MRP3: a molecular target for human glioblastoma multiforme immunotherapy.
BACKGROUND
Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3).


METHODS
We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS.


RESULTS
Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002).


CONCLUSIONS
Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.
Kuan
Chien-Tsun
CT
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA. kuan@duke.edu
Wakiya
Kenji
K
Herndon
James E
JE
Lipp
Eric S
ES
Pegram
Charles N
CN
Riggins
Gregory J
GJ
Rasheed
Ahmed
A
Szafranski
Scott E
SE
McLendon
Roger E
RE
Wikstrand
Carol J
CJ
Bigner
Darell D
DD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001932:Brain Neoplasms / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D016022:Case-Control Studies; D002478:Cells, Cultured; D005260:Female; D005909:Glioblastoma / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D007167:Immunotherapy; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism*; D009367:Neoplasm Staging; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D000911:Antibodies, Monoclonal; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; C114462:multidrug resistance-associated protein 3
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