CD74 is overexpressed in cells cultured from temozolomide resistant glioblastoma xenografts and the expression in patient samples is associated with poor overall survival

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The methylating agent temozolomide (TMZ) has demonstrated a significant cytotoxicity against glioma, and is currently used for initial treatment of glioblastoma (GBM). Over-expression of O⁶-methyguanine-DNA-methyltransferase (MGMT) is an important determinant of TMZ resistance but the expression of MGMT does not absolutely correlate with the TMZ response. We have developed a panel of GBM xenografts from patient tumors, and similar to clinical results, we have identified at least one xenograft line (GBM14) that is sensitive to TMZ despite the unmethylated MGMT promoter. To identify other gene products that are involved in TMZ resistance and sensitivity, we evaluated the gene expression in 2 xenograft lines that are sensitive to TMZ (GBM12 and 14) or resistant to TMZ (GBM 43 and 44) before and in after TMZ treatment. Global gene expression was analyzed with microarray technology using the Affymetrix Human Genome U133 2.0 Plus GeneChip Set, covering more than 54,000 gene transcripts, corresponding to almost 22,000 known genes. Differentially expressed genes were ranked according to the p-values and only the first 200 high-ranked probes (genes) were considered for further analysis. Hundred-twenty of the high-ranked 200 probes were overexpressed and 80 probes were underexpressed in the resistant relative to sensitive cells before exposure to TMZ. Among the overexpressed gene probes in the TMZ resistant cells was the MGMT, which was ranked 48 among the top 50 overexpressed genes. Interestingly, CD74, which is a membrane receptor of macrophage migration inhibitory factor (MIF), showed up first on the list. Evaluation of CD74 expression by RT-PCR revealed that CD74 is expressed in 7 (30%) of 21 glioblastoma xenografts and in 4 (27%) of 15 established glioblastoma cell lines. Immunohistochemical evaluation of CD74 expression revealed strong expression in 8 (25%) of 32 glioblastoma patient samples. Interestingly, CD74 expression was associated with poor overall survival (median survival 316 vs. 476; Log-rank p=0.05). Knockdown of CD74 by shRNA inhibited cell proliferation in U-87 glioma cells. Intriguingly, CD74 negative U-87 cells were significantly more sensitive to TMZ compare to the control CD74 expressing U-87 cells (P<0.001). Our findings underscore the importance of MGMT in the development of resistance against TMZ and also provided CD74 as a candidate gene of which further studies are currently in progress.