Indra Mohanam

The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice

Matrix Metalloproteinase‐9 (MMP‐9) consists of a prodomain, catalytic domain with 3 fibronectin‐like type II modules and C‐terminal hemopexin‐like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP‐9‐PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP‐9‐PEX. The selected clones exhibited decreased MMP‐9 activity and reduced invasive capacity. We assessed how secretion of MMP‐9‐PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP‐9‐PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary‐like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor‐2 protein level. The suppression of HMECs survival by conditioned medium from MMP‐9‐PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub‐G0/G1 content, fragmentation of DNA, caspase‐3, ‐8 and ‐9 activation and poly (ADP‐ribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP‐9‐PEX stable transfectants in nude mice with attenuation of CD31 and MMP‐9 protein expression. These results demonstrate that MMP‐9‐PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth.

Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells.

Background Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. Methods and Findings Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells. Conclusions Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.

Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells.

Background Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions. Methods We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone. Results Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells. Conclusions Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.

Correction: Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells (PLoS ONE (2014) 9:8 (e105555) DOI: 10.1371/journal.pone.0105555)

[This corrects the article DOI: 10.1371/journal.pone.0105555.].

Stabilization of HIF-1α and HIF-2α, upregulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

Abstract 5213: RUNX2 modulates the angiogenic potential of human neuroblastoma cells

Neuroblastoma is the most common extracranial pediatric solid tumor with an undifferentiated status and characterized by heterogeneous clinical courses ranging from spontaneous regression to a very aggressive malignant progression. The RUNX gene family comprises RUNX1, RUNX2, and RUNX3 transcription factors and plays pivotal roles in development, differentiation and tumorigenesis. Hypoxia is known to be a significant physiological stress in the tumor microenvironment and is associated with tumor malignant phenotype. In this study, we examined the effects of hypoxia on the Runx2 expression in human neuroblastoma cells and the role of RUNX2 on angiogenic potential of neuroblastoma cells. Runx2 has two isoforms, Runx2-I and Runx2-II. Isoform I is controlled by the P2 proximal promoter whereas isoform II is transcribed from the P1 distal promoter. Increased expression of mRNAs of isoform I and II as well as intact isoform of RUNX2 was observed in NB1691 cells exposed to hypoxia. Furthermore, hypoxia enhanced the activity of a luciferase reporter containing Runx2 P1 promoter in NB1691 cells. Hypoxic exposure caused an increase in protein levels of RUNX2 as well as survivin, a RUNX2 downstream target gene in NB1691 cells. Hypoxia also stabilized RUNX2 protein levels in NB1691 cells without altering the stability of RUNX2 mRNA. Hypoxia enhanced angiogenic potential of NB1691 cells. Knockdown of Runx2 significantly decreased VEGF mRNA and protein in NB1691 cells exposed to hypoxia. A decrease in angiogenesis was observed in chorioallantoic membrane assay performed using conditioned medium collected from RUNX2 siRNA treated hypoxic NB1691 cells compared with control. Further, transcript levels were measured by PCR array of human angiogenesis pathway and the analysis showed down regulation of EDN1, FIGF, TEK and VEGF A mRNAs in hypoxic NB1691 cells treated with RUNX2 siRNA. In conclusion, our study suggests that hypoxia upregulates RUNX2 expression in NB1691 cells and knockdown of RUNX2 decreases the angiogenic potential of hypoxic NB1691 cells. Citation Format: Manu Gnanamony, Indra Mohanam, Sanjeeva Mohanam. RUNX2 modulates the angiogenic potential of human neuroblastoma 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 5213. doi:10.1158/1538-7445.AM2015-5213

Abstract 499: RUNX2 protects human neuroblastoma cells against apoptosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Neuroblastoma is the most common extracranial pediatric solid tumor with an undifferentiated status and is characterized by heterogeneous clinical courses. The RUNX gene family comprises RUNX1, RUNX2, and RUNX3 transcription factors and is closely involved in a variety of cellular processes including development, differentiation, and tumorigenesis. Since RUNX genes play crucial roles in neuronal differentiation, we studied the expression of RUNX mRNAs in human neuroblastoma NB1691 cells. An increased RUNX2 mRNA expression was found in NB1691 cells compared with RUNX1 and RUNX3 mRNAs. Hypoxia is known to be a significant physiological stress in the tumor microenvironment and is associated with tumor malignant phenotype. NB1691 cells exhibited an increased expression of RUNX2 mRNA upon exposure to hypoxia compared with normoxic cells. Knockdown of HIF-2α, but not HIF-1α, decreased hypoxia-induced up regulation of RUNX2 mRNA in NB1691 cells. BothHIF-1α and HIF-2α mRNAs as well as proteins were downregulated in hypoxic NB1691 cells treated with RUNX2 siRNA compared with non-targeted control siRNA. GLUT1, a HIF-1α downstream target gene and Oct4, a HIF-2α downstream target gene were also found to be decreased in RUNX2 siRNA-treated NB1691 cells exposed to hypoxia. Runx2 knockdown using RNA interference in NB1691 cells resulted in decreased cell proliferation under normoxic as well as hypoxic conditions. An increased percentage of apoptotic cells was found in NB1691 cells treated with RUNX2 siRNA, and this percentage further increased in hypoxic NB1691 cells treated with RUNX2 siRNA. Further, these observations were validated by TUNEL assay. Real-time PCR analysis revealed elevated levels of Bax, Puma, Noxa and Nix in NB1691 cells treated with RUNX2 siRNA compared with cells treated with non-targeted control siRNA-treated cells under hypoxic conditions. In conclusion, our study suggests that hypoxia up regulates RUNX2 in NB1691 cells in a HIF2α- dependent manner and RUNX2 protects neuroblastoma cells against apoptosis. Citation Format: Manu Gnanamony, Indra Mohanam, Sanjeeva Mohanam. RUNX2 protects human neuroblastoma cells against apoptosis. [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 499. doi:10.1158/1538-7445.AM2014-499

Abstract 510: Intermittent hypoxia confers resistance to radiation in human neuroblastoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Neuroblastoma, the most common extracranial solid tumor of children, arises from the sympathetic nervous system, is characterized by extensive genetic, morphologic, and clinical heterogeneity and presents as benign to highly aggressive forms in various stages. Hypoxia has been linked to tumor progression and increased malignancy. Like the most common malignant solid tumors, neuroblastomas contain zones of chronic or acute hypoxia reflecting poor oxygenation. Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of therapies. Radiotherapy is commonly applied in higher stage neuroblastoma patients with MYCN oncogene amplification and metastases. The present study focuses how intermittent hypoxia modulates the effects of radiation treatment in human neuroblastoma cells. Intermittent hypoxia-conditioned cells were derived from human neuroblastoma cells that were exposed to 10 cycles of hypoxia and reoxygenation. Intermittent hypoxia stabilized the expression of HIF-l α in neuroblastoma cells. Cell survival to radiation treatment was assessed by the colony formation assay. The hypoxia-conditioned neuroblastoma cells exhibit increased survival to irradiation treatment compared with parental cells suggesting alterations in apoptotic pathways. The expression of selected genes associated with DNA damage as well as genes of BCL-2 family and caspase family were studied in parental and hypoxia-conditioned neuroblastoma cells in response to radiation treatment. Radiation caused an increase in the levels of proapoptotic proteins Bax, Bad, Bid and cleaved products of caspase-3 and −9 in parental tumor cells. Similar observations were not obtained in hypoxia-conditioned neuroblastoma cells upon radiation treatment demonstrating their increased radioresistance characteristics. We also assessed how hypoxia modulates radiation-induced in vitro angiogenesis. Human microvascular endothelial cells were exposed to conditioned medium from irradiated parental and irradiated hypoxia-conditioned tumor cells and capillary-like structure formation was studied. Further, tumor cell conditioned medium was also evaluated in chorioallantoic membrane assay for its angiogenic capabilities. These results suggest that intermittent hypoxia-conditioned neuroblastoma cells demonstrate increased survival, resistance to induction of apoptosis and angiogenic capabilities to radiation treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 510.