Rama P. Kotipatruni

Autotaxin Inhibition with PF-8380 Enhances the Radiosensitivity of Human and Murine Glioblastoma Cell Lines.

Purpose: Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is radio-resistant and recurs despite aggressive surgery, chemo, and radiotherapy. Autotaxin (ATX) is over expressed in various cancers including GBM and is implicated in tumor progression, invasion, and angiogenesis. Using the ATX specific inhibitor, PF-8380, we studied ATX as a potential target to enhance radiosensitivity in GBM. Methods and Materials: Mouse GL261 and Human U87-MG cells were used as GBM cell models. Clonogenic survival assays and tumor transwell invasion assays were performed using PF-8380 to evaluate role of ATX in survival and invasion. Radiation dependent activation of Akt was analyzed by immunoblotting. Tumor induced angiogenesis was studied using the dorsal skin fold model in GL261. Heterotopic mouse GL261 tumors were used to evaluate the efficacy of PF-8380 as a radiosensitizer. Results: Pre-treatment of GL261 and U87-MG cells with 1 μM PF-8380 followed by 4 Gy irradiation resulted in decreased clonogenic survival, decreased migration (33% in GL261; P = 0.002 and 17.9% in U87-MG; P = 0.012), decreased invasion (35.6% in GL261; P = 0.0037 and 31.8% in U87-MG; P = 0.002), and attenuated radiation-induced Akt phosphorylation. In the tumor window model, inhibition of ATX abrogated radiation induced tumor neovascularization (65%; P = 0.011). In a heterotopic mouse GL261 tumors untreated mice took 11.2 days to reach a tumor volume of 7000 mm3, however combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach a tumor volume of 7000 mm3. Conclusion: Inhibition of ATX by PF-8380 led to decreased invasion and enhanced radiosensitization of GBM cells. Radiation-induced activation of Akt was abrogated by inhibition of ATX. Furthermore, inhibition of ATX led to diminished tumor vascularity and delayed tumor growth. These results suggest that inhibition of ATX may ameliorate GBM response to radiotherapy.

Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

The role of adiponectin in obesity-associated female-specific carcinogenesis

Adipose tissue is a highly vascularized endocrine organ, and its secretion profiles may vary with obesity. Adiponectin is secreted by adipocytes that make up adipose tissue. Worldwide, obesity has been designated a serious health problem among women and is associated with a variety of metabolic disorders and an increased risk of developing cancer of the cervix, ovaries, uterus (uterine/endometrial), and breast. In this review, the potential link between obesity and female-specific malignancies is comprehensively presented by discussing significant features of the intriguing and complex molecule, adiponectin, with a focus on recent findings highlighting its molecular mechanism of action in female-specific carcinogenesis.

Generation and selection of pluripotent stem cells for robust differentiation to insulin-secreting cells capable of reversing diabetes in rodents

Induced pluripotent stem cell (iPSC) technology enables the creation and selection of pluripotent cells with specific genetic traits. This report describes a pluripotent cell line created specifically to form replacement pancreatic cells as a therapy for insulin-dependent diabetes. Beginning with primary pancreatic tissue acquired through organ donation, cells were isolated, re-programmed using non-integrating vectors and exposed to a four day differentiation protocol to generate definitive endoderm, a developmental precursor to pancreas. The best performing iPSC lines were then subjected to a 12-day basic differentiation protocol to generate endocrine pancreas precursors. The line that most consistently generated highly pure populations was selected for further development. This approach created an iPSC-variant cell line, SR1423, with a genetic profile correlated with preferential differentiation toward endodermal lineage at the loss of mesodermal potential. This report further describes an improved differentiation protocol that, coupled with SR1423, generated populations of greater than 60% insulin-expressing cells that secrete insulin in response to glucose and are capable of reversing diabetes in rodents. Created and banked following cGMP guidelines, SR1423 is a candidate cell line for the production of insulin-producing cells useful for the treatment of diabetes.

642. NDRG4 Is a Novel Therapeutic Target in Malignant Meningioma and Its Loss Induces Apoptosis

NDRG4 is a member of the N-Myc downstream regulated gene family belonging to the alpha/beta hydrolase super family. The roles of NDRG4 in meningioma tumor development have not previously been evaluated. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for meningioma patients. We have recently identified the NDRG4 protein as being overexpressed in aggressive meningiomas. We have demonstrated that NDRG4 downregulation results in decreased cell proliferation, migration and invasive properties. In follow up to our prior studies on the effects of NDRG4 gene silencing in IOMM-Lee and CH-157 MN meningioma cell lines; here we demonstrate that the predominant form of cell death is apoptosis, utilizing Annexin-V flow cytometry assay. We show that apoptosis caused by upregulation of p53, phosphorylation of p53 at ser 15, Bax activation and downregulation of Bcl-2 and BcL-xL along with cytochrome c release from mitochondria and execution of caspases after depletion of NDRG4. The proapoptotic effect of p53 was verified by the results in which a small molecule compound PFT-α, an inhibitor of p53 phosphorylation, is greatly protected against targeting NDRG4 induced apoptosis. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis. In addition, we carried out the fluorescence cytohemcial analysis to confirm the mitochondrial-mediated apoptosis by changes in mitochondrial membrane potential (Ym), using JC-1 dye. These findings bring novel insight to the roles of NDRG4 in meningioma tumor cell progression. We hypothesized that NDRG4 has played crucial role in regulating the p53 by its molecular interactions. Binding of NDRG4 with p53 was confirmed by Immunoprecipitation and double Immunocolocalization was verified by confocal microscopy. In conclusion, we show that NDRG4 is predominantly a cytosolic protein expressed in response to cell proliferation and demonstrated the suppression of NDRG4 induces the apoptosis through p53 activated and mitochondrial mediated apoptotic pathway. This therapeutic strategy may have promise in the management of meningiomas.

Abstract 3326: Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers

Therapies for poor prognosis cancers, such as lung cancer and glioblastoma, are limited due to radio-resistance and tumor recurrence. Development of molecular targeted therapy can serve as a potential method to improve the efficacy of radiation therapy in both glioblastoma and lung cancer. Ionizing radiation (IR) can activate a series of pro-survival pathways which contributes to the pathogenesis of cancer cells. Among these pathways, cytosolic phospholipase A2 (cPLA2) is an integral component which is activated by IR. Following activation, cPLA2 cleaves arachidonic acid to form phosphatidylcholine (PC) and yields lysophosphatidylcholine (LPC). Autotaxin (ATX) then converts LPC to lysophosphatidic acid (LPA). LPA acts through G-protein-coupled receptors (GPCR) such as LPA1, LPA2, and LPA3, influencing various fundamental cellular functions. Specifically, these receptors can modulate the ability of cancer cells to proliferate, differentiate, and survive. Earlier we have shown that inhibition of ATX decreases invasion and enhances radiation sensitivity of cancer cells. In the present study we investigated the role of LPA receptors and its effect on radioresistance in human A549, murine Lewis Lung Carcinoma (LLC), and rat C6 glioma cells. We analyzed the expression levels of the three LPA receptors in various cancer cell lines including lung, brain, breast and pancreatic cancer cells. We found high expression of LPA1 in LLC, C6 and A549, which we thus chose for further study. Using inhibitors of LPA1/LPA3, Ki16425 and VPC 12249, we determined the effect of LPA1 inhibition on the AKT and ERK activation. Inhibition of LPA1 attenuated phosphorylation of both AKT and ERK in irradiated lung cancer cells. This implicated LPA1 in the regulation of pro-survival signaling, and we hypothesized that LPA1 regulation could influence radioresistance in lung cancer and glioma cells. We targeted LPA1 using siRNA in LLC, C6 and A549 cells which led to an average of 50% reduction in clonogenic survival and cell proliferation after irradiation. Similar results were obtained when LLC and A549 cells were treated with 10μM Ki16425 or 10μM VPC 12249. In addition, LLC and A549 cells which were treated with combinations of radiation and LPA1 inhibitors and analyzed using cell invasion assays showed a 70% reduction in cancer cell invasion. These results indicate that LPA1 could serve as a novel target for cancer treatment due to its ability to enhance the efficacy of radiotherapy in multiple cancer cell lines. Citation Format: Arpine Khudanyan, David Dadey, Rowan Karvas, Rama Kotipatruni, Dennis Hallahan, Dinesh Thotala. Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers. [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 3326. doi:10.1158/1538-7445.AM2015-3326

Development of plasmid-lipid complexes for direct intratumoral injection.

Gene therapy holds great promise as novel therapeutics for several major disorders, such as metabolic abnormalities and cancer. However, its use is limited by the lack of efficient, safe, and specific delivery strategies. Successful development of such strategies requires interdisciplinary collaborations involving expertise in lipid chemistry, cell biology, molecular biology, nanotechnology, systems biology, medical imaging methods, and clinical medicine. This methodological chapter summarizes cationic lipid-mediated gene transfer, which could be a safe and effective means of delivering potent therapeutic genes for transgene expression or gene suppression directly into tumors and gives references to studies on plasmid DNA delivery. These observations apply to gene therapy and DNA-based macromolecular drug synthesis, as well as in vitro and in vivo drug delivery studies.

Abstract 846: Inhibition of lysophosphatidic acid receptor 1 radiosensitizes lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite advances in diagnostic and therapeutic modalities, lung cancer remains the leading cause of cancer-related mortality in the United States. In most cases, lung cancer presents as an unresectable mass requiring a combination of chemotherapy and radiotherapy (CRT) for treatment. A barrier to efficacy of this treatment is radioresistance. Radiation induced activation of pro-survival pathways are currently thought to play an important role in radioresistance. The enzyme cytosolic phospholipase A2 (cPLA2) has been identified as a key component in radiation induced pro-suvival pathways. Ionizing radiation activates cPLA2, which then cleaves phosphatidylcholine (PC) to yield lysophosphatidylcholine (LPC). LPC is then converted to lysophosphatidic acid (LPA) by autotaxin (ATX). LPA binds to distinct G-protein coupled receptors (GPCRs) on the plasma membrane; LPA1, LPA2 and LPA3, which are encoded by the endothelial differentiation gene family. These receptors are involved in the regulation of various aspects of cancer, including proliferation, migration and metastasis. Previously, we have shown that inhibition of cPLA2, as well as inhibition of ATX, was sufficient to enhance the sensitivity of cancer cells to radiation. However, the question remains as to whether inhibition of LPA receptor activity would have a similar effect. Thus, we studied the role of LPA receptors as mediators of radioresponse in human A549 and murine Lewis Lung Carcinoma (LLC) cell models. Quantitative polymerase chain reaction (QPCR) analysis of lung cancer cells showed that LPA1 was highly expressed in LLC and A549, and that both cell lines had nearly undetectable levels of LPA2 and LPA3 mRNA. Hence, we used Ki16425 and VPC12249, specific inhibitors of LPA1 and LPA3, to study the effect of LPA1 inhibition on clonogenic survival, cellular proliferation and invasion after irradiation. We found that pre-treatment of LLC and A549 cells with 10µM Ki16425 or 10µM VPC12249 significantly attenuated cell proliferation and viability after irradiation. Pre-treatment of LLC cells with 10µM Ki16425 or 10µM VPC12249 also reduced clonogenic survival. Transwell cell-invasion assays showed that the combination of LPA1 inhibitors with irradiation significantly reduced LPA-induced cell invasion in A549 and LLC cells. To investigate a potential mechanism for these effects, we also determined the activation of AKT and ERK signaling pathways. Inhibition of LPA1 resulted in reduced radiation-induced phosphorylation of both AKT and ERK. Overall, our results indicate that LPA1 is a mediator of pro-survival signaling in the adaptive response to radiation, and could serve as a viable target for enhancing the efficacy of radiation therapy of lung cancer. Note: This abstract was not presented at the meeting. Citation Format: David Y.A. Dadey, Rowan M. Karvas, Rama Kotipatruni, Jerry Jaboin, Dennis Hallahan, Dinesh Thotala. Inhibition of lysophosphatidic acid receptor 1 radiosensitizes lung cancer 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 846. doi:10.1158/1538-7445.AM2014-846

Abstract 2287: Nuclear localization of N-myc downstream regulated gene 4: A novel therapeutic target in meningioma

NDRG4 is a member of the N-Myc downregulated gene family (NDRG) belonging to the alpha/beta hydrolase super family. The roles of NDRG4 in meningioma tumor development have not previously been evaluated. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for meningioma patients. We have recently identified the N-Myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningiomas and demonstrated that NDRG4 downregulation results in decreased cell viability, migration and invasive properties. NDRG4 was initially identified as a cytoplasmic protein. In the present study, we proposed that nuclear localization of NDRG4 and the role of NDRG4 in cell proliferation. Subcellular distribution of NDRG4 was identified by western blotting in nuclear and cytosolic extracts. Immunocytofluoresence analysis revealed the NDRG4 nuclear localization. Here we demonstrate that depletion of NDRG4 induces the predominant form of cell death is apoptosis, utilizing DNA laddering and Annexin V/APC flow cytometry assay. We show that apoptosis caused by transduction of lentiviral shNDRG4 involves of p53 phoshorylation at ser 15, Bax activation. Flow cytometry analysis revealed changes in mitochondrial membrane potential (Ψm), which was observed by JC-1. Sub-cellular distribution of Bax and Cytochrome c indicated the mitochondrial-mediated apoptosis. In conclusion, we show that NDRG4 is also a nuclear protein expressed in response to cell proliferation and demonstrated the suppression of NDRG4 induces the apoptosis through p53 and Bax mediated intrinsic apoptotic pathway. Citation Format: Rama P. Kotipatruni, Xuan Ren, Robert P. Vanderwaal, Dinesh Thotala, Jerry J. Jaboin. Nuclear localization of N-myc downstream regulated gene 4: A novel therapeutic target in meningioma. [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 2287. doi:10.1158/1538-7445.AM2014-2287

Abstract 2282: Targeting oncogenic NDRG4: A promising strategy for meningioma treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Meningiomas are the second most common brain tumors, and 20-30% of these tumors are aggressive. These aggressive subtypes are characterized by the capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and have proposed to study its role in cell survival, invasion/migration and angiogenesis. We utilized shRNA-containing Lentiviral plasmids to downregulate NDRG4 mRNA and protein expression in two high grade meningioma cancer cell lines, IOMM-Lee and CH157MN. Downregulation resulted in reduction in cell survival, and G2-M cell cycle arrest. Utilizing DNA laddering and Annexin V/APC flow cytometry, we determined that the predominant form of cell death was apoptosis. NDRG4 downregulation also decreased cellular invasion and migration, as determined by a number of assays, including: spheroid migration, linear and radial wound healing assays, Boyden chamber matrigel invasion and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we utilized immortalized brain endothelial cells, bEnd.3 cells. We treated the bEnd.3 cells with conditioned media from the NDRG4 depleted IOMM-Lee and CH157MN cells, and the ability to form bEnd.3 capillary-like tubes, to proliferate and to invade was abrogated. NDRG4 is not overexpressed in bEnd.3 cells, and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the role of NDRG4 in meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment. 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 2282. doi:1538-7445.AM2012-2282