Nuclear FABP7 immunoreactivity is preferentially expressed in infiltrative glioma and is associated with poor prognosis in EGFR-overexpressing glioblastoma

Yu Liang 1 2 Andrew W Bollen 0 Ken D Aldape 3 Nalin Gupta 2 0 Department of Pathology, University of California , San Francisco, CA 94143 , USA 1 Current address: Division of Molecular Biology, Sequence Detection System & Arrays, Applied Biosystems , Foster City, CA 94404 , USA 2 Department of Neurological Surgery, Brain Tumor Research Center, University of California , San Francisco, CA 94143 , USA 3 Department of Pathology, Section of Neuropathology, University of Texas M. D. Anderson Cancer Center , Houston, TX 77030 , USA Background: We previously identified brain type fatty acid-binding protein (FABP7) as a prognostic marker for patients with glioblastoma (GBM). Increased expression of FABP7 is associated with reduced survival. To investigate possible molecular mechanisms underlying this association, we compared the expression and subcellular localization of FABP7 in non-tumor brain tissues with different types of glioma, and examined the expression of FABP7 and epidermal growth factor receptor (EGFR) in GBM tumors. Methods: Expression of FABP7 in non-tumor brain and glioma specimens was examined using immunohistochemistry, and its correlation to the clinical behavior of the tumors was analyzed. We also analyzed the association between FABP7 and EGFR expression in different sets of GBM specimens using published DNA microarray datasets and semi-quantitative immunohistochemistry. In vitro migration was examined using SF763 glioma cell line. Results: FABP7 was present in a unique population of glia in normal human brain, and its expression was increased in a subset of reactive astrocytes. FABP7 immunoreactivity in grade I pilocytic astrocytoma was predominantly cytoplasmic, whereas nuclear FABP7 was detected in other types of infiltrative glioma. Nuclear, not cytoplasmic, FABP7 immunoreactivity was associated with EGFR overexpression in GBM (N = 61, p = 0.008). Expression of the FABP7 gene in GBM also correlated with the abundance of EGFR mRNA in our previous microarray analyses (N = 34, p = 0.016) and an independent public microarray dataset (N = 28, p = 0.03). Compared to those negative for both markers, nuclear FABP7-positive/EGFR-positive and nuclear FABP7-positive/EGFR-negative GBM tumors demonstrated shortest survival, whereas those only positive for EGFR had intermediate survival. EGFR activation increased nuclear FABP7 immunoreactivity in a glioma cell line in vitro, and inhibition of FABP7 expression suppressed EGF-induced glioma-cell migration. Our data suggested that in EGFR-positive GBM the presence of nuclear FABP7 immunoreactivity increases the risk of poor prognosis Conclusion: In this study, we identified a possible mechanism as the basis of the association between nuclear FABP7 and poor prognosis of GBM. FABP7 expression can be found in all grades of astrocytoma, but neoplastic cells with nuclear FABP7 were only seen in infiltrative types of tumors. Nuclear FABP7 may be induced by EGFR activation to promote migration of GBM tumor cells. Positive nuclear FABP7 and EGFR overexpression correlated with short survival in EGFR-positive GBM patients. Therefore, nuclear FABP7 immunoreactivity could be used to monitor the progression of EGFR-overexpressed GBM. - Background GBM is the highest grade of astrocytoma and is also the most common primary brain tumor in adults. Approximately 50% of patients with GBM die within a year of diagnosis, despite the use of many aggressive treatment approaches [1]. Lack of reliable prognostic markers for these patients is a hindrance to improving therapy and individualizing therapeutic interventions. Amplification and/or overexpression of the EGFR gene, mutation of the p53 gene, and proliferation indices have all been proposed to predict survival of patients with GBM and to play a role in the pathophysiology of their tumors [2,3]; however, other studies have shown no such association with outcome [4-6]. One reason for this discrepancy is that strong clinical factors such as patient age need to be included [7,8]. Although clinical parameters such as age, Karnofsky performance status at diagnosis, and extent of resection are routinely used in clinical practice to predict the outcome of patients with GBM, none of these variables have a direct connection with tumor pathogenesis. In a previous study, gene expression profiling of a group of GBM specimens identified a cluster of about 50 named genes whose expression was inversely associated with survival [9]. In examining the annotations of "biological process" in the Gene Ontology terms for each gene [10], the annotation "neurogenesis" appeared most frequently, suggesting a common role for these genes in central nervous system development. In contrast, a number of other annotations for biological process such as "cell proliferation," "inflammatory response," and "immune response" were underrepresented in these genes. Because several of these genes are involved in cell-cell and cell-matrix interactions and cell migration, we hypothesized that their increased expression might be related to more infiltrative and aggressive tumor behavior. Based on the results of the preceding analyses and the availability of antibodies, we chose to investigate the prognostic value of one gene, FABP7, in greater detail [9]. Although FABP7 is a cytoplasmic protein, its varying subcellular localization between nucleus and cytoplasm has been reported in developing brain [11], glioma cell lines [12], and GBM specimens [9]. Increased FABP7 expression was also found in glia following nerve injury [13,14]. We separately scored FABP7 immunoreactivity in nucleus and cytoplasm, and found that nuclear FABP7 immunoreactivity was inversely correlated with survival of patients with GBM, particularly in younger cases [9]. This result is consistent with other reports that emphasize the effect of age upon various prognostic factors [7,8], and such a pattern is similar to a recent finding of an association between EGFR overexpression and poor prognosis in younger GBM patients [7]. FABP7 is a member of the multi-gene fatty acid-binding protein (FABP) family and binds to very-long-chain polyunsaturated fatty acids (C > 16) such as docosahexaenoic acid (DHA) with very high affinity in vitro [15]. FABP7 appears to have different roles in different tissue types. It is highly expressed in radial and Bergmann glial cells throughout the developing central nervous system and gradually declines in the adult [16,17]. FABP7 is required for neuron-induced glial differentiation and subsequent migration of neurons along the glial processes, but has no effect on cell proliferation and adhesion [11]. In Schwann cells, FABP7 expression is downstream of the Ras-independent EGFR signaling pathway, and it regulates interactions between Schwann cells and axons in normal peripheral nerves and peripheral nerve tumors without affecting cell proliferation and migration [13]. Differential expression of different types of FABPs is fou (...truncated)