Nahir Cortes-Santiago

Genetic and Epigenetic Modifications of Sox2 Contribute to the Invasive Phenotype of Malignant Gliomas

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.

TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1

Membrane-bound enzyme relocates to the cell nucleus to modify chromatin, inducing cancer resistance to radiotherapy. DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas

Glioblastoma recurrence after treatment with the anti–vascular endothelial growth factor (VEGF) agent bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. Our group has previously reported that Tie2-expressing monocytes (TEMs) are aberrantly present at the tumor/normal brain interface after anti-VEGF therapies and their significant role in the invasive outgrowth of these tumors. Here, we aimed to further understand the mechanisms leading to this pro-invasive tumor microenvironment. Examination of a U87MG xenogeneic glioma model and a GL261 murine syngeneic model showed increased tumor expression of angiopoietin 2 (Ang2), a natural ligand of Tie2, after anti-angiogenesis therapies targeting VEGF or VEGF receptor (VEGFR), as assessed by immunohistochemical analysis, immunofluorescence analysis, and enzyme-linked immunosorbent assays of tumor lysates. Migration and gelatinolytic assays showed that Ang2 acts as both a chemoattractant of TEMs and an enhancing signal for their tumor-remodeling properties. Accordingly, in vivo transduction of Ang2 into intracranial gliomas increased recruitment of TEMs into the tumor. To reduce invasive tumor outgrowth after anti-angiogenesis therapy, we targeted the Ang-Tie2 axis using a Tie2 decoy receptor. Using syngeneic models, we observed that overexpression of soluble Tie2 within the tumor prevented the recruitment of TEMs to the tumor and the development of invasion after anti-angiogenesis treatment. Taken together, these data indicate an active role for the Ang2-Tie2 pathway in invasive glioma recurrence after anti-angiogenesis treatment and provide a rationale for testing the combined targeting of VEGF and Ang-Tie2 pathways in patients with glioblastoma.

Translocation t(7;12) as the sole chromosomal abnormality resulting in ACTB-GLI1 fusion in pediatric gastric pericytoma.

We hereby report an unusual gastric tumor arising from the pyloric wall of the stomach in a 9-year-old child harboring the exceptionally rare translocation t(7;12) resulting in ACTB-GLI1 gene fusion. This tumor has been previously classified as pericytoma with t(7;12) and described in 6 patients, 2 of them children. We discuss the challenges in recognizing this rare entity and the importance of the molecular studies in establishing the correct diagnosis. Our case is the first report of this type arising in the stomach of a child.

Abstract 3944: Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance

Glioblastoma is the most frequent subtype of primary brain tumor in adults and are resistant to current strategies of surgery, irradiation, and chemotherapy. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 months. Glioblastomas invariably recur after therapy due to the presence of cells exhibiting a multitherapy-resistance phenotype. To decipher the aberrant DNA repair pathways that enable tumor cells to survive DNA damage upon chemotherapy and radiotherapy should guide us to provide our patients with a more efficacious therapy. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. Our group reported the expression of the TKR Tie2 in in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. We observed that Tie2 traffics from the cellular membrane to the nucleus upon IR stimuli, as assessed by immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blots. This phenomenon is ligand dependent, as increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1), were observed after ionizing radiation, and nuclear Tie2 levels were decreased using soluble Tie2, which blocks the Ang1/Tie2 interaction. Of clinical significance, the presence of Tie2 in the nucleus is significantly associated with radioresistance, as observed by cell viability and clonogenic assays. Tie2 trafficking was associated to Caveolin-1, and this functional complex was disturbed when Caveolin-1 inhibitor or siCaveolin-1 were used, resulting in decreased Tie2 nuclear levels. In addition, we observed that upon IR treatment, nuclear Tie2 bound to DNA/protein complexes, and specifically to the key DNA repair protein γH2AX, as a component of the DNA-repair foci. Based on these results, we hypothesized that Tie2 was involved in DNA damage repair and focused on one of the main pathways involved in double strand break (IR induced), the non-homologous end-joining (NHEJ) repair mechanism. By using a fluorescent reporter construct in which a functional GFP gene is reconstituted following an NHEJ event (gift from Dr. Gorbunova, University of Rochester), we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Mutation of a Tie2 nuclear localization signal significantly decreased NHEJ efficiency repair and radiosensitization of BTSCs, suggesting a role of nuclear Tie2 in DNA damage repair, specifically in the NHEJ repair mechanism. Collectively, our results should propel the development of preclinical studies of the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas. Citation Format: Mohammad B. Hossain, Nahir Cortes-Santiago, Xuejun Fan, Konrad Gabrusiewicz, Joy Gumin, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K.Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2014-3944

Abstract 3298: ABL1 is required for Tie2-mediated DNA repair in brain tumor stem cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma is the most aggressive primary brain tumor and, in spite of surgery and chemoradiotherapy, invariably recurs. The poor prognosis associated with this disease, with a median survival of 15 months, is largely caused by the striking radioresistance of these tumors. The development of new therapeutic strategies for patient with brain tumors requires the identification of key molecular pathways regulating their resistant phenotype. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. We previously reported the expression of the TKR Tie2 in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. These results were confirmed by using immunofluorescence studies using confocal microscope and subcellular fractionation followed by Western blots. Of clinical interest, the presence of Tie2 in the nucleus is associated with radioresistance, as observed after mutagenesis of a newly discovered nuclear localization signal. In addition, upon IR, we detected increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1). The blocking of the Ang1/Tie2 interaction, by the use of a soluble receptor, modulated the IR-mediated Tie2 nuclear translocation, indicating Tie2 intracellular trafficking was ligand dependent. Additionally we also found that after IR treatment, Tie2 localized in the DNA-repair foci and complexed with the H2AX, the key DNA repair protein. The data presented here clearly suggested a role of Tie2 in the DNA damage repair machinery. To test our hypothesis, we used a fluorescent reporter construct in which a functional GFP gene was reconstituted following a non-homologous end joining (NHEJ) event (gift from Dr. Gorbunova, University of Rochester), and we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Based on the recently reported role of ABL1 (cAbl) in the ATM and KAT5 mediated DNA damage repair, we explored the relationship between ABL1 and the Tie2-mediated radioresistance. Our data clearly showed that DNA repair efficiency significantly and specifically decreased by using ABL1 inhibitor but not by knocking down ABL2 expression. We further analyzed the interactions between Tie2 and chromatin and, interestingly, observed that Tie2 complexes with core histones. Collectively, our results should propel the development of preclinical studies on the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas. Citation Format: Mohammad B. Hossain, Rehnuma Shifat, David G. Johnson, Mark T. Bedford, Mien-Chie Hung, Nahir Cortes-Santiago, Konrad Gabrusiewicz, Joy Gumin, Ravesanker Ezhilarasan, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K. Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. ABL1 is required for Tie2-mediated DNA repair in brain tumor stem cells. [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 3298. doi:10.1158/1538-7445.AM2015-3298

Abstract 2136: Nuclear trafficking of Tie2 is associated with radioresistance of gliomas

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Resistance and relapse are still primary causes that result in poor effectiveness of chemo- and radiotherapy in glioblastoma multiforme (GBMs), the most frequent subtype of primary brain tumors in adults. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 month, and the disease invariably recur after therapy due to the presence of cells exhibiting a multidrug-resistance phenotype. Aberrant DNA repair pathways can enable tumor cells to survive DNA damage that is induced after chemotherapy and radiotherapy. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating the resistant phenotype of these tumors. Previous work from our laboratory showed that Tie2, a previously considered specific vascular tyrosine kinase receptor, was expressed in glioma tumors and in brain tumor stem cells. The degree of expression of Tie2 was related to the malignancy of these tumors, and played an essential role in the multidrug resistance phenotype of gliomas. Our current research is focused on deciphering the mechanisms underlying this critical function. We found that density of Tie2 receptors in the membrane decreased upon exposure to both ligand (angiopoietin 1, Ang1) and ionizing irradiation (IR). Immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blot showed that Tie2 traffics from the cellular membrane to the nucleus upon both stimuli. Western blot analysis using antibodies against N- and C-terminus, and against phosphorylated-Tie2, indicated that the nuclear Tie2 is a full-length protein, and importantly that is phosphorylated at Tyr992. Of biological importance, to find the role of Tie2 in the nucleus, we irradiated the glioma cells, U251.Tie2, and found that ionizing radiation also helps Tie2 to move to the nucleus and importantly it binds with γH2AX one of the key DNA repair protein complex. By using confocal microscopy we found that Tie2 makes foci with γH2AX after DNA damage. We also found that after genotoxic stress like ionizing radiation or DNA-damaging drugs, Tie2-expressing cells were more resistant to DNA damaging effect, as assessed by cell viability assay and comet assay. Collectively, the nuclear Tie2 plays important role in the DNA repair which will result in the design of Tie2-targeting combinational therapies for patients with glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2136. doi:1538-7445.AM2012-2136

Abstract 1020: Recruitment of Tie2-expressing monocytes is associated with the heightened invasive phenotype of gliomas after antiangiogenic therapies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Gliomas are highly vascularized tumors, and preclinical data have suggested that glioma growth critically depends on the generation of tumor associated blood vessels. Among multiple factors controlling the complex process of angiogenesis, VEGF (Vascular Endothelial Growth Factor) and its associated signaling cascade are considered of central importance. Glioma cells are a major source of VEGF, and high levels of VEGF generation have been reported to correlate with high grade malignancy and poor prognosis. Antiangiogenic therapy has been recently added to the battery of treatments to newly diagnosed malignant gliomas. However, preclinical data from our laboratory and others, as some clinical results, shown concomitantly elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness. These results warrant preclinical investigation. Here, we present evidence of accumulation of Tie2-expressing monocytes (TEMs) in the brain tumor/normal interphase regions in tumor-bearing animals treated with anti-VEGF agents. Immunohistochemistry using F4/80 or Iba1 antibodies revealed an increase number of microglia/macrophages in central and peripheral tumoral areas after antiangiogenesis treatment. Moreover, the recruitment of these tumor-associated microglia/macrophages (TAMs) was associated with an overrepresentation of TEMs. Almost 50% of Tie2-positive cells co-localized with microglia/macrophages after bevacizumab or prolonged VEGF-Trap/Aflibercept therapies. To determine the role of TEMs in the invasive phenotype of gliomas upon antiangiogenic therapies, we use monocytes isolated from buffy coats and sorted positively for Tie2 expression, and monocytic cultures polarized to M2 phenotype and forced to express Tie2. We found that TEMs express high level of MMP2, MMP9, CD44, and CXCR4, molecules related with extracellular matrix remodeling, using q-PCR, ELISA, FACS analysis and double immunofluorescence. Gelatin zymography, as well as collagenase assay confirmed the upregulation of MMP-2 and MMP-9 activity. Of interest, knocking down Tie2 by siRNA suppresses MMP9 activity emphasizes a role of Tie2 signaling in the regulation of MMP9. Additionally, in vitro invasion assay showed a pivotal role of TEMs in development of an invasive phenotype in glioma cells. Together, our results suggest a novel mechanism of escape of GBMs to anti-VEGF therapies and the necessity of targeting the TEM population in combination with anti-VEGF therapies to improve clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1020. doi:1538-7445.AM2012-1020

Abstract 3307: The multitasking of Sox2: Maintaining the stemness and inducing the malignant phenotype of gliomas

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, displays an enrichment gene expression related to a stem cell signature. Sox2, a major stemness factor involved in pluripotency and in the maintenance of neural stem cell identity, has been described as frequently overexpressed in poorly differentiated gliomas. However, the role that this molecule plays in brain tumors remains under investigation. In this study, we sought to ascertain the Sox2 potential function in the control of self-renewal of brain tumor stem cells (BTSCs) and the malignant phenotype of gliomas. Our results showed Sox2 expression in human surgical malignant gliomas. In addition, Sox2 expression was significantly higher in the BTSC lines than in established glioma cell lines. Downmodulation of Sox2 in BTSCs by using siRNA resulted in the loss of the self-renewal properties of this cell population, and modification in the pattern of expression of markers related with stem cells properties and differentiation. These results are of major importance since, in the TCGA, we observed Sox2 overexpression in more than 80% of the samples (n=188), gene amplification of SOX2 in 18% of the individuals, and hypomethylation of the promoter in about 90% of the samples when compared with normal tissue, suggesting the relevance of this transcription factor in the malignant phenotype of GBMs. In this regard, our results demonstrated that SOX2 plays a prominent role in the invasive and migratory capabilities of both BTSCs and established glioma cell lines. Thus, ectopic expression of Sox2 was sufficient to induce invasion of glioma cells and knockdown experiments demonstrated that Sox2 is essential for maintaining the invasive properties of glioma cells. Therefore, these results identify a multi-functional aspect of Sox2 as both, a stemness promoting molecule and an essential pro-invasive protein, elucidating the possibility of targeting Sox2 for glioma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2011-3307