HDMX regulates p53 activity and confers chemoresistance to 3-bis(2-chloroethyl)-1-nitrosourea.
Journal: 2010/December - Neuro-Oncology
ISSN: 1523-5866
Abstract:
Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.
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Neuro Oncol 12(9): 956-966

HDMX regulates p53 activity and confers chemoresistance to 3-Bis(2-chloroethyl)-1-nitrosourea

+3 authors
Department of Pathology, Pediatric Brain Tumor Foundation Institute and Preston Robert Tisch Brain Tumor Center at Duke (G.J., S.C., B.C., W.C.C., S.T.K., P.K., C.D., C.P., R.M., D.D.B., H.Y.) and Department of Medicine, Center for Human Genetics (S.G.G.), Duke University Medical Center, Durham, North Carolina
Corresponding Author: Hai Yan, MD, PhD, Duke University Medical Center, Box 3156, Durham, NC 27710 (ude.ekud.cm@20000nay).
These authors contributed equally to this work.
Department of Pathology, Pediatric Brain Tumor Foundation Institute and Preston Robert Tisch Brain Tumor Center at Duke (G.J., S.C., B.C., W.C.C., S.T.K., P.K., C.D., C.P., R.M., D.D.B., H.Y.) and Department of Medicine, Center for Human Genetics (S.G.G.), Duke University Medical Center, Durham, North Carolina
Received 2009 Aug 19; Accepted 2010 Mar 5.

Abstract

Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.

Keywords: chemoresistance, GBM, HDMX, oncogene, p53
Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults and is associated with a median survival of 12 months.1,2 Current therapy includes surgery, radiation, and adjuvant chemotherapy. An important component of multiple agent adjuvant chemotherapy regimens in brain and other tumors has been the DNA alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which exerts its cytotoxicity mainly by alkylating DNA at the O position of guanine and by forming DNA interstrand crosslinks. Patients with newly diagnosed malignant gliomas who were treated with BCNU wafers have noted survival benefits.3,4 The emergence of resistance to BCNU, however, has hampered its use in GBM patients.5,6 Variations in multidrug resistance genes and DNA repair proteins such as O-methylguanine-DNA methyltransferase, glutathione S-transferase, and intracellular glutathione content could all bestow BCNU chemoresistance.710

The protein encoded by TP53 controls multiple cellular functions, including cell proliferation, DNA repair, senescence, and apoptosis.11 Tumor resistance to chemotherapeutic agents has also been reported to arise through failed p53 signaling.12 Genes involved in the p53 signaling pathway are frequently altered in GBMs: TP53 mutations are found in 28% of primary GBMs;2 the murine double minute 2 protein (MDM2), a negative regulator of p53,13,14 is amplified and overexpressed in 8%–10% of GBMs;15 and p14, which inhibits p53 degradation by sequestering MDM2 to the nucleolus and rendering it inactive, undergoes deletion in 70% of GBMs.1,3,1518 Additionally, p53 may enhance chemosensitivity by promoting apoptosis through either the intrinsic or the extrinsic pathways. It may also undermine chemosensitivity by promoting cell-cycle arrest, DNA repair, differentiation, and the transcription of antiapoptotic genes.18 The role of p53 in the GBM response to BCNU, however, remains controversial. Inactivation of p53 was found to sensitize tumor cells to BCNU,79 whereas another study showed that exogenous expression of wild-type p53 resulted in an increased sensitivity to BCNU.19 In another study, TP53 status was found to be an unreliable prognostic marker of tumor sensitivity to BCNU.20

HDMX was originally identified as a homologue of MDM221,22 and HDMX is a potent antagonist of p53 transcriptional activity.23HDMX is amplified or overexpressed in 10%–20% of lung, colon, stomach, and breast cancers and in 65% of retinoblastomas.13 Amplification of the 1q32 chromosomal region encompassing HDMX has been observed in GBMs,2429 implicating HDMX as a candidate oncogene in the amplicon. Though HDMX is a negative regulator of p53,13,3032 its functional role and the mechanisms whereby it modulates p53 activity during GBM pathogenesis and its effects on the response of GBMs to chemotherapeutic agents have yet to be fully delineated.

In this study, we investigated the genomic amplification and overexpression of the HDMX gene in GBMs and their correlation with the TP53 status and MDM2 amplification. We also investigated the oncogenicity of HDMX in GBMs and how the overexpression of HDMX affected the response of GBM cells to BCNU in vitro and in vivo. We found that HDMX amplifications occurred mutually exclusively of TP53 mutation and MDM2 amplification. The overexpression of HDMX drove GBM growth and enhanced tumor cell survival against BCNU. We found that the induction of HDMX stimulated the repair of BCNU-inflicted DNA damage. These novel findings cast HDMX as an attractive therapeutic target with potential clinical benefits.

Acknowledgements

We thank Melissa J. Ehinger, Diane L. Satterfield, and Jennifer D. Funkhouser for assistance with collecting clinical samples, and Joe Zeidner for assistance in generating the GBM xenografts.

Conflict of interest statement. None declared.

Acknowledgements

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