Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

Connective tissue growth factor (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine-rich CYR61/CTGF/ nephroblastoma-overexpressed gene] family. These proteins have been implicated in various biological processes, including stimulation of cell proliferation, migration, angiogenesis and tumorigenesis. In a previous study, we found that CTGF mRNA was elevated in primary gliomas, and a significant correlation existed between CTGF mRNA levels versus tumor grade, histology and patient survival. In this study, the role of CTGF in glioma tumorigenesis was explored. Forced expression of CTGF in glioblastoma multiforme (GBM) cells accelerated their growth in liquid culture and soft agar, stimulated cells migration in Boyden chamber assays and significantly increased their ability to form large, vascularized tumors in nude mice. CTGF induced the expression of the antiapoptotic proteins, Bcl-xl, Survivin and Flip. Overexpression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum-free medium and resist antitumor drugs including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand, VELCADE (bortezomib, proteasome inhibitor) and temozolomide. Our data suggest that CTGF plays an important role in glioma progression, by supporting tumor cells survival and drug resistance.

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The whole genomic gene copy number was measured using single nucleotide polymorphism DNA microarray (SNP-Chip). Chromosome 1p36.23 was frequently deleted (19 of 55 clinical samples, 35 %) and EGFR was often amplified (35 of 55, clinical samples 66 %) in glioblastoma multiform (GBM). microRNA 34a (miR-34a) localizes in this minimum common deleted 1p36.23 region. We hypothesized that miR-34a may act as a tumor suppressor in the brain. Interestingly, we found that the mean survival time was significantly shorten (p < 0.05) in the patients whose GBM showed both EGFR amplification and miR-34a deletion compared to those with either EGFR amplification, miR-34a deletion or normal levels of EGFR and miR-34a. Expression of miR-34a was significantly lower in GBM samples compared to normal brain tissue as examined using real-time RT PCR. Forced expression of miR-34a in GBM cell lines decreased their cell growth both in liquid culture and in immunodeficient mice; and the latter was associated with decreased angiogenesis. These cells also had decreased ability to migrate as detected by Boyden chamber and had profoundly low levels of cell cycle proteins (Cyclin -A1, -D1, -D3 and -B1, CDK2, as well as, E2F1, −3, and −4) and increased expression of cyclin kinase inhibitor (CKI) proteins, p21 and p27. Furthermore, the protein expression of EGFR was decreased in the cells with overexpression of miR-34a. Taken together, GBMs often acquire a miR-34a deletion and have low expression of this microRNA. The miR-34a probably normally acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell cycle proteins and EGFR. Individuals whose GBM have both deletion of miR-34a and amplification of EGFR have a particularly poor prognosis. 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 2101.

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