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Forschungsarbeit


Identifying new molecular targets in astrocytic tumors – Expression analysis

von Jonas Feldheim

The World Health Organization classifies astrocytic tumors into subgroups based on their molecular profile. Among these, isocitrate dehydrogenase mutant diffuse astrocytoma (Low-grade astrocytoma, LGA) are well-differentiated tumors with a comparatively good prognosis. However, malignant astrocytic tumors, such as glioblastoma multiforme (GBM) do not only appear de novo but can also develop from LGA precursor lesions.

The current standard in the therapy of GBM, the most common primary malignancy of the central nervous system, includes tumor resection, radiation, and concomitant, as well as adjuvant chemotherapy with temozolomide (TZM). Although recently, the introduction of tumor-treating fields to the therapeutic scheme has shown a great potential to improve patients' outcome, the overall survival remains rather unfavorable.

Recurrence tends to be the rule, with the relapse even increasing in aggressiveness and resistance to therapy. Although recently Osswald, Jung [1] shed light on molecular mechanisms of local tumor recurrence, not much is known about the mechanisms leading to multifocal growth of astrocytic tumors. As astrocytic tumor cells spread wide in the brain of patients with unifocal tumor growth [2], the question arises which mechanisms prevent or trigger growth of these single tumor cells. Therefore, further insight into the genetic basis of GBM would be highly desirable, not only to allow personal therapy adaptations by predicting tumor growth and recurrence patterns but also to identify additional molecular targets for new therapeutic concepts.

A literature research identified such proteins that either showed promising results in preliminary work or were never examined regarding a potential role in GBM development, but due to their mode of action have a high probability to be pro-migratory and pro-invasive in GBM. Therefore, we collected tissue samples of GBM, LGA, and normal brain (NB) and analyzed the posttranscriptional and posttranslational expression of the chosen factors. By retrospectively reconstructing the clinical course of the GBM-patients, we evaluated potential correlations of the proteins' cellular expression and the clinical course (e.g., growth pattern, overall survival, relapses, and treatment). The protein, for which our analysis is most advanced, is Activating-Transcription-Factor 5 (ATF5).

ATF5, a basic leucine zipper protein, suppresses differentiation of neuroprogenitor cells into glia or neurons in the NB. It is overexpressed in GBM. A reduction of ATF5 activity by application of a dominant-negative peptide leads to p53-independent apoptotic cell death of GBM cell lines and in mouse models. This interference did not cause any visible effects on NB or cultured astrocytes [3, 4], therefore indicating potential suitability as a therapeutic target.

Our analysis indicates that ATF5, compared to NB, is significantly overexpressed in LGA and GBM independently of tumor growth pattern, relapse, or treatment (e.g., Figure 1, data not shown). Furthermore, we observed that patients with high ATF5 expression had a significantly shorter one-year- and progression-free survival (Figure 2). Since inhibition of ATF5 seems to lead to the selective death of glioma cells, but not of non-tumor cells, it might serve as a potential ubiquitous therapeutic target in astrocytic tumors, independently of WHO grading or growth pattern.

To reconstruct the clinical course of the above mentioned GBM patients, we also examined known prognostic factors, such as the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. MGMT removes alkyl groups from guanine in the DNA, potentially counteracting the therapeutic efficacy of alkylating chemotherapeutics, such as TMZ, in tumor cells [5]. Its epigenetic silencing is associated with an improved response to TMZ-chemotherapy and a longer overall survival [6]. Clinical trials indicate that TMZ-chemotherapy might only have significant effect on the survival in MGMT silenced patients.

This is interesting, as there have been a few studies describing changes of MGMT methylation during the clinical course. The current diagnostic standard demands MGMT status determination only for the primary tumor and not necessarily for the relapse.

In contrast to the common "methylated" versus "unmethylated" rating, we aimed to quantify the degree of MGMT promoter methylation of a small panel of GBM patients and to correlate the changes with the clinical outcome. In addition, we summarized the current state of knowledge by reviewing literature reporting MGMT promoter methylation changes in astrocytic brain tumors and performed a meta-analysis (Figure 3). Although the MGMT-methylation status remained stable for the majority of GBM patients, there was a change in the quantity of methylation in more than 60% of our patients. However, there was no indication of additional benefit from detecting these changes. It is unlikely that a switch of the methylation status is a response to therapy since it occurs equally to both directions. Thus, the molecular mechanisms and potential triggers remain to be further examined.

 

References

[1] Osswald, M., et al., Brain tumour cells interconnect to a functional and resistant network. Nature, 2015. 528(7580): p. 93-8.
[2] Sahm, F., et al., Addressing diffuse glioma as a systemic brain disease with single-cell analysis. Arch Neurol, 2012. 69(4): p. 523-6.
[3] Cates, C.C., et al., Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide. Oncotarget, 2016. 7(11): p. 12718-30.
[4] Angelastro, J.M., et al., Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5. Oncogene, 2006. 25(6): p. 907-16.
[5] Kaina, B., et al., MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair (Amst), 2007. 6(8): p. 1079-99.
[6] Weller, M., et al., MGMT Promoter Methylation Is a Strong Prognostic Biomarker for Benefit from Dose-Intensified Temozolomide Rechallenge in Progressive Glioblastoma: The DIRECTOR Trial. Clin Cancer Res, 2015. 21(9): p. 2057-64.


 

 

Figure 1 – Expression of ATF5 in glioma specimen: ATF5 mRNA expression of frozen tumor samples was measured using duplex qPCR with GAPDH as endogenous marker. a) Comparison of ATF5 mRNA expression in normal brain (NB, n = 10), low grade astrocytoma WHO° II (LGA, n = 40) and glioblastoma (GBM, n = 79). b) Expression analysis of ATF5 mRNA in NB (n = 10) and specimens of GBM displaying different growth patterns (primary local tumors (n = 24) and their local relapses (n = 9), primary local tumors (n = 11) and their multifocal relapses (n = 3) and primary multifocal tumors (n = 10). ANOVA, Scheffé procedure, error bars represent 95% confidence interval. c) ATF5 was stained with a DAB-based protocol in paraffin-embedded specimens of NB (top), GBM (middle) and peri-necrotic palisades of a GBM (bottom). Shown is one representative sample of n = 21; magnification 400×. d) Quantification of ATF5 protein expression based on the optical density of anti-ATF5-DAB staining in NB (n = 8) and GBM (n = 21). Unpaired two-sided t-test with division in a “high optical density” and “low optical density” group as variable, error bars represent 95% confidence interval.

Figure 2 – Kaplan-Meier analysis of patients with high and low ATF5 mRNA expression at primary surgery: GBM patients were grouped into “low ATF5 expression” and “high ATF5 expression” according to the median ATF5 mRNA overexpression compared to NB (6-fold). Both groups were compared for a) overall survival and b) progression-free survival in a Kaplan-Meier analysis (Breslow) (m = months).

Figure 3 – Forest plot: Changes of MGMT promoter methylation from the primary tumor to relapse: Studies, with at least ten patients, that report changes in MGMT promoter methylation at the relapse of astrocytic tumors, were included. Changes from “methylated” to “unmethylated” and from “unmethylated” to “methylated” are summarized in one figure and based on the cut-off value of the respective publication. Confidence intervals (95%) were calculated based on the Wilson score interval.

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