signaling by TNF-R1, in part by inducing cFLIP expression.

Urokinase receptor mediates lung fibroblast attachment and migration toward provisional matrix proteins through interaction with multiple integrins.

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

Abstract LB-340: Mechanism of bevacizumab internalization and fate by brain endothelial cells .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAntiangiogenic immunotherapy shows great promise for treatment of malignancies, including cancer. Bevacizumab is a humanized monoclonal antibody that inactivates vascular endothelial growth factor-A (VEGF-A), thereby inhibiting angiogenesis. It has received FDA approval for patients with recurrent glioblastoma (GBM); however, more than 30% of patients are non-responsive. The internalization and fate of bevacizumab in endothelial cells (ECs) may play a critical role in the response to therapy, i.e., bevacizumab may be degraded, recycled to the vessel lumen, or transcytosed to the sub-basal EC where tumor cell VEGF-A is found. We examined the internalization of bevacizumab by normal brain ECs in different conditions and found that internalization increased in a time-dependent manner (30 min - 24 hrs). Epidermal growth factor (EGF) stimulated internalization, suggesting bevacizumab enters the cells by an endocytic pathway that is induced by growth factors, such as macropinocytosis. Also, internalized bevacizumab was compartmentalized into vesicles that in part co-localized with the EGF receptor. Treatment with Amiloride (inhibitor of macropinocytosis) decreased the number of bevacizumab-containing vesicles per cell, suggesting macropinocytosis may be one mechanism of internalization. Treatment with Bafilomycin A1 (inhibitor of lysosomal acidification) caused accumulation of bevacizumab, suggesting one fate is lysosomal degradation. Ongoing experiments will determine the endocytic compartment(s) containing bevacizumab, its trafficking, and whether it is transcytosed across normal brain and tumor-associated ECs. Understanding the mechanism of internalization and trafficking of bevacizumab in brain ECs will positively impact the design and modification of monoclonal antibody therapy for GBM patients and for other cancers.nnCitation Format: Gaelle M. Muller-Greven, Cathleen Carlin, Candece L. Gladson. Mechanism of bevacizumab internalization and fate by brain endothelial cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-340. doi:10.1158/1538-7445.AM2013-LB-340

Expression (Exp) of FIP200 and Rb in breast cancer (BreastCA) metastasis (met) to the brain and effect on survival.

e21152 Background: BreastCA is the most common non-cutaneous cancer and the 2nd leading cause of cancer deaths. Although the outcome for BreastCA patients (pts) with brain met remains poor, there is significant variation in overall survival (OS). However, there are no markers or to predict OS. FIP200 is a signaling node regulating several pathways. It inhibits cell proliferation when localized to the nucleus by promoting Rb-1 and p21, and it promotes cell survival when localized to the cytoplasm through inhibition of Pyk2 and regulation of autophagy. FIP200 gene is deleted or mutated in approximately 20 % of primary BreastCA biopsies, and the level of Exp of FIP200 and Rb1 are correlated in BreastCA cell lines. The Exp of FIP200 and its cellular localization have not been examined in brain met of any cancer.nnnMETHODSnIn a single institution retrospective analysis brain tissues on 21 pts with metastatic BreastCA, obtained between 8/2000 and 3/2010 were evaluated for Exp of FIP200 and Rb1. These biopsies were selected based on the availability of adequate tissue in the blocks and a prior diagnosis (Dx) of invasive ductal BreastCA. Immunohistochemistry was used to evaluate the localization and levels of Exp of FIP200 and Rb1 which in turn were tested as predictors of prolonged OS.nnnRESULTSn37% of primary breastCA were triple negative, 25% were ER negative/HER2 positive. Median time from Dx to met in the 21 pts was 23 (0-245) months (mos), and the median OS from Dx of BreastCA was 43 (6-264) mos. Nuclear Exp of Rb1 was detected in < 30% vs. ≥ 30% of cells in 11 vs. 10 pts. The median OS was 39 (range 6-264) mos in pts with Rb1 Exp < 30% of cells and 47 (20-190) mo in those with Rb1 Exp in ≥ 30% of cells. Nuclear Exp of FIP200 was detected in <20% vs. ≥ 20% of cells in 13 vs. 8 pts. The median OS was 39 (6-264) mos in pts with FIP200 nuclear Exp in <20% of cells and 45 (43-122) mos in those with FIP200 Exp in ≥ 20% of cells. Cytoplasmic Exp of Fip200 was detected in ≥ 20% of cells in 20 of 21 cases.nnnCONCLUSIONSnThe pattern of nuclear Exp of Rb1 is different in pts with brain met from invasive intra-ductal BreastCA with a longer OS. An expanded study is underway to determine whether nuclear Rb1 and/or FIP200 predict OS and define the role of these proteins in brain met from BreastCA.