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INTRODUCTION: Osteopontin (OPN) is a ubiquitous protein associated with a wide range of normal and pathologic functions. It is a central regulator of malignant phenotype in non-small cell lung cancer (NSCLC). OPNa, the full length isoform, is disproportionately expressed in NSCLC tumors and cell lines, and increases malignant behavior when over expressed. OPNc, the shortest isoform, does not induce malignant properties when expressed in NSCLC. The only structural difference between OPNa and OPNc is the transcription of exon 4. The hypothesis of this project is that a small interfering peptide to OPN exon 4 will reduce in vitro malignant activity in NSCLC. METHODS: A 16-amino acid peptide which mimics the central sequence in OPN exon 4 and a scrambled sham were constructed and florescent labeled. Three NSCLC cell lines, H358 (low), A549 (moderate), and H460 (high endogenous OPN) were used in binding, proliferation, and invasion assays in presence of exon 4 mimic or sham. Cisplatin sensitivity was performed with peptides in A549. RESULTS: There was florescent evidence for cell surface binding of the exon 4 mimic in all of the NSCLC cell lines without binding of the sham. The exon 4 mimic significantly decreased in vitro invasion and proliferation in the A549 and H460 cell lines, with a lesser effect in H358 with low endogenous OPN. (Table) The exon 4 mimic increased cisplatin sensitivity, decreasing cell viability 2-fold compared to sham and control at cisplatin concentrations 0.1-0.8 uM/ml. CONCLUSIONS: Exon 4 of OPN has not previously been identified as a regulatory region essential for OPN's effect on malignant behavior. We have evidence for an un-described cell surface receptor to this region. A small mimicking peptide to OPN exon 4 significantly decreased in vitro malignant behavior, suggesting interference with important regulatory pathways. This novel binding site could serve as an ideal for target for antibody or small molecule therapies in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3444. doi:1538-7445.AM2012-3444

Introduction: The current inability to forecast outcomes for malignant pleural mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach, and hence compromises their prognosis. It was previously shown that microRNAs (miRs) have a role in the cancer process, and that they can serve as biomarkers for different tumors or histological types. Here we investigated whether specific miRs could differentiate a largely surgically-treated group of mesotheliomas into good or bad prognosis categories. Methods: In a retrospective study using snap frozen, immunohistochemically-proven mesothelioma surgical specimens, we looked for miRs associated with survival and time to progression. A training set of 44 and a test set of 98 mesothelioma tumor samples were analyzed by a custom ∼900 microRNA microarray platform and verified by qRT-PCR for cross-platform validation. Patients were segregated into good or bad time to progression (greater/less than 12 months) and into good or bad survival (greater/less than 15 months). Mann-Whitney tests for good vs. bad prognosis and Kaplan-Meier analysis were used to detect prognosticator miRs. The biological effect of the identified prognostic miR was tested in 2 mesothelioma cell lines. Functional implications as well as downstream targets of the potential prognostic miR were investigated in vitro. Results: In both the training and test sets, one miR was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma than in sarcomatoid cases, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Thresholds set within the training set for microarray reading were fully valid in the test-set, as was the comparable threshold using qRT-PCR. Increased expression of this specific miR predicted throughout a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulatory genes, known in mesothelioma, are mediated by this miR as was demonstrated through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. This suggests that a control mechanism might explain the clinical findings, supporting their validity. Conclusions: One miR has the potential to be a prognostic biomarker in mesothelioma and shows the ability to change proliferation, migration, invasion upon over-expression in mesothelioma cell lines. Further validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future, by allowing personalized treatment for patients who are more to benefit. 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 4053.

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 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.