Michael E. Robbins, Ph.D. - US grants

DESCRIPTION: (Adapted from the applicant's abstract): The objective of the proposed research is to gain insight into potential mediators and pathogenic mechanisms responsible for the development and progression of radiation nephropathy, thereby providing a scientific basis for the rational design and later application of interventional therapies attenuating the severity of this dose-limiting normal tissue morbidity. The working hypothesis of this proposal is that irradiation induces over expression of the intra renal renin angiotensin system (RAS) and the fibrogenic cytokine transforming growth factor-beta. These act in an autocrine and paracrine manner to alter the mesangial cell (MC) phenotype in particular, with resultant over expression of extracellular matrix (ECM) gene products leading to glomerulosclerosis, loss of functioning nephrons, and ultimate renal failure. The application of regimens designed to selectively increase or decrease the severity of radiation nephropathy will be associated with a concomitant up regulation or down regulation, respectively of the RAS, TGF-beta, and ECM gene products. The proposal will test this hypothesis using four specific aims. The ability to reduce the severity of radiation nephropathy would have significant impact in cancer therapy. Thus, a thorough understanding of the specific mediators and mechanisms involved in experimental radiation nephropathy offers the promise of ultimately developing rational therapeutic strategies targeted at reducing the severity of this lesion in human beings.

DESCRIPTION: (Adapted from the investigator's abstract) Despite multimodality therapy including surgery, radiation therapy and chemotherapy, the median survival for patients presenting with glioblastma multiforme remains < 1 year. Novel therapeutic approaches are urgently needed. The PI hypothesizes that polyunsaturated fatty acids (PUFAs) may represent an exciting alternative. PUFA-enrichment of glioma cells leads to increased free radical generation, tumor cell cytotoxicity and an increased radiosensitivity; normal cell growth and radiosensitivity are unaffected. Moreover, the antioxidant enzyme catalase in normal rat brain microvascular endothelial cells (RBMECs) and astrocytes is upregulated by PUFAs; glioma catalase levels do not increase significantly. The PI hypothesizes that this ability of PUFAs to selectively modulate catalase gene expression reflects selective activation of the peroxisomal proliferator-activated receptor (PPAR), retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) nuclear hormone receptors (NHRs) with resultant upregulation of catalase gene expression. To test this hypothesis he will I] map regions of the rat catalase promoter that contain putative PUFA responsive cis-elements that may participate in governing the expression of catalase using transcriptional reporter assays with promoter-reporter deletion constructs of the catalase gene, ii] use gel mobility shift assays to characterize the binding of NHRs to putative response elements identified in the rat catalase promoter region and determine constitutive expression of the alpha, beta and gamma isotypes of PPAR, RAR and RXR NHRs in RBMECs, primary astrocytes and glioma cells; iii] investigate the role of particular eicosanoids in mediating the PUFA-induced upregulation of catalase in RBMECs and rat astrocytes by determining if pre-incubation of these cells with PUFA in the presence of cyclooxygenase, lipoxygenase or cytochrome P-450 inhibitors abrogates the PUFA-mediated increase in catalase gene expression. In addition, he will test the significance of these PUFA-mediated effects in vivo by I] evaluating the anti-glioma properties of PUFAs using a well-defined rat glioma model; ii] evaluate the ability of PUFAs to modulate in vivo normal brain catalase levels and determine if this increase leads to reduced radiation-induced brain injury. Defining the molecular basis for the tumor cytotoxic effect of PUFAs and the potential protection of normal cells, via upregulation of catalase, will lead to the development of novel therapies that offer the promise of significantly improving long-term cure and survival rates for an aggressive and often fatal neoplasm.

DESCRIPTION: The objective of the proposed research is to define the pathogenic role that specific glomerular (ie., mesangial cells [MCs]) and tubular (i.e., tubule epithelial cells [TECs]) cells and the renin-angiotensin system (RAS) play in radiation induced kidney injury. We hypothesize that i] renal irradiation results in alterations in MC and TEC phenotype that lead to activation of the intra-renal RAS. Specific aim (SA) 1 will test the hypothesis that radiation and/or Ang II lead to modulation of gene products associated with ECM accumulation and degradation, in part, by signaling mechanisms involving reactive oxygen species (ROS). We will determine if treating MCs and TECs with radiation, Ang II and other ROS generators in vitro leads to modulation of plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases-2 (TIMP-2) matrix metalloproteinase-2 (MMP-2), TGF-B and angiotensinogen (AGT, precursor of Ang II). We also will test whether addition of antioxidants or transfection with antioxidant enzymes blunts the radiation-and ANG II-induced modulation of these various gene products. Further, we will determine if the radiation-induced upregulation of PAI-1, MMP-2, TIMP-2, TGF-B and AGT expression observed in renal cells involves activation of the redox regulated transcription factors AP-1, AP-2 or NF-kB. The ability of Ang II to alter renal cell phenotype via ROS generation also will be studied. SA 2 will study the potential role of a glomerular-based intra-renal RAS by using adenovirus-based techniques to overexpress AGT in rat MCs. Using these transfected cells we will determine if overexpression of AGT can alter radiation-induced changes in MC expression of PAI-1, TIMP-2, MMP-2, and TGF-B. In addition, we will administer AGT transfected MCs to rat kidney glomeruli in vivo, to modulate intra-renal generation of Ang II in the irradiated kidney. SA 3 will use a unique double transgenic model produced by breeding mice that express human AGT only in proximal convoluted TECs with mice expressing human renin. Resultant double transgenics exhibit increased intra-renal Ang II synthesis; systemic levels are unaffected. Human AGT expression is androgen-dependent; expression in females is dependent on exogenous androgen administration. We will determine if irradiating kidneys of male double transgenic mice leads to increased ECM accumulation and exacerbation of functional and morphologic injury. Female mice will be used to investigate the temporal aspect of intra-renal RAS by administering androgens at various times/periods after irradiation. These studies offer the promise of developing rational therapeutic strategies directed at reducing the severity of this dose-limiting morbidity in human beings.

DESCRIPTION: (Adapted from the investigator's abstract) Despite multimodality therapy including surgery, radiation therapy and chemotherapy, the median survival for patients presenting with glioblastma multiforme remains < 1 year. Novel therapeutic approaches are urgently needed. The PI hypothesizes that polyunsaturated fatty acids (PUFAs) may represent an exciting alternative. PUFA-enrichment of glioma cells leads to increased free radical generation, tumor cell cytotoxicity and an increased radiosensitivity; normal cell growth and radiosensitivity are unaffected. Moreover, the antioxidant enzyme catalase in normal rat brain microvascular endothelial cells (RBMECs) and astrocytes is upregulated by PUFAs; glioma catalase levels do not increase significantly. The PI hypothesizes that this ability of PUFAs to selectively modulate catalase gene expression reflects selective activation of the peroxisomal proliferator-activated receptor (PPAR), retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) nuclear hormone receptors (NHRs) with resultant upregulation of catalase gene expression. To test this hypothesis he will I] map regions of the rat catalase promoter that contain putative PUFA responsive cis-elements that may participate in governing the expression of catalase using transcriptional reporter assays with promoter-reporter deletion constructs of the catalase gene, ii] use gel mobility shift assays to characterize the binding of NHRs to putative response elements identified in the rat catalase promoter region and determine constitutive expression of the alpha, beta and gamma isotypes of PPAR, RAR and RXR NHRs in RBMECs, primary astrocytes and glioma cells; iii] investigate the role of particular eicosanoids in mediating the PUFA-induced upregulation of catalase in RBMECs and rat astrocytes by determining if pre-incubation of these cells with PUFA in the presence of cyclooxygenase, lipoxygenase or cytochrome P-450 inhibitors abrogates the PUFA-mediated increase in catalase gene expression. In addition, he will test the significance of these PUFA-mediated effects in vivo by I] evaluating the anti-glioma properties of PUFAs using a well-defined rat glioma model; ii] evaluate the ability of PUFAs to modulate in vivo normal brain catalase levels and determine if this increase leads to reduced radiation-induced brain injury. Defining the molecular basis for the tumor cytotoxic effect of PUFAs and the potential protection of normal cells, via upregulation of catalase, will lead to the development of novel therapies that offer the promise of significantly improving long-term cure and survival rates for an aggressive and often fatal neoplasm.

DESCRIPTION (provided by applicant): Progressive cognitive impairment occurs in up to 50% of primary and metastatic brain tumor patients who survive 6 months or longer after treatment with partial or whole-brain irradiation (WBI);~200,000 patients/year receive these treatments. A growing body of evidence suggests that oxidative stress and pro-inflammatory responses play a critical role in radiation-induced brain injury. These observations provide the rationale for investigating anti-inflammatory-based therapeutic approaches to ameliorate or prevent radiation-induced brain injury. This competitive renewal will focus on the role of the peroxisomal proliferator-activated receptors 1 and 4 (PPAR1, PPAR4) in ameliorating or preventing radiation-induced brain injury, including cognitive impairment. These PPARs are potent mediators of anti-inflammatory responses. During the current funding period, we have demonstrated that i) administration of the PPAR3 agonist, pioglitazone, prevents fractionated WBI- induced cognitive impairment in young adult male rats;ii) the irradiated brains of PPAR1 KO mice have increased microglial activation, iii] administration of the PPAR1 agonist, fenofibrate, prevents both WBI-induced microglial activation and decreased neurogenesis, and iii) pre-treatment of microglial cells with PPAR1 agonists prevents radiation-induced increases in inflammation. In this competitive renewal, we propose to extend our PPAR1 studies and initiate studies on PPAR4, increasingly recognized as a promising pharmacological target for neuroprotection. We hypothesize that administration of PPAR1 and/or PPAR4 agonists will not only ameliorate or prevent radiation-induced brain injury, including cognitive impairment, but will also inhibit brain tumor growth. To test this hypothesis, we will pursue the following Specific Aims: 1] using a fractionated WBI rat model, we will determine if administration of PPAR1 or PPAR4 agonists will ameliorate or prevent radiation-induced brain injury, including cognitive impairment;2] using PPAR4 KO mice, we will determine if i] knocking down PPAR4 will increase radiation-induced brain injury, and ii] if administering a PPAR4 agonist will ameliorate or prevent radiation-induced brain injury through PPAR4- dependent mechanisms;3] using murine hippocampal neurons and microglial cells, we will determine if incubating these cells with PPAR4 agonists modulates radiation-induced changes in cellular phenotype via inhibition of pro-inflammatory signaling pathways and/or upregulation of anti-inflammatory mediators;and 4] using human glioma cell lines and immortalized normal glial cells, we will determine if treating with PPAR1 or PPAR4 agonists leads to selective glioma cell kill. Further, we will use an in vivo orthotopic rat model to determine if administering PPAR1 or PPAR4 agonists, alone or in combination with ionizing radiation, inhibits tumor growth and increases survival times. Successful completion of these aims will serve as the foundation for translating these findings into clinical trials designed to enhance the quality of life and long-term survival of cancer patients receiving partial or WBI. PUBLIC HEALTH RELEVANCE: Approximately 100,000 cancer patients per year survive long enough after partial or WBI to develop radiation- induced injury, including cognitive impairment. No successful long-term treatments for radiation-induced brain injury are currently available nor are there any effective preventive strategies. The establishment of an interventional role for PPAR1 and/or PPAR4 in radiation-induced brain injury should lead to the rapid translation of these preclinical findings to the clinic, thereby, increasing the therapeutic window for cancer patients receiving partial or WBI as well as impacting both on their quality of life and their long-term survival.

The terrorist attacks on the World Trade Center and the Pentagon on September 11, 2001, provided a stark reminder of the potential risk of large-scale involvement of the civilian population in a radiological terrorist event. During the era of the Cold War, the threat of nuclear exposure was limited by the need for sophisticated offensive systems, i.e., intercontinental missiles, to deliver the nuclear warhead. In contrast, the current risk for radiation exposure is a more potent threat. The rise of international terrorism has occurred at the same time, as has the ability to produce easily transportable radioactive dispersal devices, so-called "dirty bombs." At the very time when there is a critical, increased national (and international) need for highly trained biomedical personnel to respond effectively to a radiological terrorist attack or nuclear incident, the number of suitably trained and qualified individuals is deceasing markedly. Government support for education and training in many areas of the radiation sciences has declined over the last 3 decades. In radiation oncology, the number of physician-scientists has decreased by more than 50% in the last decade, and the majority of radiation biologists will be retiring within the coming decade. The population of scientists, physicians and emergency responders with the necessary knowledge and expertise in the biological and/or medical effects of radiation is shrinking and aging. As noted in a recent editorial in Radiation Research, "A crisis is visible on the horizon." Ensuring the successful creation and maintenance of medical countermeasures against radiation will necessitate the creation of specialized research training programs aimed at producing basic scientists, medical physicists and clinicians that possess a common base of knowledge and expertise required to respond to any radiological assault. Support for the reestablishment of comprehensive radiation training programs that produce MDs and PhDs with a common base of knowledge in radiation chemistry, radiation physics, and radiation biology and radiation medicine is paramount. Without such support, the US will be unable to respond adequately to a radiological terrorist event involving large numbers of the civilian population. Core F, the Education/Training component of the RadCCORE proposal, will provide both the "hands-on" training and the vital educational expertise in radiation biology and radiation/health physics using the outstanding expertise available at Wake Forest and Duke Universities. Through series of didactic lectures available on CD/DVD, laboratory training experiences, as well as Annual Seminar Series and Workshops, the faculty and trainees within the RadCCORE consortium will receive the basic foundation of knowledge in. radiation biology and physics to maintain and indeed grow the required expertise in radiation sciences.

DESCRIPTION (provided by applicant): There is a growing concern that the failure to train sufficient numbers of basic- and physician-scientists actively engaged in high quality, translational research, will result in the inability of the US to maintain the rate of progress in biomedical research achieved over the past decade. This is particularly true for radiation oncology, where the number of physician-scientists and the number of PhDs in radiation biology, radiation chemistry and radiation physics has decreased significantly over the last 10 or more years. Radiation oncology, biology and physics stand at the threshold of a new, molecular-based approach that promises to utilize the extraordinary advances in radiation delivery, molecular biology and molecular imaging to develop individualized radiation regimes that will significantly affect cancer patient long-term survival and quality of life. However, ensuring the successful development and translation of these advances necessitates the creation of a cadre of basic scientists and radiation oncologists who have a common knowledge base and are dedicated to the pursuit of translational radiation oncology research. To meet these needs, we propose the establishment of a postdoctoral Training Program in Translational Radiation Oncology (TRADONC) research in the Department of Radiation Oncology, Wake Forest University School of Medicine. The major goals of this program are to i] broaden the research infrastructure of translational radiation researchers by increasing the number of well-trained basic scientists and medical physicists exposed to the clinical practice of radiation oncology, and ii] develop a cadre of clinical radiation oncologists proficient in hypothesis-driven basic, translational and clinical research as well as being proficient in the design and implementation of clinical trials. We propose to enroll two trainees each year until 6 are in the program. These trainees will be radiation oncology fellows (MDs) who have completed their residency, postdoctoral basic scientists (PhDs) or postdoctoral medical physicists (PhDs);each trainee will receive support for 3 years. A common didactic curriculum combined with individually tailored formal courses will ensure a common scientific and clinical knowledge base. Moreover, all trainees will receive hands-on experience in both basic laboratory cancer research and clinical research under the guidance of a diverse, interdisciplinary faculty. Over time, this will increase the number of basic research scientists, medical physicists and radiation oncologists able to compete for extramural research funding, as well as increase thenumber and quality of multidisciplinary, translational radiation oncology studies that integrate the lab and clinic

[unreadable] DESCRIPTION (provided by applicant): The NCI has identified long-term survival from cancer as one of the new areas of public health emphasis; the late effects of cancer treatments are of particular importance. Progressive dementia occurs in some 20-50% of brain tumor patients who are long-term survivors after treatment with brain irradiation. The need to both understand and minimize the side effects of brain irradiation is exacerbated by the ever- increasing number of patients with brain metastases that require treatment with large field or whole brain irradiation (WBI); some 200,000 cancer patients/year receive large field or WBI. At the present time, there are no successful treatments for radiation-induced brain injury, nor are there any known effective preventive strategies. Data support a role for the renin-angiotensin system (RAS) in radiation-induced late effects in kidney, lung; both angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II (Ang II) receptor antagonists (ATRA) have proved effective. However, the pathogenic mechanism(s) involved remains unknown. Recent studies have identified a functioning RAS in the brain that is involved in cognition, memory, anxiety and stress. We hypothesize that WBI upregulated the intrinsic brain RAS, leading to a chronic and persistent oxidative stress/inflammatory response that results in the development and progression of radiation-induced brain injury, including cognitive impairment. To test this hypothesis we will pursue the following in vitro and in vivo Specific Aims: In Aims 1 and 2 we will test the hypothesis that inhibiting Ang II in normal brain cells will reduce the severity of pro-inflammatory changes in brain cell phenotype and/or function. We will use well-defined models of primary rat astrocytes, rat brain microvascular endothelial cells and rat microglia. In aims 3 and 4 we will test the hypothesis that inhibiting the intrinsic brain RAS using RAS blockers targeted at either ACE (ACEI), or the Ang II receptors (AT1RA and AT2RA) will ameliorate the development and progression of radiation-induced brain injury in vivo. Rats will receive a clinically relevant fractionated course of WBI, and acute (Specific Aim 3) and chronic (Specific Aim 4) changes in components of the RAS and pro-inflammatory mediators will be determined, as well as chronic changes in cognitive function. The establishment of an interventional role for Ang II blockers in modulating radiation-induced brain injury should lead to the rapid translation of these findings to the clinic, with the promise of increasing the therapeutic window for cancer patients receiving large field or WBI as well as improving their quality of life. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Every year as many as 200,000 patients receive whole brain irradiation (WBI). Most of these patients are treated not for primary brain tumors, but for brain metastases from melanomas, breast cancer or lung cancer that increase in middle age. As patient survival after WBI has increased, 20-50% of the long term survivors develop radiation-induced brain injury, including cognitive impairment. Radiation-induced cognitive impairment occurs in the absence of gross structural pathology, suggesting that this phenomenon is likely preceded by subtle changes in the myelin or the synaptic connections among neurons that underlie cognitive function. However, there have been, i] no quantitative studies of radiation-induced changes in myelin and synapses, ii] no published studies of radiation-induced cognitive impairment with a clinically relevant fractionated course of radiation, and iii] no attempts to ameliorate or prevent this radiation-induced structural or cognitive damage in middle-aged animals. The overall goal of the present grant is to answer the following questions. What changes in myelin and synapses occur when middle-aged rats are given a clinically relevant dose of fractionated WBI? Are the subtle changes in myelin and synapses early predictors of the ensuing radiation-induced cognitive impairment? Will modulation of the intrinsic brain renin-angiotensin system (RAS) ameliorate or prevent this radiation-induced brain injury, including cognitive impairment? Accordingly, the hypotheses to be tested are that, 1) modifications in myelin and synapses are early predictors of radiation-induced brain injury associated with cognitive impairment, and 2) both the structural modifications and the cognitive impairment induced by WBI can be ameliorated or prevented by modulating the intrinsic brain RAS. To test these hypotheses, we will, i] quantify the changes in myelin, synapses and cognition after treating 12 month old F344XBN rats with a clinically relevant course of WBI, and ii] determine if inhibition of the intrinsic brain RAS by administration of ACE inhibitors or Ang II type 1 receptor antagonists will modulate the structural and/or cognitive damage after 12 month old F344XBN rats are treated with fractionated WBI. These studies should provide information that is likely to lead to treatments that ameliorate radiation-induced brain injury, including cognitive impairment;thereby, addressing one of NCI's new areas of public health emphasis.