Reuben Antony

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR‑410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N-BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the over-expression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

Abstract 1153: Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Medulloblastoma (MB) is the most common malignant brain tumor in children. Treatment of medulloblastoma includes surgery, radiation and chemotherapy. Despite long-term survival for the majority of children, 50% of medulloblastomas recur. Recently, medulloblastomas are classified into four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4 based on constitutive activation of WNT/β-catenin, SHH, and NOTCH signaling pathways that regulate cancer stem cell pluripotency, tumor growth, progression and metastasis. Previously, we investigated the molecular machinery underlying uPAR and WNT/β-catenin interdependent signaling pathways in medulloblastoma stem cells. Further, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. Complementing to this we demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. Here, we show that Hand-1 expression is observed to be downregulated in all the molecular subgroups of medulloblastoma. We also showed that the pluripotency marker, Oct-3/4 (encoded by the Pou5f1 gene) interacts with β-catenin and Hand-1 that ultimately regulated the uPAR expression. Our preliminary study demonstrated that Oct-3/4 preferentially cooperated with β-catenin in medulloblastoma to maintain tumor progression. Immunohistostaining of clinical specimens also confirmed our aforementioned results. Taken together, our studies show that overexpression of Hand-1 in medulloblastoma cells may effectively inhibit uPAR-induced invasion and metastatic ability, thereby regulating Oct-3/4/β-catenin balance. Citation Format: Swapna Asuthkar, Maheedhara R. Guda, Andrew J. Tsung, Reuben Antony, Dzung H. Dinh, Kiran K. Velpula. Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1153.

Abstract 2480: Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells

Neuroblastoma is the most common extra-cranial solid tumor in children. Despite aggressive therapy prognosis of high risk neuroblastoma remains guarded and also unpredictable because of heterogeneity of tumor response to treatment. Radio-resistance has been recognized as a significant contributor to aggressive clinical behavior of neuroblastoma. Characterization of genetic alterations have been used for predicting clinical outcome in neuroblastomas and one of the most studied genetic markers in aggressive neuroblastoma is nMyc/MYCN. The MYCN gene belongs to the MYC family of transcription factors and nMyc expression is to a large extent inversely correlated with cMyc expression and nMyc expression is also associated poor clinical outcomes. In this study, we have attempted to correlate the expression of nMyc in neuroblastoma cells NB1691 and SKNBE2 to radiation resistance. We exposed neuroblastoma cells NB1691 and SKNBE2 to chronic radiation doses with a cumulative high energy X-ray radiation dose of 25Gy achieved by dosing at 5Gy increments over a period of 15 days. Cells surviving after the 25Gy cumulative radiation dose were selected and designated as NB1691R and SKNBE2R. Expression levels of nMyc, cMyc and uPA were determined. We observed that the radiation tolerant cells showed at least 164 fold increase in the expression of uPA in NB1691R cells and at least 97 fold increase in SKNBE2R cells. We also observed that the expression levels of nMyc decreased in both NB1691R and SKNBE2R cells to almost undetectable levels. Further, the expression levels of cMyc revealed that NB1691R cells showed a 208 fold increase and SKNBE2R a 71 fold increase when compared to parental cells. We further determined whether the expression of uPA can modulate the expression of nMyc. We silenced uPA expression in NB1691 cells and observed that downregulation of uPA moderately rescued nMyc expression. Cell cycle analysis revealed that acute radiation of NB1691 cells induced accumulation of cells in the G2/M phase and un-radiated NB1691R cells continued to show cells accumulated in the G2/M phase similar to acute radiated NB1691 cells. Our study shows that: 1. Chronic radiation exposure suppresses the expression of nMyc and induces the expression of cMyc. 2. cMyc expression was accompanied with the expression of uPA and suppression of uPA expression moderately rescued the expression of nMyc. Our preliminary findings suggest that chronic exposure to radiation alters the nMyc/cMyc ratio in neuroblastoma cells and induces the expression of the pro-invasive molecule uPA. Citation Format: Manu Gnanamony, David Pinson, Reuben Antony, Karen S. Fernandez, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2480.

Hand1 overexpression inhibits medulloblastoma metastasis.

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis.

Overcoming barriers to hospice care for children with cancer: Promoting active participation of the oncology team.

131 Background: The concept of pediatric hospice care is a natural progression of the hospice philosophy as it has been applied to adults, although, there are unique challenges presented by children. At our institution hospice service for children are offered by the adult team. We evaluated the number of children with cancer eligible for hospice between July 2009 and May 2015 and also explored the barriers that prevented hospice enrollment and the experiences of families, and hospice staff. METHODS Retrospective Review, Programmatic Initiative. RESULTS Thirty-two children diagnosed with cancer died in a 6 year period. Diagnosis breakdown was: Solid tumors 17/32 (53%), brain tumors 8/32 (25%), leukemia/lymphoma 6/32 (19%), 1 rare hematology condition. Twenty-one patients (65%) were 0 - 9.99 years and 11(35%) between 10 - 19.99 years. Fifteen patients (47%) were enrolled in hospice and all died at home except one. 18/32 (56%) children died in the hospital, of those 10/18 (55%) their parents chose the hospital as the site for their children passing, 6/18 (33%) died from an unexpected acute episode that resulted in death, and 2/18 (11%) patients had an acute episode that lead to un-planned patient hospitalization. The identified barriers to establishing and delivering hospice care were: a) family unwillingness to work with the hospice team b) family desire to have the patient die in the hospital c) hospice services not available in the patients residential area and, d) low level of comfort in the care of young children on the part of the hospice staff. Families expressed mixed feelings regarding hospice services. CONCLUSIONS Hospice services for children with cancer are underutilized. There are family, institutional and community barriers to hospice enrolment for children with cancer. The great majority of patients still die in the hospital. We propose to improve our regional pediatric hospice service by strengthening collaboration between pediatric oncology, palliative care and hospice to better serve children with cancer at the end of life with a programmatic initiative involving the oncology team. We aim to evaluate the impact of this initiative on families, hospice staff and the oncology team.

Abstract 4160: SPARC overexpression combined with irradiation reduces VEGF-A dependent angiogenesis in vitro and in vivo via up regulation of miR410 in neuroblastoma cells

Neuroblastoma (NBL) is the third most common malignancy in children and accounts for more than 15% of cancer-related deaths in children in the US. Approximately 50% of patients present with advanced-stage and/or high risk disease. Despite significant intensification of conventional chemotherapy and the addition of immunotherapy to treatment regimens, the long-term survival rates for these children remain less than 40%. Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, belongs to the matricellular family of secreted proteins. The biological functions of SPARC are known to be variable in human cancers. In neuroblastoma however, SPARC expression has been found to be associated with impaired tumor growth and decreased tumor related angiogenesis. In the present study we have attempted to elucidate the mechanisms of this angiogenesis reduction. We overexpressed SPARC adenovirus (human) with and without ionizing radiation (5Gy) in two neuroblastoma cell lines SK-N-BE2 (p53mt) and NB1691 (p53wt) and observed suppression of Vascular Endothelial Growth factor A (VEGFA) in both NB1691 (5-fold) and SK-N-BE(2) (2-fold) cells. To determine angiogenic potential of these neuroblastoma cells after SPARC overexpression, we used the dorsal skin fold model assay from which we observed that controls developed tumor cell induced vasculature (TV) which was clearly differentiated from pre-existing vasculature (PV). Radiation treatment alone of neuroblastoma cells caused the development of smaller and leaky capillaries. Overexpression of SPARC reduced the tumor cell induced vasculature to 18±6% of controls in NB1691 cells and to 48±5% of controls in SKNBE2 cells. Addition of radiation treatment to SPARC overexpressed cells further reduced tumor induced vasculature to 7±2% in NB1691 and to 15±3% of controls in SKNBE2 cells. Interestingly irradiation treatment alone increased the number of new vessel formation in both NB1691 (110±7% of controls) and SKNBE2 (136±10% of controls) cells. Previously, we observed that SPARC overexpression decreased VEGF-A expression in both NB1691 and SKNBE2 cells in vitro. To further determine the mechanism by which VEGF-A is suppressed via SPARC overexpression we determined the expression levels of VEGF-A targeting microRNAs. From the microRNA analysis we observed that miR410 was consistently overexpressed in SPARC overexpressed cells both in vitro and in vivo. We also observed that miR410 targets VEGF-A mRNA at 4 different sites, more than any other microRNA that targets VEGF-A. Our results indicate that: 1. SPARC overexpression can sensitize neuroblastoma cells to irradiation even in a varied p53 environment and; 2. Overexpression of SPARC suppresses angiogenesis by specifically inducing the expression of VEGF-A targeting microRNA miR410. Citation Format: Smita Tanpure, Jerusha Boyineni, Reuben Antony, Karen Fernandez, Julian Lin, David Pinson, Christopher S. Gondi. SPARC overexpression combined with irradiation reduces VEGF-A dependent angiogenesis in vitro and in vivo via up regulation of miR410 in 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 4160. doi:10.1158/1538-7445.AM2015-4160

Abstract 3956: Combination of SPARC and radiation suppresses HSP27 and induces p21(CIP1/WAF1) in neuroblastoma tumor cells

Neuroblastoma (NBL) is the third most common malignancy in children and accounts for more than 15% of cancer-related deaths in children in the US. Approximately 50% of patients present with advanced-stage and/or high risk disease. Despite significant intensification of conventional chemotherapy regimens, the long-term survival rates for these children remain less than 40%. A somewhat improved outcome has been obtained with multimodality therapeutic approaches including chemotherapy, surgery, autologous bone marrow transplantation, radiation therapy and use of biological agents. However, long-term survival rates for children with high-risk disease remain poor and survivors experience significant immediate and late toxicities which further limits conventional chemotherapy dose intensification. Novel biological therapies are therefore much needed. Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, belongs to the matricellular family of secreted proteins, which also includes thrombospondin 1 and 2, osteopontin, and tenascins C and X. The biological functions of SPARC are known to be variable in human cancers. In neuroblastoma however, SPARC expression has been found to be associated with impaired tumor growth and angiogenesis. Our previous studies showed that overexpression of SPARC induced autophagy-mediated apoptosis in neuroblastoma cells. In the present study we have attempted to decipher the mechanisms of this apoptotic induction. We used two neuroblastoma cell lines SK-N-BE2 (p53mt) and NB1691 (p53wt) and transfected them with plasmid overexpressing Myc-DDK-tagged SPARC with and without ionizing radiation. We observed that overexpression of SPARC accompanied with ionizing radiation (5Gy) suppressed HSP27 in NB1691 (5-fold) and SK-N-BE(2) (2-fold) cells. Further we also observed that expression of p21(CIP1/WAF1) was increased in SPARC overexpressed radiated (5Gy) cells; 4-fold in NB1691 cells and 0.5-fold SK-N-BE(2) cells. Levels of cyclin B were also found to be increased in both SK-N-BE2 (3-fold) and NB1691 (5-fold) after exposure to radiation. However SPARC overexpression in radiated cells caused a decrease in levels of Cyclin B in NB1691 (-3 fold) and SK-N-BE(2) (-2 fold) cells. Cell cycle analysis revealed that radiation induced G2-phase arrest in both NB1691 and SK-N-BE(2) cells. This G2 phase arrest was followed by decreased mitochondrial membrane potential (Δψ) in SPARC overexpressed cells when compared to controls. We further observed that suppression of native SPARC by siRNA in both SK-N-BE2 and NB1691 cells altered their sensitivity to radiation as determined by MTT assay. Our results demonstrate that SPARC expression regulates radiation sensitivity in NB1691 (p53wt) and to a lesser extent in SK-N-BE2 (p53mt) cells via modulation of the heat shock protein HSP27 and the DNA damage response molecule p21(CIP1/WAF1). Citation Format: Christopher S. Gondi, Smita Tanpure, Reuben Antony, Karen S. Fernandez, Meena Gujrati, Julian Lin. Combination of SPARC and radiation suppresses HSP27 and induces p21(CIP1/WAF1) in neuroblastoma tumor cells. [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 3956. doi:10.1158/1538-7445.AM2014-3956