Expression of CD74 in high grade gliomas: a potential role in temozolomide resistance

Abstract

Temozolomide (TMZ) is the most effective chemotherapeutic agent for glioblastoma (GBM). Resistance to this methylating agent is linked to DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). However, in recent studies MGMT status was not completely accurate as a predictor of TMZ response in GBM, suggesting other mechanisms of resistance. As part of an effort aimed at discovery of genes involved in TMZ resistance in GBM, the expression of CD74 was evaluated in GBM patient samples and the influence of CD74 on TMZ response was evaluated in GBM tumor models. Reverse transcription-polymerase-chain reaction (RT-PCR) demonstrated differential expression of CD74 mRNA among the GBM xenografts; 8 of 20 (40%) expressed CD74 mRNA. In a preliminary evaluation of whether CD74 expression might influence TMZ response, CD74 mRNA expression levels were inversely associated with in vivo TMZ resistance in 20 GBM xenograft lines (median survival 122 vs. 62.5 days; r = -0.48, P = 0.032). In follow up to this observation, CD74 shRNA knock down in U87 cells significantly suppressed in vitro proliferation and increased TMZ sensitivity as compared to a non-specific control shRNA. Consistent with an effect on proliferation and survival, silencing of CD74 by shRNA was associated with reduced Akt and Erk1/2 activation in response to stimulation by CD74 ligand macrophage-migration inhibition factor (MIF). Lastly, expression of CD74 protein was assessed in patient samples [nine anaplastic astrocytoma (AA), and 62 GBM] by immunohistochemistry, and appreciable expression was observed in 28% of samples. Collectively, these findings suggest that CD74 is expressed in a subset of high grade gliomas and may contribute to TMZ resistance.

Fig 1

Expression of CD74 mRNA in 4 GBM xenograft lines relative to temozolomide sensitivity. A) The heat-map results of a microarray analysis of the CD74 probe set for the TMZ sensitive (GBM12 and GBM14) and TMZ resistant (GBM43 and GBM44) lines are shown. B) Total RNA from the same lines were reverse transcribed and PCR amplified using primers specific for human CD74 (upper panel). Beta-actin was used as internal control (lower panel)

Fig 2

Expression of CD74 and MIF mRNA in a panel of GBM xenografts. A) Total RNA isolated for 20 different GBM xenograft lines was subject to RT-PCR using primers specific for CD74, MIF and beta-actin. B) Expression of CD74 mRNA in GBM xenografts was quantitated using qRT-PCR. The level of CD74 expression is calculated relative to control U87 GBM cells.

Fig 3

CD74 expression relative to TMZ response in 20 GBM xenograft lines. A) Relative CD74 expression levels, determined from qRT-PCR, are plotted relative to the survival benefit for TMZ therapy in a panel of 20 GBM xenografts Survival benefit for each tumor line is defined as the ratio of median survival of TMZ treated versus placebo treated mice. B) Kaplan-Meier survival estimate curves associating CD74 mRNA expression with TMZ response for individual mice with intracranial GBM xenografts treated in the survival experiments.

Fig 4

Expression of CD74 in established GBM cell lines and effective targeting of CD74 expression by shRNA. A) RT-PCR of CD74 and MIF expression in established GBM cell lines, relative to beta-actin control. B) Western blotting results for CD74 and beta-actin from U87 clones stably transfected with non-specific shRNA (NT shRNA) or CD74 shRNA (2 independent clones).

Fig 5

Effect of silencing CD74 expression on proliferation and TMZ sensitivity in U87 GBM cells. U87 clones expressing CD74 shRNA or control non-specific targeting (NT) shRNA were evaluated by direct counting using trypan blue cell. A, Average cell number at each time-point is shown over the course of 7 day incubation. The error bars represent the standard error of the mean for two independent studies performed with triplicate samples. P-values shown are for comparing differences in proliferation at day 7. B, the effects of graded doses of TMZ on survival of the indicated U87 clones following 7 day incubation. Error bars represent the standard error of the mean for data generated from 4 independent experiments. P-values are shown for comparing control vs. CD74 shRNA transfected clones at a 60 μM concentration of TMZ. C - D, MIF induced activation of ERK1/2, AKT and Src in U87 cells is mediated by CD74. NT shRNA and CD74 shRNA U87 clones were serum deprived for 24 hours, stimulated with MIF (25 ng/ml) for the indicated duration prior to processing for western blotting. Membranes were probed for the indicated phospho-proteins and total proteins. Results are representative of 2 independent experiments.

Fig 6

Immunohistochemical expression of CD74 in GBM patient samples. A) Displays a representative case with strong CD74 expression with the corresponding H&E stain. B) Shows a representative CD74 negative case with the corresponding H&E stain. Specific CD74 staining of tumor cells is seen.