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Supplementary Table S1 from Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Supplementary Fig. S1 from Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Supplementary Table S2 from Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)

In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non–small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerase-negative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogenously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT- and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long (∼14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN. (Cancer Res 2006; 66(7): 3584-92)

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy. Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) contribute to malignant growth in several tumor types including thoracic malignancies while a role in MPM remains widely undefined. Therefore, aim of the present study was to investigate the expression and impact of FGFs and FGFRs in MPM and to evaluate their potential suitability as new therapeutic targets. Material und Methods: Expression of all known FGF and FGFR genes was assessed by expression array analysis and confirmed by qRT PCR in MPM cell lines (n=10) and normal mesothelial cells. Selected FGFs were also investigated by immunohistochemistry on tissue samples. FGFR-specific tyrosine kinase inhibitors and an adenovirus expressing dominant-negative FGFR1 were used to block FGF signal transduction in MPM cell models. MTT and clonogenic assays as well as spheroid formation assays and videomicroscopy were performed to analyze cell growth, survival and migration. The effect on downstream signal transduction was assessed by immunoblotting with phosphorylation site-specific antibodies. Results: FGFR1 as well as the ligands FGF2 and FGF18 were highly expressed in almost all MPM cell lines investigated. Moreover, immunohistochemistry demonstrated high FGF expression also in tissue sections. Stimulation with exogenous FGF2 in contrast, led to a remarkable increase in cell migration and clonogenic growth. In contrast, inhibition of FGFR1 by the specific small molecule kinase inhibitor PD166866 lead to decreased proliferation, survival, migration and spheroid formation in the majority of cell lines tested. Adenoviral expression of dominant-negative FGFR1 further confirmed these results. In addition, concomitant FGFR inhibition increased the efficacy of currently used chemotherapeutic agents such as cisplatin. Also combination of FGFR inhibition and radiation lead to enhanced cytotoxicity. Conclusion: Taken together, these data suggest that FGFR signals contribute to proliferation, survival, migration and chemotherapy resistance of MPM cells and their inhibition should be further evaluated as a potential new treatment strategy in MPM. 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 1067. doi:1538-7445.AM2012-1067

Aims: Malignant pleural mesothelioma is a devastating cancer with increasing incidence. Despite the fact that for establishing the diagnosis a number of immunohistochemical markers are needed, there is only one pathological prognosticator accepted and utilized, namely the histological subtype. Accordingly, we have investigated the prognostic significance of the Ki76 labeling index in malignant pleural mesothelioma. Patients and Methods: Ki67 index has been determined by assessing the labeled nuclei per high power field and correlated in a retrospective manner with patients’ clinical history and outcome. The prognostic power of the routine proliferation marker Ki67 was first determined in a test cohort (TC) of paraffin-embedded tissue samples from 55 patients. The independence of prognostic power against basic clinicopathological characteristics has been determined by multivariate analysis. Next, we measured the prognostic power in an independent validation cohort (VC) of 42 patients from another center. Results: Median Ki67 index was found to be 10 for both the test and validation. Patients whose tumor sample was categorized by low Ki67 index (≥ 10) had significantly longer survival times than those with higher Ki76 indices (in the test cohort: hazard ratio [HR] 2.49, 95% confidence interval [CI] 1.30-4.79, p 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 1712. doi:1538-7445.AM2012-1712