Kerry L. Sanders

Induction of MCP-1 expression in airway epithelial cells: Role of CCR2 receptor in airway epithelial injury

The repair of an injured bronchial epithelial cell (BEC) monolayer requires proliferation and migration of BECs into the injured area. We hypothesized that BEC monolayer injury results in monocyte chemoattractant protein-1 (MCP-1) production, which initiates the repair process. BECs (BEAS-2B from ATCC) were utilized in this study. MCP-1 interacts with CCR2B receptor (CCR2B), resulting in cell proliferation, haptotaxis, and healing of the monolayer. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to verify the presence of CCR2B. CCR2B was not merely present but also inducible by interleukin-2 (IL-2) and lipopolysaccharide (LPS). We demonstrated by immunohistochemistry that BECs express MCP-1 after injury and that receptor expression can be regulated by exposure to IL-2 and LPS. Haptotactic migration of cells was enhanced in the presence of MCP-1 and reduced in the presence of CCR2B antibody. This enhanced or depressed ability of the BECs to perform haptotactic migration was shown to be statistically significant (P < 0.05) when compared to controls. Finally, BECs proliferate in response to MCP-1 as proven by electric cell-substrate impedance sensing (ECIS) technology. MCP-1-specific antibodies were shown to neutralize the MCP-1-mediated BEC proliferation. This cascade of events following injury to the bronchial epithelium may provide insight into the mechanism of the repair process.

Polar Production of Interleukin-8 by Mesothelial Cells Promotes the Transmesothelial Migration of Neutrophils: Role of Intercellular Adhesion Molecule–1

Migration of polymorphonuclear neutrophils (PMNL) from the vascular compartment into the pleural space occurs rapidly during the development of parapneumonic effusions. This study investigated the polarized secretion of interleukin (IL)-8 in activated pleural mesothelial cells (PMC) and the migration of PMNL across resting, activated PMC monolayers. Results show that PMC produce IL-8 in a polar manner. When PMC were stimulated with Staphylococcus aureus or IL-1beta at the basal or at the apical surface, significantly (P< .05) more IL-8 was released toward the apical surface. This polarized production of IL-8 was confirmed by in situ hybridization. PMNL migration was higher from the basilar to apical than from the apical to basilar surface of PMC. Neutralizing antibodies against IL-8 and intercellular adhesion molecule (ICAM)-1 significantly (P< .001) blocked PMNL migration across activated monolayers. Thus, during pleural inflammation, PMC regulate the influx of PMNL into the pleural space by polar production of IL-8 and expression of ICAM-1.

B-Tubulin Mutations Are Associated with Resistance to 2-Methoxyestradiol in MDA-MB-435 Cancer Cells

2-Methoxyestradiol is an estradiol metabolite with significant antiproliferative and antiangiogenic activity independent of estrogen receptor status. To identify a molecular basis for acquired 2-methoxyestradiol resistance, we generated a stable 2-methoxyestradiol-resistant (2ME2R) MDA-MB-435 human cancer cell line by stepwise exposure to increasing 2-methoxyestradiol concentrations. 2ME2R cells maintained in the presence of the drug and W435 cells maintained in the absence of the drug showed 32.34- to 40.07-fold resistance to 2-methoxyestradiol. Cross-resistance was observed to Vinca alkaloids, including vincristine, vinorelbine, and vinblastine (4.29- to 6.40-fold), but minimal resistance was seen to colchicine-binding agents including colchicine, colcemid, and AVE8062A (1.72- to 2.86-fold). No resistance was observed to paclitaxel and epothilone B, polymerizing agents (0.89- to 1.14-fold). Genomic sequencing identified two different heterozygous point mutations in the class I (M40) isotype of beta-tubulin at amino acids 197 (Dbeta197N) and 350 (Kbeta350N) in 2ME2R cells. Tandem mass spectrometry confirmed the presence of both wild-type and the mutant beta-tubulin in 2ME2R cells at the protein level. Consistently, treatment of parental P435 cells with 2-methoxyestradiol resulted in a dose-dependent depolymerization of microtubules, whereas 2ME2R cells remained unaffected. In contrast, paclitaxel affected both cell lines. In the absence of 2-methoxyestradiol, 2ME2R cells were characterized by an elevated level of detyrosination. Upon 2-methoxyestradiol treatment, levels of acetylated and detyrosinated tubulins decreased in P435 cells, while remaining constant in 2ME2R cells. These results, together with our structure-based modeling, show a tight correlation between the antitubulin and antiproliferative effects of 2-methoxyestradiol, consistent with acquired tubulin mutations contributing to 2-methoxyestradiol resistance.

Mycobacteria Induces Pleural Mesothelial Permeability by Down-Regulating β-Catenin Expression

Patients with pulmonary tuberculosis develop pleural effusions with a high protein content. Pleural mesothelial adherens junctions promote mesothelial cell-cell adhesion and contribute to pleural integrity. In the present study we have investigated the effect of mycobacterium (BCG) on mesothelial cell adherens junction proteins and pleural permeability. BCG enhanced pleural mesothelial cell (PMC) release of vascular endothelial growth factor (VEGF), and decreased electrical resistance across the PMC monolayer. Neutralizing antibodies to VEGF significantly restored the drop in PMC electrical resistance caused by BCG. BCG infection down regulated β-catenin (adherens junction protein) expression and caused increased permeability across confluent mesothelial monolayer. Our results suggest that in TB pleurisy, mycobacteria cause VEGF release from mesothelial cells and leads to protein exudation by altering mesothelial adherens junction proteins.

Pleural Mesothelial Cell (PMC) Defense Mechanisms Against Malignancy

Tumors such as ovarian, lung, and breast have been found to have a predilection for the pleura. Pleural mesothelial cells (PMCs) play an active role in pleural inflammation via release of cytokines. However, mechanisms whereby PMCs defend themselves against invading malignant cells are unknown. In the present study, we hypothesized that PMCs release the antiangiogenic factor endostatin and inhibit malignant cell invasion. We evaluated the endostatin levels in malignant (MAL) and congestive heart failure (CHF) pleural fluids (PF). Endostatin expression by PMC was also demonstrated by Western analysis and confocal microscopy. Our results demonstrate that CHF PF contained significantly higher levels of endostatin when compared with MAL PF. PMCs alone released a significantly greater amount of endostatin when compared with ovarian cancer cells (OCCs). When the PMC were cocultured with OCCs without contact, there was an increase in the endostatin production. However, when the PMCs were cocultured in direct contact with OCCs the endostatin levels significantly decreased. Endostatin production was upregulated in the presence of tumor cells but not when OCCs were adherent to underlying PMC monolayer. Immunofluorescent staining of PMCs for endostatin correlated with endostatin release. These findings suggest that PMCs play a key role in the antiangiogenesis process by producing endostatin in the pleural space, and thus preventing tumor spread and metastasis in the pleura.

Inflammatory Cytokines Mediate C–C (Monocyte Chemotactic Protein 1) and C–X–C (Interleukin 8) Chemokine Expression in Human Pleural Fibroblasts

Current knowledge implicates pleural mesothelial cells as mainly responsible for inflammatory responses in the pleural space. However, a vast body of recent evidence underscores the important role of fibroblasts in the process of inflammation in several types of tissues. We hypothesize that HPFBs (human pleural fibroblasts) play an important role in pleural responses and also when activated by bacterial endotoxin LPS (lipopolysaccharide), IL-1 β (interleukin-1 β), or TNF-α (tumor necrosis factor-α) release of C–C and C–X–C chemokines—specifically, MCP-1 and IL-8. Our results show that pleural fluid–isolated human fibroblasts release IL-8 and MCP-1 upon stimulation with IL-1 β, TNF-α, and LPS in both a concentration- and time-dependent manner. RT–PCR (reverse-transcriptase–polymerase chain reaction) studies have also confirmed IL-8– and MCP-1–specific mRNA expression in activated pleural fibroblasts. On the time-dependent response curve, IL-8 was found in maximum concentrations at 144 hr, whereas MCP-1 continued to increase even after 196 hr following stimulation. IL-1 β induced the maximum release of IL-8 (800-fold) and MCP-1 (164-fold), as compared to the controls. TNF-α induced a 95-fold increase in IL-8 and an 84-fold increase in MCP-1 levels, as compared to the controls. Collectively, our results show that human pleural fibroblasts contribute to the inflammatory cascade in the pleural space.

Establishment and characterization of a novel cell line derived from human thymoma AB tumor

Thymomas are low-grade epithelial tumors of the anterior mediastinum. The complexity of the disease and the lack of in vitro and in vivo models hamper the development of better therapeutics. In this study, we report a novel cell line, designated as IU-TAB-1, which was established from a patient with stage II thymoma (World Health Organization-type AB). The IU-TAB-1 cell line was established in vitro and characterized using histological and immunohistochemical staining, fluorescence-activated cell sorting, cytogenetic analyses and functional assays including in vitro and a NOD/SCID xenograft model. A whole-genome gene expression analysis (Illumina) was performed on the IU-TAB-1 cell line and 34 thymomas to determine the clinical relevance of the cell line. The IU-TAB-1 cell line was positive for epithelial markers (pan-cytokeratin and EpCAM/CD326) including thymic epithelial (TE) surface markers (such as CD29, CD9, CD54/ICAM-1, CD58 and CD24) and p63, and negative for B- and T-cell lineage markers. Gene expression profiling demonstrated overlapping and distinct genes between IU-TAB-1 and primary thymomas including the primary tumor (from which the cell line was derived). IU-TAB-1 cells are tumorigenic when implanted in immunodeficient mice with tumors reaching a volume of 1000 mm3 at around 130 days. The established cell line represents a biologically relevant new tool to investigate the molecular pathology of thymic malignancies and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.

Pleural mesothelial cells modulate polymorphonuclear leukocyte apoptosis in empyema

In bacterial empyema, the recruited polymorphonuclear leukocytes (PMN) represent important phagocytic cells involved in antibacterial defense. In this study we demonstrate that pleural fluids (PF) obtained from patients with empyema (EMP) contains significantly higher levels of granulocyte colony stimulating factor (GM-CSF), and PMN incubated in empyema (EMP) pleural fluid (PF) showed significantly less apoptosis than congestive heart failure (CHF) PF. Staphylococcus aureus-stimulated PMC released significantly (P < 0.001) higher levels of GM-CSF than resting PMC. Staphylococcus aureus-stimulated PMC (SPMC)-CM significantly (P < 0.001) inhibited PMN apoptosis. In SPMC-CM-incubated PMN the antiapoptotic gene Bcl-xL mRNA and protein expression was up-regulated; Bak mRNA and protein expression was down-regulated compared to control PMN. The active caspases activity significantly decreased. When SPMC-CM and EMP PF were immunodepleted with GM-CSF antibody, PMN apoptosis was significantly higher. The delay in apoptosis of PMN is in part attributable to the release of cytokine GM-CSF by activated PMC. These findings suggest that S. aureus-activated PMC extend PMN life span by modulating Bcl-xL and Bak gene expression and active caspases activity during acute inflammation and empyema.

Gene Expression Analysis Reveals Distinct Pathways of Resistance to Bevacizumab in Xenograft Models of Human ER-Positive Breast Cancer.

Bevacizumab, the recombinant antibody targeting vascular endothelial growth factor (VEGF), improves progression-free but not overall survival in metastatic breast cancer. To seek further insights in resistance mechanisms to bevacizumab at the molecular level, we developed VEGF and non-VEGF-driven ER-positive MCF7-derived xenograft models allowing comparison of tumor response at different timepoints. VEGF gene (MV165) overexpressing xenografts were initially sensitive to bevacizumab, but eventually acquired resistance. In contrast, parental MCF7 cells derived tumors were de novo insensitive to bevacizumab. Microarray analysis with qRT-PCR validation revealed that Follistatin (FST) and NOTCH were the top signaling pathways associated with resistance in VEGF-driven tumors (P<0.05). Based on the presence of VEGF, treatment with bevacizumab resulted in altered patterns of metagenes and PAM50 gene expression. In VEGF-driven model after short and long-term bevacizumab treatments, a change in the intrinsic subtype (luminal to myoepithelial/basal-like) was observed in association with increased expression of genes implicated with cancer stem cell phenotype (P<0.05). Our results show that the presence or absence of VEGF expression affects the response to bevacizumab therapy and gene pathways. In particular, long-term bevacizumab treatment shifts the cancer cells to a more aggressive myoepithelial/basal subtype in VEGF-expressing model, but not in non-VEGF model. These findings could shed light on variable results to anti-VEGF therapy in patients and emphasize the importance of patient stratification based on the VEGF expression. Our data strongly suggest consideration of patient subgroups for treatment and designing novel combinatory therapies in the clinical setting.

Abstract 4490: INK128, a novel TORC1/2 inhibitor, demonstrates potent oral antitumor activity in a VEGF-reinforced murine model of breast cancer and enhances efficacy of bevacizumab

Background: Aberrant activation of the mammalian target of rapamycin (mTOR) signaling plays an important role in breast cancer progression and represents a potential therapeutic target for breast cancer. mTOR, existing in two distinct multi-protein complexes, TORC1 and TORC2, plays a key role in several signaling pathways controlling tumor growth, hypoxia, metabolism as well as angiogenesis. Through rational drug design we have identified INK128, a potent and selective small molecule active-site TORC1/2 kinase inhibitor, with excellent drug-like properties. In this study, we evaluated the potential therapeutic impact of INK128 alone or in combination with bevacizumab, the recombinant humanized VEGF targeting antibody, on tumor growth and angiogenesis using a genetically engineered xenograft model of breast cancer. Methods: MCF-7 cells transfected with vector (ML20) or VEGF (MV165) were implanted into mammary fat pads of athymic mice. Treatment of INK128 alone (0.3 mg/kg or 1 mg/kg/daily to 3 mg/kg thrice weekly for 4 weeks) or vehicle control was started when tumors were palpable. Target proteins of PI3K/Akt/ mTORC1/2 signaling were analyzed by immunoblotting, while Ki-67, VEGF, VEGFR2, and carbonic anhydrase IX (CA IX) were assessed by immunohistochemistry (IHC). The potential of INK128 in combination with bevacizumab was also evaluated in this model. Results: INK128 inhibited primary tumor growth significantly in MV165 xenografts in all doses tested (p=0.014), compared with vehicle control, while the impact of INK128 on ML20 xenografts was only significant at 3 mg/kg dose (p=0.05). Treatment with INK128 decreased the phosphorylation of Akt S473 , pS6 S240/244 , and p4EBP1 T37/45 in both ML20 and MV165 xenograft tumors. Inhibition of the PRAS40 T246 phosphorylation is more prominent in MV165 tumors in a dose-dependent manner. INK128 blocked phosphorylation of NDRG1 potently. Among the markers analyzed by IHC, only CA IX cytoplasmic expression in MV165 tumors was significantly reduced in the 3 mg/kg group as compared to control (p=0.008). Combination therapy with INK128 and bevacizumab reduced the tumor growth in both ML20 (p=0.040) and MV165 (p=0.002) xenografts significantly, when compared to vehicle groups, while the impact of inhibition was greater in MV165 than in ML20. Combination therapy was superior to INK128 alone in MV165 (p=0.009) but not in ML20 (p=0.145); in contrast, combination therapy was superior to bevacizumab in both ML20 (p=0.043) and MV165 (0.005). Conclusions: INK128 inhibits TORC1 and TORC2-dependent signaling in VEGF-overexpressing xenograft model. Addition of INK128 to bevacizumab, a VEGF-targeting antibody, provided further benefit in xenograft models, even with transgenically elevated levels of VEGF, suggesting a promising approach in breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4490.