Activation of Neural and Pluripotent Stem Cell Signatures Correlates with Increased Malignancy in Human Glioma

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal- and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies

Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas

Mutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas (<10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.American Cancer Society [RSG-10-126-01-CCE]American Cancer SocietyNCINCI [5R01-CA140316]Pediatric Brain Tumor Foundation Institute GrantPediatric Brain Tumor Foundation Institute GrantSoutheastern Brain Tumor Foundation GrantSoutheastern Brain Tumor Foundation GrantVoices Against Brain Cancer Foundation GrantVoices Against Brain Cancer Foundation GrantJames S. McDonnell Foundation GrantJames S. McDonnell Foundation GrantV FoundationV FoundationAccelerate Brain Cancer Cure Foundation GrantAccelerate Brain Cancer Cure Foundation GrantNIHNIH [5P50 NS20023, 5P50 CA108785, CA057345, CA129825, R37 011898]Sanofi-AventisSanofiAventi

Correlation of <i>LOX</i>, <i>HIF1A</i> and <i>BMP1</i> expression levels in astrocytomas of different malignant grades.

<p>Correlation was assessed in pilocytic astrocytomas (AGI: A, B and C), low-grade astrocytomas (AGII: D, E and F), anaplastic astrocytomas (AGIII: G, H and I) and glioblastomas (GBM: J, K and L). Statistically significant values were obtained in AGI cases for <i>BMP1</i> and <i>HIF1A</i> correlation (C) and in GBM cases for <i>LOX</i> and <i>BMP1</i> correlation (J), <i>LOX</i> and <i>HIF1A</i> correlation (K) and <i>HIF1A</i> and <i>BMP1</i> expression levels (L). In AGIII samples, <i>HIF1A</i> and <i>LOX</i> expression levels were negatively correlated (H). The statistically significant correlations are shown in red. Correlations between gene expression values were assessed using the non-parametric Spearman-rho correlation test.</p

LOX expression and localization in astrocytomas and non-neoplastic brain tissues.

<p>Immunohistochemistry was performed in 6 non-neoplastic brain tissues (NN), 6 pilocytic astrocytoma (AGI), 6 low-grade astrocytoma (AGII), 6 anaplastic astrocytoma (AGIII), and 6 glioblastoma (GBM) cases. The images show representative cases of each type of sample (400x magnification for all cases). Arrows indicate the endothelial cells staining.</p

LOX immunostaining analysis in astrocytomas and non-neoplastic brain tissues.

<p>The graphs illustrate semi-quantitative immunohistochemistry labeling scores (ILS) that refer to the product of staining intensity and the percentage of positive cells stained with anti-LOX in the cytoplasm (A), nucleus (B) and endothelial cells (C). The horizontal bars show the median ILS of each group. The difference in protein expression among the groups was statistically significant for nuclear staining (<i>p</i> = 0.01) and endothelial cell staining (<i>p</i> = 0.0008) as determined by a Kruskal-Wallis test. A post-hoc Dunn’s test was used to calculate the differences in expression among the groups (*<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.0001).</p

Effect of <i>LOX</i> knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.

<p>Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after <i>LOX</i> silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 10⁴ cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 10³ cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **<i>p</i><0.001; ***<i>p</i><0.0001).</p

Gene expression levels in astrocytomas of all malignant grades relative to non-neoplastic samples.

<p>Transcript levels of <i>LOX</i> (A), <i>BMP1</i> (B), and <i>HIF1A</i> (C) were determined in 19 non-neoplastic brain tissue samples (NN), 23 pilocytic astrocytomas (AGI), 26 low-grade astrocytomas (AGII), 18 anaplastic astrocytomas (AGIII) and 84 glioblastomas (GBM). Total RNA was reverse-transcribed into cDNA and analyzed by quantitative real-time PCR (RT-qPCR) using the SYBR Green method. The differences in expression levels among the groups were statistically significant for <i>LOX</i>, <i>BMP1</i> and <i>HIF1A</i> (Kruskal-Wallis test, <i>p</i><0.0001). A post-hoc Dunn’s test was used to calculate the differences in expression between NN and each astrocytoma group (*<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.0001). <i>IDH1</i> mutational status was determined for diffusely infiltrating cases and the distribution of <i>LOX</i> (D), <i>BMP1</i> (E) and <i>HIF1A</i> (F) expression levels in <i>IDH1</i> wild-type and mutated background. Horizontal bars show the median of each group. AGII and GBM cases with <i>IDH1</i>-mutated presented lower <i>LOX</i> expression levels when compared to <i>IDH1</i> wild-type cases. AGIII cases with wild-type <i>IDH1</i> presented lower <i>HIF1A</i> expression levels when compared to <i>IDH1</i>-mutated cases (Mann-Whitney test, * <i>p</i> <0.05 and t test **, <i>p</i> <0.005).</p