Marileila Varella Garcia

Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

SummaryWe have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.

Abstract 5064: CORO1B amplification and expression status identifies an aggressive subset of chromosome 11q13 amplified HNSCC

Head and neck squamous cell carcinoma (HNSCC) is highly invasive cancer type that occurs with greater incidence in West Virginia and the rest of Appalachia. The chromosome 11q13 region is amplified in approximately 30% of late stage HNSCC cases and is associated with a poor patient prognosis. The core 11q13 region encodes the well-studied tumor genes CCND1 (cyclin D1) and CTTN (cortactin). The CORO1B (coronin 1B) gene flanks the 11q13 core and is amplified in 9-14% of all HNSCC, but how CORO1B amplification contributes to HNSCC is unknown. Coronin 1B governs cell motility by negatively regulating F-actin stability through displacement of cortactin at actin related protein 2/3 (Arp 2/3) branch points, generating dynamic turnover of Arp2/3-F-actin networks required for productive cell movement. Kaplan-Meier analysis of two independent patient cohorts indicates that HNSCC cases with CORO1B amplification have significantly reduced overall survival. Cox hazard ratios also indicate the CORO1B amplification is strongly associated with increased mortality in both cohorts. Automated quantitative analysis (AQUA) of coronin 1B expression in HNSCC tissue microarrays indicates that coronin 1B overexpression decreases median survival of HNSCC patients from 81 to 29 months. Reduced expression of coronin 1B by RNAi-mediated knockdown in established HNSCC cell lines decreases several actin-mediated invasive processes, including invadopodia formation and extracelluar matrix degradation. Collectively these results suggest that elevated coronin 1B expression levels in HNSCC function to enhance tumor invasion, potentially through accelerated downregulation of cortactin stabilizing activity at Arp2/3-F-actin junctions. Importantly, CORO1B amplification and coronin 1B expression status may serve a role as a precision biomarker for identification of a subset of late-stage HNSCC patients that would benefit from more aggressive forms of initial clinical treatment. Citation Format: Jessica L. Allen, Elyse L. Walk, Kristen L. Rhodes, Colleen J. Beatty, Steven M. Markwell, Brenen W. Papenberg, Erik T. Interval, Hong Wu, Marileila Varella Garcia, James E. Bear, Scott A. Weed. CORO1B amplification and expression status identifies an aggressive subset of chromosome 11q13 amplified HNSCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5064.