Therapeutic Target In Malignant Pleural Mesothelioma Research Articles

POLD1 Is Required for Cell Cycle Progression by Overcoming DNA Damage in Malignant Pleural Mesothelioma.

The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways. The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines. High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G1/S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines. POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G1/S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM.

Antipodoplanin antibody enhances the antitumor effects of CTLA-4 blockade against malignant mesothelioma by natural killer cells.

Combination immunotherapy with multiple immune checkpoint inhibitors (ICIs) has been approved for various types of malignancies, including malignant pleural mesothelioma (MPM). Podoplanin (PDPN), a transmembrane sialomucin-like glycoprotein, has been investigated as a diagnostic marker and therapeutic target for MPM. We previously generated and developed a PDPN-targeting Ab reagent with high Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, the effects of anti-PDPN Abs on various tumor-infiltrating immune cells and their synergistic effects with ICIs have remained unclear. In the present study, we established a novel rat-mouse chimeric anti-mouse PDPN IgG2a mAb (PMab-1-mG2a ) and its core-fucose-deficient Ab (PMab-1-mG2a -f) to address these limitations. We identified the ADCC and CDC activity of PMab-1-mG2a -f against the PDPN-expressing mesothelioma cell line AB1-HA. The antitumor effect of monotherapy with PMab-1-mG2a -f was not sufficient to overcome tumor progression in AB1-HA-bearing immunocompetent mice. However, PMab-1-mG2a -f enhanced the antitumor effects of CTLA-4 blockade. Combination therapy with anti-PDPN Ab and anti-CTLA-4 Ab increased tumor-infiltrating natural killer (NK) cells. The depletion of NK cells inhibited the synergistic effects of PMab-1-mG2a -f and CTLA-4 blockade invivo. These findings indicated the essential role of NK cells in novel combination immunotherapy targeting PDPN and shed light on the therapeutic strategy in advanced MPM.

Chronic Inflammation, Oxidative Stress and Metabolic Plasticity: Three Players Driving the Pro-Tumorigenic Microenvironment in Malignant Mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal and rare cancer, even if its incidence has continuously increased all over the world. Asbestos exposure leads to the development of mesothelioma through multiple mechanisms, including chronic inflammation, oxidative stress with reactive oxygen species (ROS) generation, and persistent aberrant signaling. Together, these processes, over the years, force normal mesothelial cells' transformation. Chronic inflammation supported by "frustrated" macrophages exposed to asbestos fibers is also boosted by the release of pro-inflammatory cytokines, chemokines, growth factors, damage-associated molecular proteins (DAMPs), and the generation of ROS. In addition, the hypoxic microenvironment influences MPM and immune cells' features, leading to a significant rewiring of metabolism and phenotypic plasticity, thereby supporting tumor aggressiveness and modulating infiltrating immune cell responses. This review provides an overview of the complex tumor-host interactions within the MPM tumor microenvironment at different levels, i.e., soluble factors, metabolic crosstalk, and oxidative stress, and explains how these players supporting tumor transformation and progression may become potential and novel therapeutic targets in MPM.

Identification of CD24 as a potential diagnostic and therapeutic target for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is currently incurable due to the lack of an effective early diagnostic method and specific medication. The CDKN2A (p16) and NF2 genes are both frequently mutated in MPM. To understand how these mutations contribute to MPM tumor growth, we generated NF2/p16 double-knockout (DKO) cell clones using human MeT-5A and HOMC-B1 mesothelial cell lines. Cell growth and migration activities were significantly increased in DKO compared with parental cells. cDNA microarray analysis revealed differences in global gene expression profiles between DKO and parental cells. Quantitative PCR and western blot analyses showed upregulation of CD24 concomitant with increased phosphorylation of AKT, p70S6K, and c-Jun in DKO clones. This upregulation was abrogated by exogenous expression of NF2 and p16. CD24 knockdown in DKO cells significantly decreased TGF-β1 expression and increased expression of E-cadherin, an epithelial–mesenchymal transition marker. CD24 was highly expressed in human mesothelioma tissues (28/45 cases, 62%) and associated with the loss of NF2 and p16. Public data analysis revealed a significantly shorter survival time in MPM patients with high CD24 gene expression levels. These results strongly indicate the potential use of CD24 as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM.

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

Abstract 2136: Doublecortin-like Kinase 1 (DCLK1) correlates with the cell proliferation in malignant pleural mesothelioma

Abstract Background: Malignant pleural mesothelioma (MPM) is a refractory disease with poor prognosis despite multimodality therapy. Because standard therapy is unsatisfactory, it is expected to establish a novel strategy such as molecular-targeting therapy for MPM. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein, is one of the tumor-specific stem cell marker and is revealed that the cell survival is reduced by inhibiting this protein in pancreatic and colorectal cancers. DCLK1 is known to have several isoforms named as isoform #1-#4, although the significance of them in MPM has not been clarified. We hypothesized that DCLK1 was a potential therapeutic target for MPM.&amp;lt;Materials and Methods: Immunohistochemical staining was performed using surgically resected formalin-fixed paraffin-embedded tissue of MPM to evaluate the expression of DCLK1. Additionally, MPM cell lines were analyzed to characterize the protein and mRNA expression of DCLK1 by western blotting and RT-PCR. The expression of DCLK1 isoforms were selectively knocked down by small interfering RNA (siRNA) and cell viability was analyzed by colony formation and cell count assays. Results: Immunohistochemical staining demonstrated that the expression of DCLK1 in MPM specimens was positive in 24 out of 27 samples (89%). Western blotting and RT-PCR revealed that DCLK1 was expressed in seven out of nine MPM cell lines. The results of RT-PCR revealed that isoform #2 and isoform #4 of DCLK1 were expressed in H2052, isoform #4 was expressed in H28, and isoform #2 was expressed in YUMC44 cell lines. Expressions of DCLK1 isoforms were selectively suppressed by isoform-specific siRNAs. Colony formation and cell count assays revealed that siRNA-mediated knockdown of both isoforms #2 and #4 suppressed cell growth compared to knockdown by negative control siRNA and knockdown of only isoform #2 or #4. Conclusion: Our results demonstrate that simultaneous knockdown of both isoforms #2 and #4of DCLK1 results in the inhibition of cell growth and suggest its potential as a therapeutic target in MPM. Citation Format: Akihiro Miura, Hiromasa Yamamoto, Hiroyuki Tao, Ken Suzawa, Syunsaku Miyauchi, Kota Araki, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Kazuhiko Shien, Junichi Soh, Kazunori Okabe, Shuta Tomida, Masakiyo Sakaguchi, Shinichi Toyooka. Doublecortin-like Kinase 1 (DCLK1) correlates with the cell proliferation in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2136.

BAP1 gene mutations in Egyptian patients with advanced sporadic malignant pleural mesothelioma (MPM): relation with clinical outcomes and survival

BackgroundMalignant Pleural Mesothelioma (MPM) is a lethal cancer with few therapeutic options. Patients with MPM have a poor prognosis, with estimated 1 year median survival and currently no treatment is curative. The BRCA associated protein 1 (BAP1) has the highest prevalence of protein-altering mutations identified in MPM. AimsAssessment of the frequency and pattern of BAP1 gene mutations in Egyptian patients with advanced sporadic MPM in relation to disease progression and survival rates in order to identify a novel therapeutic target for MPM. MethodsThis prospective, cohort study included 122 patients who were diagnosed and treated as advanced MPM. BAP1 gene mutations were assessed from circulating tumor cells (CTCs) by polymerase chain reaction (PCR) and sequencing and these mutations have been confirmed using the tumor tissue. BAP1 immunohistochemistry was performed using the Dako Envision visualization system. The relationship between BAP1 gene mutations, PFS and OS rates was assessed using the log rank test. The relationship between BAP1 gene mutations, clinical response and patient's clinicopathological characteristics was assessed using chi-square test. ResultsForty seven (38.5%) MPM cases showed one or more mutations in BAP1 gene. The presence of BAP1 mutations associated significantly with BAP1 protein expression (p < 0.001), the incidence of organ metastasis (p = 0.04), PFS after second line treatment (p = 0.04) and clinical response after second line treatment (p = 0.01) only. ConclusionBAP1 gene mutations are relatively common in Egyptian patients with advanced sporadic MPM. BAP1 mutations are associated with disease progression especially after second line therapy and the incidence of organ metastasis.

Abstract 30: S100A11 is a prominent therapeutic target in malignant pleural mesothelioma

Abstract [Background &amp; Purpose] Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells that show a serious malignancy in thoracic cavity with a median survival time of 9-12 months. The therapeutic approaches in clinical standard are limited to surgery and chemotherapy for early and advanced stages, respectively. The strategies unfortunately provide only palliation, thus further advanced approach is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancer tissues. It has been reported that S100A11 plays an important role in progression of many cancers derived from thyroid, ovarian and lung tissues, but its roles and signature in MPM are poorly understood. In this study, we investigated the impact of S100A11 in MPM cell lines and resected tumors. [Material &amp; Methods] We explored the expression of S100A11 in 7 MPM cell lines (HP-1, H28, MSTO-211H, H2052, H290, H2452, and YUMC44) and 2 normal mesothelial cell lines (Met-5A and LP-9). We analyzed the effect of S100A11 on cell proliferation, colony formation, migration, invasion and downstream signaling. The efficacy of anti-S100A11 antibody on cell proliferation and downstream signaling was also determined. [Results] We found that S100A11 protein was consistently upregulated in 7 MPM cell lines at a significant level in comparison to 2 normal mesothelial cells. The same phenomenon was also confirmed in MPM tissue sections by immunohistochemistry, i.e., S100A11 was strongly stained at MPM cells, but not at surrounding normal lung cells. Specific knockdown of S100A11 by small interfering RNA turned the aggressive cells into the attenuated phenotypes for proliferation, invasion and migration. Those were notably observed in both 2 MPM cell lines (H2052 and H2452). Interestingly, we found that MPM cell lines but not normal cells actively secreted the S100A11 protein. To pursue this, we next tried to inhibit the function of the secreted S100A11. Administration of S100A11 neutralizing antibody significantly inhibited the proliferation of 4 MPM cell lines (H2052, H2452, H28, and H290). The antibody had no effect on the proliferation of only one MPM cell line, MSTO-211H, which showed no secretion of S100A11. Taken together, these results suggest that S100A11 secreted from MPM cells can be a prominent target for effective MPM therapy. [Conclusion] Our results suggest that S100A11 is a possible therapeutic target in MPM. Citation Format: Hiroki Sato, Hiromasa Yamamoto, Kei Namba, Hidejiro Torigoe, Takahiro Yoshioka, Kazuhiko Shien, Junichi Soh, Shinichi Toyooka. S100A11 is a prominent therapeutic target in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2017-30

Abstract 5191: A combination of Stat3 inhibitor and radiation for the treatment of malignant pleural mesothelioma

Abstract Background: Trimodality therapy, including surgery, is the treatment for patients with malignant pleural mesothelioma (MPM). However, the 5-year survival rate remains less than 40%. To improve the survival rate, we investigated Stat3 as a potential therapeutic target for MPM. Methods:Cell viability was assessed using Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cells (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) were seeded into 96-well plates. After treatment with a Stat3 inhibitor (BBI-608 : napabucasin), Cell Counting Kit-8 solution was added to each well and absorbance was measured using a microplate reader. The level of phosphorylated Stat3 (p-Stat3) in cell lysates was measured by ELISA. ROS level in cells was measured by FACS analysis. In the in vivo study, H226 cells were injected subcutaneously in the flank region of nude mice randomly assigned to four treatment groups (5 mice/group)—(1) treatment with vehicle control, (2) treatment with Stat3 inhibitor, (3) radiation therapy (RT), and (4) Stat3 inhibitor and RT. Tumor size was measured twice/week. Results: Napabucasin decreased the viability of MPM cells (epithelioid, sarcoma, biphasic) in a dose-dependent manner. p-Stat3 levels decreased by 90% after treatment with 10 μM napabucasin in H226 cells. Napabucasin treatment induced more ROS production in MPM cells than did Pemetrexed. Moreover, combinatorial treatment with napabucasin and radiation (2 Gy, 5 Gy) induced more ROS production than did RT alone. In the mouse model, treatment with napabucasin alone significantly suppressed tumor growth (p &amp;lt; 0.05), but napabucasin and RT completely suppressed tumor growth. Conclusion:We showed that napabucasin inhibited proliferation of the MPM cells (epithelial, biphasic, sarcomatoid) and demonstrated that combination therapy with napabucasin and radiation completely suppressed tumor growth in a mouse model. Treatment with the napabucasin alone and the combination of napabucasin and RT induced high ROS production in MPM cells. Collectively, the combination of Stat3 inhibitor with Radiation Therapy (START) is a promising approach for the treatment of MPM.TheA combination of of Stat3 inhibitor and radiation with for the treatment of malignant pleural mesothelioma Citation Format: Seiji Matsumoto, Hiroshi Doi, Akihiro Fukuda, Tohoru Nakamichi, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Nobuyuki Kondo, Seiki Hasegawa. A combination of Stat3 inhibitor and radiation for the treatment of malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2017-5191

MicroRNAs as potential biomarkers in malignant pleural mesothelioma

MicroRNAs as potential biomarkers in malignant pleural mesothelioma Eric Santoni-Rugiu, Morten Andersen, Morten Grauslund Laboratory of Molecular Pathology, Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Abstract: Malignant pleural mesothelioma (MPM), a highly lethal cancer strictly related to asbestos exposure, is usually characterized by delayed diagnosis, resistance to current therapies, and dismal prognosis. MPM is difficult to distinguish histologically from nonmalignant reactive mesothelial proliferations (RMPs) as there are no clinically validated immunohistochemical markers yet and the main diagnostic criterion remains deep invasion into the pleura and underlying fat tissue, which is often not appreciable in small pleural biopsies. In this regard, microRNAs (miRNAs), given their size and stability, are particularly attractive biomarkers in formalin-fixed paraffin-embedded tissue specimens for routine pathology. Moreover, circulating miRNAs appear to be promising biomarkers for early detection and monitoring of patients with MPM. Here, we review the studies mostly performed by miRNA arrays and reverse transcription-quantitative polymerase chain reaction in formalin-fixed paraffin-embedded or frozen tissue samples, MPM cell lines, and blood/plasma/serum samples that have highlighted the potential of miRNAs as biomarkers in MPM. Certain studies have pointed to the ability of miRNAs to distinguish the different histological MPM subtypes or separate MPM from lung adenocarcinoma, and other investigations have revealed that miRNAs can aid in differentiating MPM from RMP or have prognostic value in predicting the patient outcome. Mechanistic aspects of the involvement of miRNAs in mesothelioma genesis and possible use of miRNAs as future therapeutic targets in MPM are also emphasized. Finally, limitations of the data currently obtained due to the drawbacks of reverse transcription-quantitative polymerase chain reaction, heterogeneity of MPM tissue samples, and differences in methodological platforms as well as in types of specimens utilized in different studies are discussed. Because of these inherent weaknesses of collected data, further studies assessing the expression and distribution of miRNAs by in situ hybridization in combination with the codetection of their respective targets by immunohistochemistry and further validation of miRNAs&rsquo; targets in vitro are warranted to fully reveal the potential clinical utility of miRNAs in MPM. Keywords: malignant pleural mesothelioma, miRNAs, cancer biomarkers, diagnosis, prognosis, future therapy

106P PROTEOMIC ANALYSIS SHOWED HEAT SHOCK 70 KDA PROTEIN AND 78 KDA GLUCOSE-REGULATED PROTEIN WERE UP-REGULATED IN HUMAN MALIGNANT PLEURAL MESOTHELIOMA CELLS

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

107P IMPACT OF PREINTERVENTIONAL PLASMA FIBRINOGEN AS A PROGNOSTIC MARKER IN MALIGNANT PLEURAL MESOTHELIOMA

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

Effects of hyaluronic acid and CD44 interaction on the proliferation and invasiveness of malignant pleural mesothelioma

Hyaluronic acid (HA) has been proposed as a biochemical marker of malignant pleural mesothelioma (MPM). The present study focused on the implications of HA and CD44 interaction in the proliferation and invasiveness of MPM. The proliferation and invasive activity was evaluated in two human mesothelioma cell lines, ACC-MESO-1 and K921MSO, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the transwell chamber model. The knockdown of CD44 gene expression was accomplished by transfection of the cells with small interfering RNA. Flow cytometry revealed that both the ACC-MESO-1 and K921MSO cell lines highly expressed CD44. Treatment with HA enhanced the proliferation in both mesothelioma cell lines in comparison to cells without HA treatment. The treatment with HA (25 μg/ml) also significantly upregulated the invasion of both types of cells. The silencing of CD44 significantly abrogated the effect of HA treatment on the proliferation of ACC-MESO-1 cells and significantly suppressed the proliferation of K921MSO cells. HA-CD44 binding is important for the migration and proliferation of mesothelioma cells. Therefore, the HA-CD44 interaction is a potentially useful therapeutic target in MPM.

Role of protein kinase C β and vascular endothelial growth factor receptor in malignant pleural mesothelioma: Therapeutic implications and the usefulness of Caenorhabditis elegans model organism

Purpose:To examine the role of both protein kinase C (PKC)-β and vascular endothelial growth factor receptor (VEGFR)-2 in malignant pleural mesothelioma (MPM) using respective inhibitors, enzastaurin and KRN633.Materials and Methods:MPM cell lines, control cells, and a variety of archived MPM tumor samples were used to determine the protein expression levels of PKC-β, VEGFR-2, VEGF, and p-AKT. Effects of enzastaurin and KRN633 on phosphorylation status of key signaling molecules and viability of the mesothelioma cells were determined. The common soil nematode, Caenorhabditis elegans, was treated with enzastaurin to determine its suitability to screen for highly potent kinase inhibitors.Results:PKC-β1, PKC-β2 and VEGFR-2/KDR were overexpressed in MPM cell lines and MPM tumor tissues. Enzastaurin treatment resulted in significant loss in viability of VEGF induced cell proliferation; however, the effect of KRN633 was much less. Enzastaurin also dramatically decreased the phosphorylation of PKC-β, its downstream target p-AKT, and surprisingly, the upstream VEGFR-2. The combination of the two drugs at best was additive and similar results were obtained with respect to cell viability. Treatment of C. elegans with enzastaurin resulted in clear phenotypic changes and the worms were hypermotile with abnormal pattern and shape of eggs, suggesting altered fecundity.Conclusions:PKC-β1 and VEGFR-2 are both excellent therapeutic targets in MPM. Enzastaurin was better at killing MPM cells than KRN633 and the combination lacked synergy. In addition, we show here that C. elegans can be used to screen for the next generation inhibitors as treatment with enzastaurin resulted in clear phenotypic changes that could be assayed.

Abstract 2302: Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) inducing gene, suppresses growth of pleural mesothelioma cell lines

Abstract Background and Objective: Malignant pleural mesothelioma (MPM) is a highly aggressive disease, usually caused by asbestos exposure. The underlying molecular mechanism contributing to aggressive phenotype of MPM remains to be fully elucidated. Epithelial-to-mesenchymal transition (EMT), an early developmental program, causes a wide variety of human epithelial cancers to have the ability to invade surrounding tissues and metastasize to distant sites. MPM cells usually exhibit mesenchymal characteristics probably because of their mesodermal origin. Such characteristics include fibroblast-like morphology and expression of master EMT-inducing genes. Thus, EMT may contribute to aggressive phenotype of MPM. Among EMT-inducing genes, ZEB1, a repressor of E-cadherin, has emerged as a key player in the progression of several epithelial cancers. To explore the potential of ZEB1 as a therapeutic target for MPM, we examined the effect of ZEB1 knockdown on growth of MPM cell lines. In addition, we evaluated the effect of ZEB1 knockdown on the levels of IL-6, which is known to induce growth of mesothelioma cells. Materials and Methods: 18 human MPM cell lines and one non-tumorigenic mesothelial cell line were used. Transient and stable knockdown of ZEB1 were done in two MPM cell lines. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin, and Vimentin were done. Soluble IL-6 in cellular supernatants was measured by enzyme-linked immunosorbent assay. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. FACS with PI staining was done to examine apoptosis and cell cycle. Apoptosis was also evaluated by western blot of cleaved caspase-3. Results: The majority of MPM cells expressed higher levels of ZEB1 than a non-tumorigenic mesothelial cell. We performed ZEB1 knockdown experiments in two MPM cells, ACC-MESO-1(MESO-1) and H2052, which express high and moderate levels of ZEB1. Transient knockdown of ZEB1 caused MESO-1 but not H2052 cells to reexpress E-cadherin. Transient ZEB1 knockdown suppressed cell proliferation and liquid colony formation in the two lines. Most importantly, the ZEB1 knockdown dramatically suppressed soft agar colony formation in the two lines. We did not see apoptosis or cell cycle arrest in either of the two lines. Stable knockdown of ZEB1 induced morphologic changes suggestive of mesenchymal-to-epithelial transition (MET) in MESO-1 cells but not in H2052 cells. ZEB1 stable knockdown resulted in a decreased IL-6 production in MESO-1 cells, suggesting that growth inhibition by ZEB1 knockdown may in part due to decreased IL-6 production. Conclusion: These results suggest that ZEB1 serves as an attractive therapeutic target for MPM. 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 2302.

Abstract 3193: EPHB2 expression parallels malignant behavior in mesothelioma

Abstract Purpose: Erythropoietin-producing hepatocellular (EPH) receptors and their Ephrin (EFN) ligands are members of the transmembrane receptor tyrosine kinase (RTK) family. EPHB2 is a RTK, promotes uncontrolled cell growth, migration and tumor progression. Preliminary gene array data indicate EPHB2 overexpression in malignant pleural mesothelioma (MPM). Here, we aim to verify EPHB2 overexpression in MPM tumors and cell lines and hypothesize that EPHB2 silencing decreases malignant behavior in MPM cell lines. Methods: EPHB2 mRNA expression was evaluated by RT-PCR in eight MPM and three benign mesothelial cell lines, and seven pairs of MPM tumors and matched normal tissue. Functional significance of EPHB2 was evaluated by transfecting EPHB2-shRNA and empty vector controls into two MPM cell lines, HP-1 and H2595. Expression of EPHB2, BCL2L1, survivin, Caspase2, Caspase8, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were evaluated by RT-PCR following silencing. Pooled populations of transfected cells were evaluated in scratch closure, cell proliferation by CellTitre BlueTM, MatrigelTM Invasion and colorimetric TUNELTM apoptotic assays. Comparisons were made between EPHB2 silenced and control cell lines. Results: EPHB2 was overexpressed in all MPM cell lines, but not in the benign mesothelial cell lines and in all tumors compared to matched normal tissue. Targeted knockdown of EPHB2 in MPM cell lines reduced its expression and subsequently reduced MMP-2, VEGF, BCL2L1 and survivin expression, and increased Caspase2 and Caspase8 expression. Inhibition of EPHB2 significantly decreased scratch closure, proliferation and invasion compared to the controls, and significantly increased apoptotic cells, likely related to Caspase2 and Caspase8 mediated cell death. Conclusion: Silencing of EPHB2 reduced the malignant behavior in MPM cell lines. EPHB2 appears to play an important role in MPM pathogenesis and could potentially serve as a novel therapeutic target for MPM treatment.Results of Functional AssaysAssayHP-1 ControlHP-1 EPHB2shRNAp ValueH2595 ControlH2595 EPHB2shRNAp ValueScratch Assay (% closure)69.237.6&amp;lt;0.00556.945.7&amp;lt;0.03Cell Proliferation Assay (OD value)9.56.4&amp;lt;0.058.75.7&amp;lt;0.05Matrigel Invasion Assay (cells invaded)14678&amp;lt;0.0057643&amp;lt;0.005TUNEL Apoptotic Assay (% apoptotic cells)074n/a088.5n/a 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 3193.

Integrative decomposition procedure and Kappa statistics set up ATF2 ion binding module in malignant pleural mesothelioma (MPM)

Activating transcription factor 2 (ATF2) is a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. However, the information concerning ATF2 ion-mediated DNA binding module and function of ATF2 in malignant pleural mesothelioma (MPM) has never been addressed. In this study, by using GRNInfer and GVedit based on linear programming and a decomposition procedure, with integrated analysis of the function cluster using Kappa statistics and fuzzy heuristic clustering in MPM, we identified one ATF2 ion-mediated DNA binding module involved in invasive function including ATF2 inhibition to target genes FALZ, C20orf31, NME2, PLOD2, RNF10, and RNASEH1, upstream RNF10 and PLOD2 activation to ATF2, upstream RNASEH1 and FALZ inhibition to ATF2 from 40 MPM tumors and 5 normal pleural tissues. Remarkably, our results showed that the predominant effect of ATF2 occupancy is to suppress the activation of target genes on MPM. Importantly, the ATF2 ionmediated DNA binding module reflects ‘mutual’ positive and negative feedback regulation mechanism of ATF2 with up-and down-stream genes. It may be useful for developing novel prognostic markers and therapeutic targets in MPM.

Activated Src kinase is expressed in malignant pleural mesothelioma tumors; dasatinib inhibition leads to cytotoxicity, cell cycle inhibition, and prevention of invasion and migration

7713 Background: Malignant pleural mesothelioma (MPM) is a lethal disease with few effective therapeutic options. We sought to determine whether Src, a non-receptor tyrosine kinase, could be a new therapeutic target in MPM and to establish the potential therapeutic use of pharmacologic Src inhibitors in this disease. Methods: We analyzed four MPM cell lines (MSTO-211H, NCI-H28, NCI-H2052, and NCI- H2452) for immunohistochemical (IHC) expression of total and phosphorylated Src (Tyr 419, Tyr 530). These cell lines were treated with dasatinib, a Src inhibitor, and evaluated for apoptosis, cell cycle analysis, and migration and invasion. Downstream signaling events were studied by Western blot analysis. We also conducted IHC analyses with total Src, phosphorylated Src Tyr 419 (p-Src Tyr 419), and phosphorylated Src Tyr 530 on 46 archived MPM tumor specimens and correlated the biomarker results with the clinical outcome. Results: All four MPM cell lines expressed total and activated Src (p-Src Tyr 419). Three of the four cell lines were sensitive in vitro to cytotoxicity by dasatinib with inhibition of migration and invasion, cell cycle inhibition, and apoptosis. Treatment with dasatinib inhibited several pathways downstream of Src. In the archived MPM tumor specimens, Src protein was highly expressed on IHC analysis in tumor cells, but that expression did not correlate with overall or progression-free survival. However, expression of activated Src (p-Src Tyr 419) on the tumor cell membrane was higher in patients with stage 4 disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Tyr 419. MPM nodal involvement at N1 was associated with the highest membrane expression of inactive Src (p-Src Tyr 530) (P = 0.02), whereas N2 disease was associated with the lowest expression. No gene mutations in Src exon 12 were found in the cell lines or tumor specimens. Conclusions: Activated Src may have an important role in survival, metastasis, and invasion in MPM, and targeting Src may be an important therapeutic strategy. No significant financial relationships to disclose.

The inter-relationship of hypoxia and phospho-Akt in the pathobiology of malignant pleural mesothelioma

17057 Background: Poor outcomes with conventional oncological treatments for malignant pleural mesothelioma (MPM) have driven the imperative to advance understanding of the disease pathobiology. Carbonic Anhydrase (CA)-IX is a surrogate marker of hypoxia over-expressed in solid tumours. Protein Kinase B, or Akt, known to have important intracellular roles including resistance to apoptosis, is activated in human malignancies and upregulated in hypoxic conditions. This study examined the expression of CA-IX and phosphorylated Akt (pAkt) in tumour samples from patients with MPM, correlating expression with established prognostic factors. The role of pAkt in the survival of an MPM cell line exposed to hypoxic conditions was also examined. Methods: Full local regional ethics committee approval was obtained for this work. Tumour samples from 200 patients with MPM were stained using pAkt and CA-IX specific antibodies. The effect of hypoxia on Akt and pAkt was evaluated in the JU77 mesothelioma cell line in the presence or absence of the phosphatidylinositol-3-kinase inhibitor, LY294002. Results: There was a positive association between the level of CA-IX and pAkt staining, implying that intra-tumoural hypoxia may be a stimulus for Akt phosphorylation. On multivariate analysis increased expression of nuclear pAkt was found to be associated with a poor survival. In-vitro cell culture work showed that, although pAkt is expressed in normoxic conditions in the JU77 cell line, the rates of apoptosis were significantly increased in hypoxic conditions when the phosphorylation of Akt was blocked by LY294002. Conclusions: This work provides evidence for the anti-apoptotic role of pAkt in hypoxic conditions in solid human malignancies. Phospho-Akt may represent a novel therapeutic target in MPM. No significant financial relationships to disclose.

P-413 Wnt2 as a new therapeutic target in malignant pleuralmesothelioma

P-413 Wnt2 as a new therapeutic target in malignant pleuralmesothelioma