Tamador Elsir

Transcription factor PROX1: its role in development and cancer.

The homeobox gene PROX1 is critical for organ development during embryogenesis. The Drosophila homologue, known as prospero has been shown to act as a tumor suppressor by controlling asymmetric cell division of neuroblasts. Likewise, alterations in PROX1 expression and function are associated with a number of human cancers including hematological malignancies, carcinomas of the pancreas, liver and the biliary system, sporadic breast cancer, Kaposiform hemangioendothelioma, colon cancer, and brain tumors. PROX1 is involved in cancer development and progression and has been ascribed both tumor suppressive and oncogenic properties in a variety of different cancer types. However, the exact mechanisms through which PROX1 regulates proliferation, migration, and invasion of cancer cells are by large unknown. This review provides an update on the role of PROX1 in organ development and on its emerging functions in cancer, with special emphasis on the central nervous system and glial brain tumors.

Expression of PROX1 Is a common feature of high-grade malignant astrocytic gliomas.

PROX1 is a prospero-related transcription factor that plays a critical role in the development of various organs including the mammalian lymphatic and central nervous systems; it controls cell proliferation and differentiation through different transcription pathwaysand has both oncogenic and tumor-suppressive functions. We investigated PROX1 expression patterns in 56 human astrocytic gliomas of different grades using immunohistochemistry. An average of 79% of cells in World Health Organization Grade IV (glioblastoma, n = 15) and 57% of cells in World Health Organization Grade III (anaplastic astrocytoma, n = 13) were strongly PROX1 positive; low-grade diffuse astrocytomas (Grade II, n = 13) had 21% of cells that were strongly positive; Grade I tumors (n = 15) had 1.5%; and non-neoplastic brain tissue (n = 15) had 3.7% of cells that were PROX1 positive. Double immunolabeling showed that PROX1+ cells in glioblastomas frequently coexpressed early neuronal proteins MAP2 and &bgr;III-tubulin but not the mature neuronal marker protein NeuN. Analyses of coexpression with proliferation markers suggest that PROX1+ cells have a marginally lower rate of proliferation than other tumor cells but are still mitotically active. We conclude that PROX1 may constitute a useful tool for the diagnosis and grading ofastrocytic gliomas and for distinguishing Grade III and Grade IV tumors from Grade I and Grade II tumors.

A study of embryonic stem cell-related proteins in human astrocytomas: identification of Nanog as a predictor of survival.

Recent studies suggest that the regulatory networks controlling the functions of stem cells during development may be abnormally active in human cancers. An embryonic stem cell (ESC) gene signature was found to correlate with a more undifferentiated phenotype of several human cancer types including gliomas, and associated with poor prognosis in breast cancer. In the present study, we used tissue microarrays of 80 low‐grade (WHO Grade II) and 98 high‐grade human gliomas (WHO Grades III and IV) to investigate the presence of the ESC‐related proteins Nanog, Klf4, Oct4, Sox2 and c‐Myc by immunohistochemistry. While similar patterns of co‐expressed proteins between low‐ and high‐grade gliomas were present, we found up‐regulated protein levels of Nanog, Klf4, Oct4 and Sox2 in high‐grade gliomas. Survival analysis by Kaplan‐Meier analysis revealed a significant shorter survival in the subgroups of low‐grade astrocytomas (n = 42) with high levels of Nanog protein (p = 0.0067) and of Klf4 protein (p = 0.0368), in high‐grade astrocytomas (n = 85) with high levels of Nanog (p = 0.0042), Klf4 (p = 0.0447), and c‐Myc (p = 0.0078) and in glioblastomas only (n = 71) with high levels of Nanog (p = 0.0422) and of c‐Myc (p = 0.0256). In the multivariate model, Nanog was identified as an independent prognostic factor in the subgroups of low‐grade astrocytomas (p = 0.0039), high‐grade astrocytomas (p = 0.0124) and glioblastomas only (p = 0.0544), together with established clinical variables in these tumors. These findings provide further evidence for the joint regulatory pathways of ESC‐related proteins in gliomas and identify Nanog as one of the key players in determining clinical outcome of human astrocytomas.

PROX1 is a predictor of survival for gliomas WHO grade II

Background:The clinical course of World Health Organisation grade II gliomas remains variable and their time point of transformation into a more malignant phenotype is unpredictable. Identification of biological markers that can predict prognosis in individual patients is of great clinical value. PROX1 is a transcription factor that has a critical role in the development of various organs. PROX1 has been ascribed both oncogenic and tumour suppressive functions in human cancers. We have recently shown that PROX1 may act as a diagnostic marker for high-grade gliomas. The aim of this study was to address the prognostic value of PROX1 in grade II gliomas.Methods:A total of 116 samples were evaluated for the presence of PROX1 protein. The number of immunopositive cells was used as a variable in survival analysis, together with established prognostic factors for this patient group.Results:Higher PROX1 protein was associated with poor outcome. In the multivariate analysis, PROX1 was identified as an independent factor for survival (P=0.024), together with the presence of mutated isocitrate dehydrogenase 1 R132H protein, and with combined losses of chromosomal arms 1p/19q in oligodendrocytic tumours.Conclusion:PROX1 is a novel predictor of survival for grade II gliomas.

GABA-A Channel Subunit Expression in Human Glioma Correlates with Tumor Histology and Clinical Outcome

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the CNS and is present in high concentrations in presynaptic terminals of neuronal cells. More recently, GABA has been ascribed a more widespread role in the control of cell proliferation during development where low concentrations of extrasynaptic GABA induce a tonic activation of GABA receptors. The GABA-A receptor consists of a ligand-gated chloride channel, formed by five subunits that are selected from 19 different subunit isoforms. The functional and pharmacological properties of the GABA-A channels are dictated by their subunit composition. Here we used qRT-PCR to compare mRNA levels of all 19 GABA-A channel subunits in samples of human glioma (n = 29) and peri-tumoral tissue (n = 5). All subunits except the ρ1 and ρ3 subunit were consistently detected. Lowest mRNA levels were found in glioblastoma compared to gliomas of lower malignancy, except for the θ subunit. The expression and cellular distribution of the α1, γ1, ρ2 and θ subunit proteins was investigated by immunohistochemistry on tissue microarrays containing 87 gliomas grade II. We found a strong co-expression of ρ2 and θ subunits in both astrocytomas (r = 0.86, p<0.0001) and oligodendroglial tumors (r = 0.66, p<0.0001). Kaplan-Meier analysis and Cox proportional hazards modeling to estimate the impact of GABA-A channel subunit expression on survival identified the ρ2 subunit (p = 0.043) but not the θ subunit (p = 0.64) as an independent predictor of improved survival in astrocytomas, together with established prognostic factors. Our data give support for the presence of distinct GABA-A channel subtypes in gliomas and provide the first link between specific composition of the A-channel and patient survival.

NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape.

Glioblastoma (grade IV glioma) is the most common and aggressive adult brain tumor. A better understanding of the biology of glioblastoma cells is crucial to identify molecular targets stimulating cell death. NPM1 (nucleophosmin) is a multifunctional chaperone that plays an important role in cancer development. Herein, NPM1 was analyzed by immunohistochemistry in human astrocytic gliomas. NPM1 was detected in all tumors but with a significantly higher staining intensity in grade IV than in low grade tumors. Depletion of NPM1 had only modest effects on the viability of U251MG, U1242MG, and U343MGa Cl2:6 glioma cells, despite alterations in nucleolar morphology. Glioma cell cultures depleted of NPM1 exposed to micromolar levels of actinomycin D were more prone to cell death (apoptosis) compared to cultures retaining NPM1. We had previously found that NPM1 binds to linker histone H1.5. Here we could show that silencing of H1.5 triggered glioma cell apoptosis as evidenced by a marked increase in both the numbers of cleaved caspase-3+ cells and in the amounts of cleaved PARP. Enforced expression of NPM1 suppressed apoptosis in H1.5 depleted glioma cells. Although our studies would suggest little effectiveness of targeting NPM1 alone there could be potential using it as a combination treatment.

FGF2 as a potential prognostic biomarker for proneural glioma patients

Abstract Background. The survival of high-grade glioma patients is poor and the treatment of these patients can cause severe side effects. This fosters the necessity to identify prognostic biomarkers, in order to optimize treatment and diminish unnecessary suffering of patients. The aim of this study was to identify prognostic biomarkers for high-grade glioma patients. Methods. Eleven proteins were selected for analysis due to their suggested importance for survival of patients with other types of cancers and due to a high variation in protein levels between glioma patients (according to the Human Protein Atlas, www.proteinatlas.org). Protein expression patterns of these 11 proteins were analyzed by immunohistochemistry in tumor samples from 97 high-grade glioma patients. The prognostic values of the proteins were analyzed with univariate and multivariate Cox regression analyses for the high-grade glioma patients, including subgroup analyses of histological subtypes and immunohistochemically defined molecular subtypes. Results. The proteins with the most significant (univariate and multivariate p < 0.05) correlations were analyzed further with cross-validated Kaplan-Meier analyses for the possibility of predicting survival based on the protein expression pattern of the corresponding candidate. Random Forest classification with variable subset selection was used to analyze if a protein signature consisting of any combination of the 11 proteins could predict survival for the high-grade glioma patients and the subgroup with glioblastoma patients. The proteins which correlated most significantly (univariate and multivariate p < 0.05) to survival in the Cox regression analyses were Myc for all high-grade gliomas and FGF2, CA9 and CD44 for the subgroup of proneural gliomas, with FGF2 having a strong negative predictive value for survival. No prognostic signature of the proteins could be found. Conclusion. FGF2 is a potential prognostic biomarker for proneural glioma patients, and warrants further investigation.

The transcriptional regulatory function of p53 is essential for suppression of mouse skin carcinogenesis and can be dissociated from effects on TGF-β-mediated growth regulation†

Transcriptional regulation by p53 is critical for p53‐mediated tumour suppression; however, p53‐mediated transactivation has been dissociated from p53‐mediated biological processes including apoptosis, DNA repair, and differentiation. We compared the effects of a mutant allele, p53QS − val135, containing a double mutation in the amino‐terminus abrogating transactivation activity and a modification at amino acid 135 partially affecting DNA binding, to complete loss of p53. We applied in vitro endpoints correlated with epithelial tumourigenesis and an in vivo assay of tumour phenotype to assess whether loss of p53‐mediated transcriptional regulation underlies the malignant phenotype of p53−/−/v‐rasHa‐overexpressing keratinocytes. Transactivation deficiency of p53QS‐val135 was confirmed by reporter gene assays in fibroblasts and differentiating keratinocytes. Ras oncogene‐induced senescence was lost in both p53QS − val135/QS − val135 and p53−/− keratinocytes. Similarly, p53QS − val135/QS − val135, like p53−/−, cooperated with v‐rasHa to enhance malignant conversion. The tumours arising in p53QS − val135/QS − val135 keratinocytes displayed strong nuclear p53 expression; thus, the p53QS − val135 allele was maintained and the deficient transactivation function of the expressed p53QS mutant protein was supported by absence of p21waf1 in these tumours. The p53QS − val135 allele did not confer a dominant‐negative phenotype, as p53+/QS − val135 keratinocytes senesced normally in response to v‐rasHa expression and formed benign tumours. While p53−/− keratinocytes displayed diminished response to TGF‐β, p53QS − val135/QS − val135 and p53+/+ keratinocytes responded equivalently, indicating that the requirement for p53 in maximizing TGF‐β‐mediated growth regulation is independent of its transactivation domain and that the ability of keratinocytes to respond to TGF‐β is insufficient to suppress the malignant phenotype in this model. Furthermore, TGF‐β enhances p53QS‐induced activation of a dual p53‐TGF‐β responsive reporter in a keratinocyte cell line. These findings support an essential role for p53‐mediated transcriptional regulation in suppressing malignancies arising from ras‐induced skin tumours, consistent with previous findings in spontaneous carcinogenesis in other organs, and highlight the potential importance of senescence for tumour suppression in vivo. Copyright

PROX1 is a novel pathway-specific prognostic biomarker for high-grade astrocytomas; results from independent glioblastoma cohorts stratified by age and IDH mutation status.

PROX1 is a transcription factor with an essential role in embryonic development and determination of cell fate. In addition, PROX1 has been ascribed suppressive as well as oncogenic roles in several human cancers, including brain tumors. In this study we explored the correlation between PROX1 expression and patient survival in high-grade astrocytomas. For this purpose, we analyzed protein expression in tissue microarrays of tumor samples stratified by patient age and IDH mutation status. We initially screened 86 unselected high-grade astrocytomas, followed by 174 IDH1-R132H1 immunonegative glioblastomas derived from patients aged 60 years and older enrolled in the Nordic phase III trial of elderly patients with newly diagnosed glioblastoma. Representing the younger population of glioblastomas, we studied 80 IDH-wildtype glioblastomas from patients aged 18-60 years. There was no correlation between PROX1 protein and survival for patients with primary glioblastomas included in these cohorts. In contrast, high expression of PROX1 protein predicted shorter survival in the group of patients with IDH-mutant anaplastic astrocytomas and secondary glioblastomas. The prognostic impact of PROX1 in IDH-mutant 1p19q non-codeleted high-grade astrocytomas, as well as the negative findings in primary glioblastomas, was corroborated by gene expression data extracted from the Cancer Genome Atlas. We conclude that PROX1 is a new prognostic biomarker for 1p19q non-codeleted high-grade astrocytomas that have progressed from pre-existing low-grade tumors and harbor IDH mutations.

Abstract 2082: Control and function of the PROX1 transcription factor in malignant glioma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Malignant gliomas remain to be fatal and highly resistant to therapy. PROX1 is a transcription factor critical for embryonic development, with a role in cell cycle control and progenitor cell differentiation. PROX1 is highly relevant to study in relation to brain tumors due to its role in normal CNS development, where it leads to progenitor cell differentiation and initiation of neurogenesis. We have previously shown that PROX1 is expressed in human gliomas where there is a positive and statistically significant correlation between PROX1 expression and glioma tumor grade. We have also evaluated the prognostic value of PROX1 in a cohort of patients with grade II gliomas and found that a high number of PROX1 positive cells correlated significantly with poor outcome, a clinically important finding, as such predictors do not exist at present. The mechanism behind PROX1 overexpression in high-grade glioma and its functional consequences remain unclear. To this end we set out to study PROX1 effects on the behavior of glioma cells. We found that glioma cell lines in general expressed only low levels of PROX1 protein and mRNA. However, PROX1 was readily detected in the U343MGa Cl2:6 glioma cell line. This cell line also expresses GFAP and SOX2 and has features in common with glioma stem cells. Depletion of PROX1 in U343MGa Cl2:6 cells using siRNA had a marginal positive effect on cell proliferation in a short term assay. Furthermore, ectopic PROX1 impaired the ability of glioma cells to proliferate as indicated by reduced colony formation and decreased BrdU incorporation. We present experimental evidence showing that PROX1 induces p27. By regulating p27, PROX1 may block the activity of Cdk2/Cyclin E complex, and thus inhibit cell cycle progression. The functional consequences of PROX1 expression on the stemness, migration, and invasion phenotypes of glioma cells remain to be further investigated and preliminary results will be presented. Citation Format: Kaveh M. Goudarzi, Tamador Elsir, Monica Nister, Mikael S. Lindstrom. Control and function of the PROX1 transcription factor in malignant glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2082. doi:10.1158/1538-7445.AM2015-2082