The p53 Negative Regulator MDM4 Is Amplified and Over-Expressed in Hepatoblastoma
Angshumoy Roy, Kayuri U Patel, Kristy L Hamilton, Xinyan Lu, Milton J Finegold, Dolores Lopez-Terrada. Baylor College of Medicine, Houston, TX
Background: The p53 tumor suppressor pathway is inactivated in virtually all cancers. In many tumors, amplification or over-expression of MDM4 and MDM2 abolish the p53-mediated oncogenic stress response by inactivating the wild-type p53 protein. Restoring p53 function using inhibitors to disrupt the MDM4/MDM2/p53 interactions is a promising new therapeutic strategy.
Hepatoblastoma (HB) is a highly aggressive neoplasm of childhood. While most HBs are managed with surgery and chemotherapy, no effective treatment exists for refractory and recurrent tumors. Since TP53 mutations are rare in HB, we posited that a systematic evaluation of MDM4 and MDM2 amplification and over-expression in HB may reveal a common mechanism of p53 inactivation and a potential therapeutic target.
Design: Archival specimens (n=26) were obtained with IRB approval. In cases with double minutes (dmins) on cytogenetics, spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH) was used to fine-map the minimum genomic interval. FISH analyses with MDM4 and a chromosome 1q control probe were performed on 26 FFPE samples. 100 cells were counted and scored as amplification (>5 copies), copy-gain (3-4 copies), chromosome 1 polysomy (>2 copies of both probes), or normal.
Real-time qPCR for MDM4, MDM2 and p21 was performed on 21 frozen samples. Data in triplicate was normalized to GAPDH and plotted as fold-change compared to normal liver. MDM4 expression was evaluated by immunohistochemistry.
Results: In 2 HB cases with dmins, SKY and aCGH analyses mapped to a ∼1 Mb region on Chromosome 1q32.1 containing MDM4. MDM4 FISH analysis identified genomic amplification in 2 cases and copy gain in 8 cases (10/26 cases, 38.4%). Five additional cases had 1-2 extra copies of MDM4 and chromosome 1 polysomy. MDM4 expression was 2- to 53-fold up-regulated in 8/21 (38%) cases, including two cases without amplification or copy gain. Nuclear MDM4 protein was detected in amplified cases. MDM2 expression was however increased in only 1 case. Expression of the p53 transcriptional target, p21, was >2-fold down-regulated in 7/21 (33%) cases, suggesting p53 pathway suppression downstream of MDM4 over-expression.
Conclusions: MDM4 amplification/copy gain and over-expression, as detected by interphase FISH and qPCR, is a common mechanism by which wild-type p53 can get inactivated in HBs. In contrast, MDM2 over-expression is a rare event in our series. Our current studies are evaluating the efficacy of small molecule inhibitors in restoring p53 function in the HepG2 hepatoblastoma cell line.
Monday, March 19, 2012 2:45 PM
Platform Session: Section E, Monday Afternoon