Lung cancer can be divided into two major forms: small-cell lung cancer and non-small cell lung cancer. Both non-small cell lung cancers and small cell lung cancers have developed in survivors of the atomic bombs in Japan. Similarly, both types of lung cancer arise in smokers. Cancers arising in never smokers preferentially develop in the distal airways and are of the adenocarcinoma histological subtype, which is a type of non-small cell lung cancer. Recently, genomic sequencing technology has been utilized to identify the most commonly mutated genes in adenocarcinomas. Based on this analysis, the two most commonly mutated genes in adenocarcinomas are Trp53, which encodes the tumor suppressor p53, and the oncogene Kras. The Duke NSCOR is utilizing sophisticated genetically engineered mouse models to study the role of p53 and Kras in non-small cell lung cancer. For example, we are studying how mutations in Kras in different kinds of cells in the lung affect lung cancer development with exposure to space radiation. We are also studying mice with an additional copy of p53 or inducible p53 suppression to investigate the timing and mechanism by which p53 suppresses Kras-driven lung adenocarcinoma progression after space radiation exposure. In addition, we are developing a mouse model of radiation-induced small-cell lung cancer. Together these studies will provide new insights into how lung cancer forms, where lung cancers develop, and how Kras and p53 mutation promote lung cancers. As we answer these questions using experiments with space radiation, we expect that our results will not only help us understand how lung cancer develops on Earth, but will also provide new insights into preventing and treating lung cancer.

In addition to studying lung cancer development, the Duke NSCOR is also studying lung progenitor cell injury and repair after exposure to either terrestrial or space radiation. Injury and inflammation of the lung are key components of many diseases in people including emphysema, asthma, and lung fibrosis. Furthermore, patients receiving radiotherapy for either primary lung cancer or other neoplasms of the thoracic region (e.g., breast cancer) undergo lung tissue remodeling and declining lung function that is directly related to the dose and location of radiation exposure. By exploring which lung cells are injured by space radiation and how these injured lung cells are repaired, we anticipate that this knowledge may also lead to a better understanding of how lung diseases besides cancer develop and strategies that may be employed to moderate the effects of radiotherapy on lung tissue remodeling. This information may ultimately be used to develop novel approaches for the prevention and treatment of these lung diseases, and the improvement of public health.

Project 1. The role of the tumor suppressor p53 in space radiation-induced lung cancer. David Kirsch, M.D., Ph.D., Lead

We proposed to study the role and timing of the tumor suppressor p53 in radiation-induced lung cancer using mice with an extra copy of p53 (Aim 1) and reversible knockdown of p53 (Aim 2). In addition, we proposed to develop a model of radiation-induced small cell lung cancer (Aim 3).

For Aim 1, we analyzed lung tumor development in the lung cancer prone KrasLA1 mice, bearing normal levels of p53 or an extra copy of p53. We observed that an extra copy of p53 suppressed lung tumor initiation in the absence of radiation, without affecting tumor grade or proliferation. Although radiation exposure did not impact lung tumor initiation in mice with wildtype expression of p53, our results suggest that space radiation may increase tumor grade. In contrast, mice with an extra copy of p53 had enhanced lung tumor burden following either terrestrial or space radiation. These results suggest that an extra copy of p53 can promote radiation induced lung tumorigenesis.

For Aim 2, we utilized an in vivo knockdown system that enabled temporal regulation of p53 expression in mice. We found that when p53 expression was permanently decreased following irradiation, mice developed soft-tissue sarcomas. Space radiation increased the sarcoma incidence as compared to terrestrial radiation. Furthermore, we showed that temporarily blocking p53 expression during radiation exposure was sufficient to ameliorate acute hematologic toxicity while simultaneously reducing lymphoma development. Our results suggest that the p53 response to radiation promotes radiation-induced lymphomagenesis and that inhibiting p53 could be a promising approach to prevent hematopoietic injury following exposure to large doses of radiation.

For Aim 3, we developed a mouse model of radiation-induced small cell lung cancer. Using this model, we found that space radiation was more effective than terrestrial radiation at accelerating lung and brain tumor development. Radiation dose and quality also impacted tumor incidence and histological subtype. Consistent with data in humans, our results suggest that female mice may have a higher excess relative risk of lung and brain tumor development following irradiation.

Project 2. The role of cell of origin in space radiation-induced lung cancer. Mark Onaitis, M.D., Lead

We proposed to investigate the cell of origin of K-RasG12D-induced lung cancer in response to space radiation. Our aims include studying the effects of radiation on mice in which K-RasG12D is inducibly expressed in different cell types of the lung: Club cells (formerly known as Clara cells) (Aim 1), basal cells (Aim 2), and Type II cells (Aim 3).

For Aim 1, we irradiated and analyzed tumor formation in CC10-CreER; lsl K-RasG12D mice. When mice were exposed to fractionated space radiation, they had more extensive lung tumor formation, but no change in tumor number or distribution as compared to unirradiated controls. Using microarray data from the tumors of mice described above, we also identified the potential targets EphA3, Zbtb16, and Pla2g7 that may be responsible for the differences in tumor initiation and progression that we observe. Analysis of tumors formed following terrestrial radiation exposure is ongoing.

For Aim 2, we generated K5-CreER; lsl K-RasG12D mice and treated them with tamoxifen. Unfortunately, these mice quickly developed tumors in the forestomach and lip causing respiratory occlusions and morbidity. Therefore, we have not been able to characterize the impact of space radiation in this model.

For Aim 3, we generated SPC-CreER; lsl K-RasG12D mice and treated them with tamoxifen. Unfortunately, this Cre-driver was leaky and many of the mice developed confluent tumors in the lung prior to radiation exposure. Therefore, we have not been able to characterize the impact of space radiation in this model.

K-RasG12D mutant mice, especially those in Aims 2 and 3, developed widespread tumors causing death of the mouse within 24 weeks post tamoxifen administration. As an alternative approach, we crossed the small cell lung cancer model (developed by the Kirsch lab) with the CC10-CreER (Aim 1); K5-CreER (Aim 2); and SPC-CreER (Aim 3) genes in order to assess the effects of radiation in a less penetrant model. The Rbfl/fl;p53fl/+ irradiated mice, as well as their sham controls, are currently being monitored for tumor formation.

Project 3. Effects of space radiation and p53 signaling on lung progenitor cells. Barry Stripp, Ph.D., Lead

The focus of this project was to compare effects of terrestrial and space radiation radiation on the clonogenic behavior and repair capacity of lung epithelial progenitor cells and to determine the impact of p53 deficiency on these responses. We have developed novel mouse models to functionally investigate progenitor cell behavior both in vivo and in vitro following radiation exposure.

For Aim 1 we used in vivo lineage tracing and novel in vitro models that recapitulate epithelial-stromal interactions seen in small airways, to determine how radiation exposure impacts clonal expansion of epithelial progenitor cells. Lineage tracing coupled with morphometry in tissue sections showed that exposure to radiation was associated with dose-dependent increases in clone sizes within airways. By utilizing a whole-mount imaging system to quantitate patch size across an entire lung lobe, we found that terrestrial gamma-ray exposure increased the frequency of medium, but not large patches, whereas exposure to HZE particles increased both the total size and frequency of medium and large patches. However, in vivo clonal expansion of epithelial progenitor cells was not associated with a significant change in the epithelial proliferative index.

Our ability to couple lineage tracing of epithelial progenitor cells with an in vitro assay in which epithelial cells are co-cultured with stromal support cells in a 3D matrix has provided a sensitive measure of moderate- to low-dose effects. We found that both terrestrial and space radiation exposures caused a dose-dependent decrease in freshly isolated airway epithelial cells immediately following radiation. When we isolated airway epithelial cells two months after space, but not terrestrial, radiation we saw that colony forming efficiency was still significantly decreased. Using qRT-PCR and immunofluorescent staining for markers of senescence, we found that HZE particle exposure leads to senescence of progenitors, which leaves fewer progenitor cells to maintain the lung, leading to larger clonal expansion.

For Aim 2, we used both in vitro and in vivo experiments to assess the role of p53 airway epithelial repair following radiation exposure. We used lineage tracing methods in mice deficient for p53 and assessed clonal expansion following space radiation exposure. We found that, while wild type mice had significantly increased clone sizes post-radiation, p53 deficient mice did not undergo clonal expansion, indicating that the senescence-induced patch expansion we observe following radiation exposure is p53-dependent. Additionally, we exposed p53 deficient cells to terrestrial radiation, plated them in our in vitro 3-D co-culture assay, and assessed colony forming ability as compared to wild type controls. p53 null cells had a higher colony forming ability at baseline as well as following radiation exposure. Lastly, through collaborative interaction with the Kirsch lab, we demonstrated that p53 gene dose dramatically impacts homeostatic behavior of epithelial progenitor cells, suggesting that inter-individual differences in regulation of the p53 pathway may impact epithelial behavior in the normal lung that influences responses to radiation exposure.

Core A: Administrative Core. David Kirsch, M.D., Ph.D., Lead

The Administrative Core (Core A) provided overall management of the NSCOR award by ensuring that projects made satisfactory progress. During the fifth year of funding, the Administrative Core monitored project progress by conducting Duke NSCOR meetings once a month and multiple teleconferences with NASA funded investigators. Minutes were recorded at these meetings in order to ensure that tasks were completed in a timely manner. In addition, the Administrative Core worked with Project leads and Core leads to consider the strengths of our NSCOR to develop ideas and concepts for the competing renewal application, which is now pending.

Core A program coordinator Ms. Cooley made travel arrangements for the Duke NSCOR team to travel to Brookhaven National Laboratory biannually in order to expose mice to 56Fe and 28Si ions. Travel arrangements were also made for the annual Radiation Research Society Meeting. Moreover, the Administrative Core arranged for the NASA Human Research Program (HRP) Investigators' Workshop.

Duke NSCOR administrators served as liaisons between the project groups to guide BNL (Brookhaven National Laboratory) and Duke training and credentialing of new investigators, ensure timely and accurate submission and renewal of IACUC protocols, NSCOR progress reports, as well as applications for NASA Space Radiation Laboratory (NSRL) Beam Time. Core A provided budget oversight for the Duke NSCOR. Lisa Hall monitored project expenditures. Mrs. Hall met monthly with Dr. Kirsch to review spending and fiscal matters for each NSCOR project and Core. Marcia Painter assisted with the financial accounting for the Duke NSCOR.

Core B: Physics Core. Terry Yoshizumi, Ph.D., Lead

The Physics Core (Core B) provided comprehensive measurements of radiation dose (dosimetry) and oversaw the radiation safety of experiments performed by investigators in the Duke NSCOR for experiments with X-rays. By performing routine dosimetry measurements on the standard small animal X-Ray irradiator, the Physics Core provided quality control for radiation exposure experiments. Members of the physics core participated and presented physics reports at regularly-scheduled NSCOR meetings. The Core ensured the timely incorporation of new dosimetry technology to provide state-of-the-art dosimetry support. The Physics Core successfully collaborated with the X-ray Irradiator manufacturer addressing kV x-ray dosimetry.

Core C: Education Core. Rochelle Schwartz-Bloom, Ph.D., Lead

The Education Core (Core C) developed a problem-based unit (Raising Interest in Science Education: Research & Development) to teach high school students about radiation in space by incorporating principles of physics, chemistry, and biology. The unit contains a hypothetical scenario in which a group of young astronauts are selected to travel to Mars in the year of 2040. The astronauts must learn about the types of radiation they will encounter in space (compared to on Earth), the damage these high energy particles and cosmic rays can cause to their DNA molecules, how their bodies can deal with the damage using a protein called p53, and what would happen if their p53 gene has a mutation. They also learn how mutations in p53 genes can increase the risk of cancer, especially of the lung. The astronauts will meet some “virtual” scientists (the Principal Investigators of projects 1-3) who study these topics and whose research findings are crucial to the development of a successful space program that includes a trip to Mars. This past year after the final revisions of the unit (and after its beta-testing by high school students), the website was developed for dissemination. The URL is http://rise.duke.edu/radiation [Ed. note 3/11/21: URL no longer connects; suggest contacting PI for further information].

Lung cancer can be divided into two major forms: small-cell lung cancer and non-small cell lung cancer. Both non-small cell lung cancers and small cell lung cancers have developed in survivors of the atomic bombs in Japan. Similarly, both types of lung cancer arise in smokers. Cancers arising in never smokers preferentially develop in the distal airways and are of the adenocarcinoma histological subtype, which is a type of non-small cell lung cancer. Recently, genomic sequencing technology has been utilized to identify the most commonly mutated genes in adenocarcinomas. Based on this analysis, the two most commonly mutated genes in adenocarcinomas are Trp53, which encodes the tumor suppressor p53, and the oncogene Kras. The Duke NSCOR is utilizing sophisticated genetically engineered mouse models to study the role of p53 and Kras in non-small cell lung cancer. For example, we are studying how mutations in Kras in different kinds of cells in the lung affect lung cancer development with exposure to space radiation. We are also studying mice with an additional copy of p53 or inducible p53 suppression to investigate the timing and mechanism by which p53 suppresses Kras-driven lung adenocarcinoma progression after space radiation exposure. In addition, we are developing a mouse model of radiation-induced small-cell lung cancer. Together these studies will provide new insights into how lung cancer forms, where lung cancers develop, and how Kras and p53 mutation promote lung cancers. As we answer these questions using experiments with space radiation, we expect that our results will not only help us understand how lung cancer develops on Earth, but will also provide new insights into preventing and treating lung cancer.

In addition to studying lung cancer development, the Duke NSCOR is also studying lung progenitor cell injury and repair after exposure to either terrestrial or space radiation. Injury and inflammation of the lung are key components of many diseases in people including emphysema, asthma, and lung fibrosis. Furthermore, patients receiving radiotherapy for either primary lung cancer or other neoplasms of the thoracic region (e.g. breast cancer) undergo lung tissue remodeling and declining lung function that is directly related to the dose and location of radiation exposure. By exploring which lung cells are injured by space radiation and how these injured lung cells are repaired, we anticipate that this knowledge may also lead to a better understanding of how lung diseases besides cancer develop and strategies that may be employed to moderate the effects of radiotherapy on lung tissue remodeling. This information may ultimately be used to develop novel approaches for the prevention and treatment of these lung diseases, and the improvement of public health.

Project 1. The role of the tumor suppressor p53 in space radiation-induced lung cancer. David Kirsch, M.D., Ph.D., Lead

We proposed to study the role and timing of the tumor suppressor p53 in radiation-induced lung cancer using mice with an extra copy of p53 (Aim 1) and reversible knockdown of p53 (Aim 2). In addition, we proposed to develop a model of radiation-induced small cell lung cancer (Aim 3).

For Aim 1, we completed irradiation and analysis of lung tumor development in KrasLA1 mice predisposed to lung cancer bearing normal levels of p53 or an extra copy of p53. We observed that p53 suppresses lung tumor initiation in the absence of radiation, but that an extra copy of p53 does not affect the proliferation of low grade lung tumors or the expression of pERK, which is a negative prognostic marker. Exposure to neither terrestrial radiation nor space radiation impacted lung tumor initiation in mice with wildtype expression of p53. However, our results suggest that space radiation may increase the grade of lung tumors.

For Aim 2, we utilized an in vivo knockdown system that enables temporal regulation of p53 expression in the lungs of mice. We have begun to use this system in combination with the model of radiation-induced lung cancer that we developed in Aim 3 to decrease p53 expression temporarily during radiation exposure or permanently during and following radiation exposure to investigate the timing of p53-mediated tumor suppression. Preliminary results suggest that permanent knockdown of p53 reduces the survival of mice following radiation.

For Aim 3, we find that fractionated exposure to terrestrial and space radiation accelerates lung tumor formation in a genetically engineered mouse model of small cell lung cancer and adenocarcinoma. This model will be valuable for determining the relative biological effectiveness with which terrestrial and space radiation cause lung cancer to develop. In the coming year, we plan to expose these mice to varying doses of terrestrial and space radiation to determine whether space radiation is more effective at causing lung cancer.

Project 2. The role of cell of origin in space radiation-induced lung cancer. Mark Onaitis, M.D., Lead

We proposed to study the cell of origin of K-RasG12D-induced lung cancer in response to space radiation. Our aims include studying the effects of 600 MeV/n 56Fe (HZE radiation) on mice in which K-RasG12D is inducibly expressed in different cell types of the lung: Club cells (formerly known as Clara cells) (Aim 1), basal cells (Aim 2), and Type II cells (Aim 3).

For Aim 1, we have irradiated CC10-CreER; lsl K-RasG12D mice with 5 fractions of 0.2 Gy 600MeV/n 56Fe and have CC10-CreER; lsl K-RasG12D controls that were sham irradiated and the lungs analyzed 8 weeks post irradiation. Our data suggest that HZE radiation increases tumor burden, but does not affect the distribution or numbers of tumors. We are currently irradiating more mice with a single fraction of 0.1 Gy 600 MeV/n 56Fe for a low dose comparison. Additionally, another cohort will be irradiated with a single fraction of 0.1Gy 300MeV/n 28Si to compare another ion species. These tumors will all be compared for relative biological effectiveness to terrestrial radiation-induced tumors using mice exposed to 320kVp X-rays at Duke University.

For Aim 2, we generated K5-CreER; lsl K-RasG12D mice and treated them with tamoxifen. Unfortunately, these mice quickly developed tumors in the forestomach and lip causing respiratory occlusions and morbidity. Therefore, we have not been able to characterize the impact of HZE radiation in this model.

For Aim 3, we generated SPC-CreER; lsl K-RasG12D mice and treated them with tamoxifen. Unfortunately, we found that this model was leaky in that many of the mice developed confluent tumors in the lung even before radiation exposure. Therefore, we have not been able to characterize the impact of HZE radiation in this model.

Because the K-RasG12D mutant mice develop widespread tumors causing death of the mouse within 24 weeks after tamoxifen administration, as an alternative approach, we have begun irradiating an Rbflox/flox; p53flox/+ model with the CC10-CreER (Aim 1); K5-CreER (Aim 2); and SPC-CreER (Aim 3) genes in order to assess the effects of radiation in a less penetrant model.

Project 3. Effects of space radiation and p53 signaling on lung progenitor cells. Barry Stripp, Ph.D., Lead

The focus of this project is to compare direct and non-targeted effects of X-rays and HZE radiation on the clonogenic and repair capacity of lung epithelial progenitor cells, and to determine the impact of p53 deficiency on these responses. We have shown that region-specific progenitor cells maintain the specialized epithelium of mouse and human airways and have developed novel mouse models to functionally investigate their behavior in vivo and in vitro. An important feature of our in vitro model used to assess the clonogenic behavior of epithelial progenitor cells is the use of a three-dimensional culture environment in which epithelial cells are co-cultured with stromal support cells.

For Aim 1 we have used in vivo lineage tracing and novel in vitro models that recapitulate epithelial-stromal interactions seen in small airways, to determine how either 320 kVp X-ray or 600 MeV/n 56Fe particles (HZE) impact clonal expansion of epithelial progenitor cells. Lineage tracing coupled with morphometry was used to establish that whole body exposures to either X-ray or HZE were associated with dose-dependent increases in the probability that epithelial progenitor cells expanded to yield large clone sizes within airways. However, in vivo clonal expansion of epithelial progenitor cells was not associated with a significant change in the epithelial proliferative index. Ongoing experiments are using double labeling methods to define the effects of radiation dose and type on the pool size of epithelial progenitor cells in vivo. Moreover, we have initiated pilot experiments to determine how lung injury from either ozone or influenza virus impacts the rate of progenitor cell expansion following radiation exposure. We predict that the range of radiation effects on epithelial progenitor cells from different radiation doses and qualities of radiation will be amplified by environmental triggers that cause epithelial cell injury.

In vitro experiments performed over the past funding period have revealed direct effects of either X-ray or HZE exposure on lung progenitor cells following whole-body exposures. Our ability to couple lineage tracing of epithelial progenitor cells with in vitro clonal behavior has provided a sensitive measure of moderate to low-dose effects. We found that both X-ray and HZE exposures caused a dose-dependent decrease on freshly isolated airway epithelial cells immediately following radiation. Interestingly, when we isolate airway epithelial cells two months after HZE exposure, we see that colony forming efficiency is still significantly decreased. We are currently investigating if the sustained decrease in colony forming efficiency occurs following low-LET radiation exposure as well.

For Aim 2, we used both in vitro and in vivo experiments to assess the role of p53 airway epithelial repair following radiation exposure. We used lineage tracing methods in mice deficient for p53 and assessed clonal expansion following HZE exposure. We found that, while wild type mice had significantly increased clone sizes post-radiation, the p53 deficient mice did not undergo clonal expansion. We are working to uncover the mechanism by which clonal expansion is triggered following radiation exposure and how p53 modulates this response. Additionally, we exposed p53 deficient cells to low-LET radiation, plated them in our in vitro 3-D co-culture assay, and assessed colony forming ability as compared to wild type controls. p53 null cells had a higher colony forming ability at baseline as well as following radiation exposure. We are currently performing live imaging of these cells to determine the mechanism by which colony forming ability is increased when p53 is lost.

Core A: Administrative Core. David Kirsch, M.D., Ph.D., Lead ; Duke NSCOR Administrators: Michelle Cooley, Lisa Hall, Marcia Painter The Administrative Core (Core A) provides overall management of the NSCOR award by ensuring that projects make satisfactory progress. During the fourth year of funding, the Administrative Core has monitored project progress by conducting Duke NSCOR meetings once a month and multiple teleconferences with NASA funded investigators. Minutes were recorded at these meetings in order to ensure that tasks were completed in a timely manner. In addition, we have scheduled our annual Internal Advisory Committee Meeting for December 16th, 2014, which will include two outside experts in lung biology and space radiation. During this meeting, our Project leads will present their current work and the Advisory Committee will provide feedback. Ideas for our NSCOR renewal will also be discussed at this meeting.

Core A made travel arrangements for the Duke NSCOR team to travel to Brookhaven National Laboratory in Spring and Fall 2014 in order to expose mice to 56Fe and 28Si ions. Travel arrangements were also made for the annual Radiation Research Society Meeting in September. Moreover, the Administrative Core arranged for the two day NASA HRP Investigators' Workshop in Galveston, Texas in February.

Duke NSCOR administrators served as liaisons between the project groups to guide BNL and Duke training and credentialing of new investigators, ensure timely and accurate submission and renewal of IACUC protocols, NSCOR progress reports, as well as application for NSRL Beam Time Request for 2015. Core A provided budget oversight for the Duke NSCOR. Lisa Hall monitored project expenditures. Mrs. Hall met monthly with Dr. Kirsch to review spending and fiscal matters for each NSCOR project and Core. Marcia Painter assisted with the financial accounting for the Duke NSCOR.

Core B: Physics Core. Terry Yoshizumi, Ph.D., Lead

The Physics Core (Core B) provides comprehensive measurements of radiation dose (dosimetry) and oversees the radiation safety of experiments performed by investigators in the Duke NSCOR for experiments with X-rays. By performing routine dosimetry measurements on the standard small animal X-Ray irradiator, the Physics Core provided quality control for radiation exposure experiments. Members of the physics core participate and present physics reports at regularly-scheduled NSCOR meetings. The Core ensures the timely incorporation of new dosimetry technology to provide state-of-the-art dosimetry support. The Physics Core has established collaboration arrangements with X-ray Irradiator manufacturers in developing industry standards for quality assurance and performance improvement.

Core C: Education Core. Rochelle Schwartz-Bloom, Ph.D., Lead

The Education Core (Core C) has developed a problem-based unit to teach high school students about radiation in space by incorporating principles of physics, chemistry, and biology. The unit contains a hypothetical scenario in which a group of young astronauts are selected to travel to Mars in the year of 2040. The astronauts must learn about the types of radiation they will encounter in space (compared to on Earth), the damage these high energy particles and cosmic rays can cause to their DNA molecules, how their bodies can deal with the damage using a protein called p53, and what would happen if their p53 gene has a mutation. They also learn how mutations in p53 genes can increase the risk of cancer, especially of the lung. The astronauts will meet some “virtual” scientists (the PIs of projects 1-3) who study these topics and whose research findings are crucial to the development of a successful space program that includes a trip to Mars. This past year after completion of the unit, it was field-tested in a local high school AP biology class for impact on content knowledge. Students demonstrated significantly increased knowledge of biology and physics principles after working with the unit.

Lung cancer can be divided into two major forms: small-cell lung cancer and non-small cell lung cancer. Both non-small cell lung cancers and small cell lung cancers have developed in survivors of the atomic bombs in Japan. Similarly, both types of lung cancer arise in smokers. Cancers arising in never smokers preferentially develop in the distal airways and are of the adenocarcinoma histological subtype, which is a type of non-small cell lung cancer. Recently, genomic sequencing technology has been utilized to identify the most commonly mutated genes in adenocarcinomas. Based on this analysis, the two most commonly mutated genes in adenocarcinomas are Trp53, which encodes the tumor suppressor p53, and the oncogene Kras. The Duke NSCOR is utilizing sophisticated genetically engineered mouse models to study the role of p53 and Kras in non-small cell lung cancer. For example, we are studying how mutations in Kras in different kinds of cells in the lung affect lung cancer development with exposure to space radiation. We are also studying mice with an additional copy of p53 or inducible p53 suppression to investigate the timing and mechanism by which p53 suppresses Kras-driven lung adenocarcinoma progression after space radiation exposure. In addition, we are developing a mouse model of radiation-induced small-cell lung cancer. Together these studies will provide new insights into how lung cancer forms, where lung cancers develop, and how Kras and p53 mutation promote lung cancers. As we answer these questions using experiments with space radiation, we expect that our results will not only help us understand how lung cancer develops on earth, but will also provide new insights into preventing and treating lung cancer.

In addition to studying lung cancer development, the Duke NSCOR is also studying lung progenitor cell injury and repair after exposure to either terrestrial or space radiation. Injury and inflammation of the lung are key components of many diseases in people including emphysema, asthma, and lung fibrosis. Furthermore, patients receiving radiotherapy for either primary lung cancer or other neoplasms of the thoracic region (e.g. breast cancer) undergo lung tissue remodeling and declining lung function that is directly related to the dose and location of radiation exposure. By exploring which lung cells are injured by space radiation and how these injured lung cells are repaired, we anticipate that this knowledge may also lead to a better understanding of how lung diseases besides cancer develop and strategies that may be employed to moderate the effects of radiotherapy on lung tissue remodeling. This information may ultimately be used to develop novel approaches for the prevention and treatment of these lung diseases, and the improvement of public health.

Project 1. The role of the tumor suppressor p53 in space radiation-induced lung cancer. David Kirsch, M.D., Ph.D., Lead

We proposed to study the role and timing of the tumor suppressor p53 in radiation-induced lung cancer using mice with an extra copy of p53 (Aim 1) and reversible knockdown of p53 (Aim 2). In addition, we proposed to develop a model of radiation-induced small cell lung cancer (Aim 3).

For Aim 1, we completed irradiation and analysis of lung tumor development in KrasLA1 mice predisposed to lung cancer bearing normal levels of p53 or an extra copy of p53. We observed that p53 suppresses lung tumor initiation in the absence of radiation, but that an extra copy of p53 does not affect the proliferation of low grade lung tumors or the expression of pERK, which is a negative prognostic marker. Exposure to neither terrestrial radiation nor space radiation impacted lung tumor initiation in our model. However, our results suggest that space radiation may increase the grade of lung tumors.

For Aim 2, we have validated an in vivo knockdown system that enables temporal regulation of p53 expression in the lungs of mice. We plan to use this system in combination with the model of radiation-induced lung cancer that we have developed in Aim 3 to decrease p53 expression temporarily during radiation exposure or permanently during and following radiation exposure to investigate the timing of p53-mediated tumor suppression.

For Aim 3, we confirmed that exposure to terrestrial and space radiation accelerates lung tumor formation in a genetically engineered mouse model of small cell lung cancer and adenocarcinoma. This model will be valuable for determining the relative biological effectiveness with which terrestrial and space radiation cause lung cancer to develop. In the future, we plan to expose these mice to varying doses of terrestrial and space radiation to determine whether space radiation is more effective at causing lung cancer.

Project 2. The role of cell of origin in space radiation-induced lung cancer. Mark Onaitis, M.D., Lead

We proposed to study the cell of origin of K-RasG12D-induced lung cancer in response to space radiation. Our aims include studying the effects of 600 MeV/n 56Fe (HZE radiation) on mice in which K-RasG12D is inducibly expressed in different cell types of the lung: Clara cells (Aim 1), basal cells (Aim 2), and Type II cells (Aim 3).

For Aim 1, we have now irradiated 29 CC10-CreER; lsl K-RasG12D mice and have 29 CC10-CreER; lsl K-RasG12D controls that were sham irradiated and the lungs analyzed 8 weeks post irradiation. Preliminary results suggest that HZE radiation may increase tumor burden, but the results are not yet statistically significant (p=0.089). We will continue to irradiate more cohorts of these mice in the next year to increase our power. Additionally, for comparison of 600 MeV/n 56Fe to terrestrial forms of radiation, we have so far exposed 13 CC10-CreER; lsl K-RasG12D mice to 320 kVp X-rays at Duke University. Analysis of tumors formed from X-ray exposed mice is ongoing.

For Aim 2, we generated K5-CreER; lsl K-RasG12D mice mice and treated them with tamoxifen. Unfortunately, these mice quickly developed tumors in the forestomach and lip. Therefore, we have not been able to characterize the impact of HZE radiation in this model.

For Aim 3, we have now radiated 14 SPC-CreER; lsl K-RasG12D mice and have 6 SPC-CreER; lsl K-RasG12D mice that were sham irradiated as controls. The mice have been sacrificed and the lungs fixed. Unfortunately, we found that this model was leaky in that many of the mice developed confluent tumors in the lung even before radiation exposure. Therefore, we have not been able to characterize the impact of HZE radiation in this model.

Because the K-RasG12D mutant mice develop widespread tumors causing death of the mouse within 24 weeks after tamoxifen administration, as an alternative approach, we have begun irradiating CC10-CreER; NF1flox/flox; Ink4A/Arf flox/flox mice in order to assess the effects of radiation in a less penetrant model.

Project 3. Effects of space radiation and p53 signaling on lung progenitor cells. Barry Stripp, Ph.D., Lead

The focus of this project is to compare direct and non-target effects of X-rays and HZE radiation on the clonogenic and repair capacity of lung epithelial progenitor cells, and to determine the impact of p53 deficiency on these responses. We have shown that region-specific progenitor cells maintain the specialized epithelium of mouse and human airways and have developed novel mouse models to functionally investigate their behavior in vivo and in vitro. An important feature of our in vitro model used to assess the clonogenic behavior of epithelial progenitor cells is the use of a three-dimensional culture environment in which epithelial cells are co-cultured with stromal support cells to restore critical elements of the in vivo microenvironment.

For Aim 1 we have used in vivo lineage tracing and novel in vitro models that recapitulate epithelial-stromal interactions seen in small airways, to determine how either 320 kVp X-ray or 600 MeV/n 56Fe particles (HZE) impact clonal expansion of epithelial progenitor cells. Lineage tracing coupled with morphometry was used to establish that whole body exposures to either X-ray or HZE were associated with dose-dependent increases in the probability that epithelial progenitor cells expanded to yield large clone sizes within airways. However, in vivo clonal expansion of epithelial progenitor cells was not associated with a significant change in the epithelial proliferative index. Ongoing experiments are using double labeling methods to define the effects of radiation dose and type on the pool size of epithelial progenitor cells in vivo, and to determine how lung injury resulting from either ozone or influenza virus impacts the rate of progenitor cell expansion following IR exposure. We are currently analyzing the results from the Spring 2013 BNL run, where we exposed mice to HZE and then exposed them to ozone upon return to Duke. These studies are important as we show that the effects of IR exposure are latent within the epithelium of airways. We predict that the effects of radiation exposure and differences between dose and quality of radiation, on epithelial progenitor cells will be amplified by environmental triggers that cause epithelial cell injury.

In vitro experiments performed over the past funding period have revealed direct effects of either X-ray or HZE exposure on lung progenitor cells following whole-body exposures. Our ability to couple lineage tracing of epithelial progenitor cells with in vitro clonal behavior has provided a sensitive measure of moderate to low-dose effects. In vitro exposure of either isolated epithelial progenitor cells or stromal cells used in 3D co-cultures has provided preliminary insights into direct versus non-target effects of radiation exposure on the clonogenic behavior of epithelial progenitor cells. In collaborative studies with Dr. Jerry Shay and the UTSW NSCOR, we are coupling in vitro exposure models with drug screens to identify radio-protective molecules and pathways impacting progenitor cell responses to either X-ray or HZE radiation.

For Aim 2, we have established lines of mice allowing application of either in vivo or in vitro assays to assess behavioral changes of epithelial progenitor cells to IR exposure that accompany loss of p53 function. Initial experiments are focusing on X-ray exposures with HZE exposures at NSRL/BNL planned for fall 2013 and spring 2014. We will be looking at in vivo clonal expansion following HZE in fall 2013 and are currently breeding mice to study in vitro colony forming efficiency for spring 2014.

Core A: Administrative Core. David Kirsch, M.D., Ph.D., Lead. Duke NSCOR Administrators: Michelle Cooley, Erin Dillard (Jan-August), Lisa Hall (September to present), Marcia Painter

The Administrative Core (Core A) provides overall management of the NSCOR award by ensuring that projects make satisfactory progress. During the third year of funding, the Administrative Core has monitored project progress by conducting Duke NSCOR meetings once to twice a month, an annual Internal Advisory Committee Meeting, and multiple teleconferences with NASA. Minutes were recorded at these meetings in order to ensure that tasks were completed in a timely manner. Core A made travel arrangements for the Duke NSCOR team to travel to Brookhaven National Laboratory in Spring and Fall 2013 in order to expose mice to 56Fe ions. Travel arrangements were also made for the annual Radiation Research Society Meeting. Moreover, the Administrative Core arranged travel to the day-long meeting in Arlington, VA for the NSCOR Mid-Term Review.

Duke NSCOR administrators served as liaisons between the project groups to guide BNL and Duke training and credentialing of new investigators, ensure timely and accurate submission and renewal of IACUC protocols, NSCOR progress reports as well application for the NSRL Beam Time Request for 2014. Core A provided budget oversight for the Duke NSCOR. Erin Dillard monitored project expenditures. Ms. Dillard met monthly with Dr. Kirsch to review spending and fiscal matters for each NSCOR project and Core. Marcia Painter assisted with the financial accounting for the Duke NSCOR.

Core B: Physics Core. Terry Yoshizumi, Ph.D., Lead

The Physics Core (Core B) provides comprehensive measurements of radiation dose (dosimetry) and oversees the radiation safety of experiments performed by investigators in the Duke NSCOR for experiments with X-rays. By performing routine dosimetry measurements on the standard small animal X-Ray irradiator, the Physics Core provided quality control for radiation exposure experiments. Members of the physics core participate and present physics reports at regularly-scheduled NSCOR meetings. The Core ensures the timely incorporation of new dosimetry technology to provide state-of-the-art dosimetry support.

Forthcoming publications:

1. Stanton IN, Belley MD, Nguyen G, Rodrigues A, Li Y, Kirsch DG, Yoshizumi TT, and Therien MJ. Europium-Doped Yttrium Oxide Nano-Scintillators That Display a Linear Emission Intensity to X-Ray Radiation Flux; Integration into a Fiber-Optic Dosimeter Prototype. Analytical Chemistry 2013 (under review).

2. Belley MD, Wang C, Nguyen G, Gunasingha R, Chao NJ, Chen BJ, Dewhirst MW, Yoshizumi TT. Towards an Organ Based Dose Prescription Method for the Improved Accuracy of Murine Dose in Orthovoltage X-ray Irradiators. Medical Physics 2013 (under review).

Core C: Education Core. Rochelle Schwartz-Bloom, Ph.D., Lead

The Education Core (Core C) is developing an online problem-based unit to teach high school students about radiation in space by incorporating principles of physics, chemistry, and biology. The unit contains a hypothetical scenario in which a group of young astronauts are selected to travel to Mars in the year of 2040. The astronauts must learn about the types of radiation they will encounter in space (compared to on earth), the damage these high energy particles and cosmic rays can cause to their DNA molecules, how their bodies can deal with the damage using a protein called p53, and what would happen if their p53 gene has a mutation. They also learn how mutations in p53 genes can increase the risk of cancer, especially of the lung. The astronauts will meet some “virtual” scientists (the PIs of projects 1-3) who study these topics and whose research findings are crucial to the development of a successful space program that includes a trip to Mars. The educational unit will be field-tested in local high schools for impact on content knowledge and interests in science.

Lung cancer can be divided into two major forms: small-cell lung cancer and non-small cell lung cancer. Both non-small cell lung cancers and small cell lung cancers have developed in survivors of the atomic bombs in Japan. Similarly, both types of lung cancer arise in smokers. Cancers arising in never smokers preferentially develop in the distal airways and are of the adenocarcinoma histological subtype, which is a type of non-small cell lung cancer. Recently, genomic sequencing technology has been utilized to identify the most commonly mutated genes in adenocarcinomas. Based on this analysis, the two most commonly mutated genes in adenocarcinomas are Trp53, which encodes the tumor suppressor p53, and the oncogene Kras. The Duke NSCOR is utilizing sophisticated genetically engineered mouse models to study the role of p53 and Kras in non-small cell lung cancer. For example, we are studying how mutations in Kras in different kinds of cells in the lung affect lung cancer development with exposure to space radiation. We are also studying mice with an additional copy of p53 or inducible p53 suppression to investigate the timing and mechanism by which p53 suppresses Kras-driven lung adenocarcinoma progression after space radiation exposure. In addition, we are developing a mouse model of radiation-induced small-cell lung cancer. Together these studies will provide new insights into how lung cancer forms, where lung cancers develop, and how Kras and p53 mutation promote lung cancers. As we answer these questions using experiments with space radiation, we expect that our results will not only help us understand how lung cancer develops on earth, but will also provide new insights into preventing and treating lung cancer.

In addition to studying lung cancer development, the Duke NSCOR is also studying lung progenitor cell injury and repair after exposure to either terrestrial or space radiation. Injury and inflammation of the lung are key components of many diseases in people including emphysema, asthma, and lung fibrosis. Furthermore, patients receiving radiotherapy for either primary lung cancer or other neoplasms of the thoracic region (e.g. breast cancer) undergo lung tissue remodeling and declining lung function that is directly related to the dose and location of radiation exposure. By exploring which lung cells are injured by space radiation and how these injured lung cells are repaired, we anticipate that this knowledge may also lead to a better understanding of how lung diseases besides cancer develop and strategies that may be employed to moderate the effects of radiotherapy on lung tissue remodeling. This information may ultimately be used to develop novel approaches for the prevention and treatment of these lung diseases, and the improvement of public health.

Project 1. The role of the tumor suppressor p53 in space radiation-induced lung cancer. David Kirsch, M.D., Ph.D., Lead

We have proposed to study the role and timing of the tumor suppressor p53 in radiation-induced lung cancer using mice with an extra copy of p53 (Aim 1) and reversible knockdown of p53 (Aim 2). In addition, we are developing a model of radiation-induced small cell lung cancer (Aim 3).

For Aim 1, we have confirmed an increased induction of p53 transcriptional targets in mice with an additional copy of p53 (super p53 mice) after both X-ray and 600 MeV/n 56Fe (HZE) irradiation. We have bred mice with 2 or 3 copies of p53 with mice that are predisposed to lung cancer and have exposed 77 of these mice to space radiation at BNL and 23 mice to X-ray irradiation at Duke. We have analyzed the majority of these mice for lung tumor burden six months following radiation. Our preliminary results suggest that space radiation exposure increases the occurrence of lung cancer in these mice. In addition, an extra copy of p53 appears to protect mice from lung tumor initiation. In the coming year, we plan to increase the size of our mouse cohorts to verify these initial findings.

For Aim 2, we have bred mice predisposed to lung cancer in which p53 expression can be knocked down using a doxycycline-inducible RNA interference system. Using these mice, we will temporarily knockdown p53 during space radiation exposure to investigate the temporal role of p53 in HZE radiation-induced lung cancer. We will irradiate our first cohort of these mice in November 2012.

For Aim 3, we have irradiated 42 mice with fractionated space radiation at BNL and are following them for the development of small cell lung cancer. We have already observed several mice with lung tumors after radiation. In the coming year, we plan to irradiate mice with varying susceptibility to small cell lung cancer with both single dose and fractionated HZE and low-LET (X-ray) radiation to evaluate the effect of space radiation exposure on lung tumor initiation.

Project 2. The role of cell of origin in space radiation-induced lung cancer. Mark Onaitis, M.D., Lead

We have proposed to study the cell of origin of K-RasG12D-induced lung cancer in response to space radiation. Our aims include studying the effects of HZE radiation on mice in which K-RasG12D is inducibly expressed in different cell types of the lung: Clara cells (Aim 1), basal cells (Aim 2), and Type II cells (Aim 3).

For Aim 1, we have now radiated 27 CC10-CreER; lsl K-RasG12D mice and have 28 CC10-CreER; lsl K-RasG12D controls that were sham irradiated. Analysis of tumor formation in these mice is ongoing. We will continue to irradiate more cohorts of these mice in the next year.

For Aim 2, we have had difficulty breeding mice. Recently, we identified successful breeding pairs. Therefore, we are now expanding this colony and will begin irradiating these mice this year.

For Aim 3, we have now radiated 14 SPC-CreER; lsl K-RasG12D mice and have 6 SPC-CreER; lsl K-RasG12D mice that were sham irradiated as controls. The mice have been sacrificed and the lungs fixed. We are currently analyzing sections of the lungs to assess for phenotypic differences. Many more of these mice will be irradiated in the next year. Because the K-RasG12D mutant mice develop widespread tumors causing death of the mouse within 24 weeks after tamoxifen administration, as an alternative approach, we have begun irradiating CC10-CreER; floxed NF1 or floxed Ink4A/Arf mice in order to assess the effects of radiation in a less penetrant model.

Project 3. Effects of space radiation and p53 signaling on lung progenitor cells. Barry Stripp, Ph.D., Lead

The focus of this project is to compare direct and non-target effects of X-rays and HZE radiation on the clonogenic and repair capacity of lung epithelial progenitor cells, and to determine the impact of p53 deficiency on these responses. We have shown that region-specific progenitor cells maintain the specialized epithelium of mouse and human airways and have developed novel mouse models to functionally investigate their behavior in vivo and in vitro. An important feature of our in vitro model used to assess the clonogenic behavior of epithelial progenitor cells is the use of a three-dimensional culture environment in which epithelial cells are co-cultured with stromal support cells to restore critical elements of the in vivo microenvironment.

For Aim 1 we have used in vivo lineage tracing and novel in vitro models that recapitulate epithelial-stromal interactions seen in small airways, to determine how either 320 kVp X-ray or 600 MeV/n 56Fe particles (HZE) impact clonal expansion of epithelial progenitor cells. Lineage tracing coupled with morphometry was used to establish that whole body exposures to either X-ray or HZE were associated with dose-dependent increases in the probability that epithelial progenitor cells expanded to yield large clone sizes within airways. However, in vivo clonal expansion of epithelial progenitor cells was not associated with a significant change in the epithelial proliferative index. Ongoing experiments are using double labeling methods to define the effects of radiation dose and type on the pool size of epithelial progenitor cells in vivo, and to determine how lung injury resulting from either ozone or influenza virus impacts the rate of progenitor cell expansion following IR exposure. These studies are important as we show that the effects of IR exposure are latent within the epithelium of airways. We predict that the effects of radiation exposure and differences between dose and type of IR, on epithelial progenitor cells will be amplified by environmental triggers that cause epithelial cell injury. In vitro experiments performed over the past funding period have revealed direct effects of either X-ray or HZE exposure on lung progenitor cells following whole-body exposures. Our ability to couple lineage tracing of epithelial progenitor cells with in vitro clonal behavior has provided a sensitive measure of moderate to low-dose effects. In vitro exposure of either isolated epithelial progenitor cells or stromal cells used in 3D co-cultures has provided preliminary insights into direct versus non-target effects of radiation exposure on the clonogenic behavior of epithelial progenitor cells. In collaborative studies with Dr. Jerry Shay and the UTSW NSCOR we are coupling in vitro exposure models with drug screens to identify radio-protective molecules and pathways impacting progenitor cell responses to either X-ray or HZE radiation.

For Aim 2, we have established lines of mice allowing application of either in vivo or in vitro assays to assess behavioral changes of epithelial progenitor cells to IR exposure that accompany loss of p53 function. Initial experiments are focusing on X-ray exposures with HZE exposures at NSRL/BNL planned for 2013.

Core A: Administrative Core. David Kirsch, M.D., Ph.D., Lead. Duke NSCOR Administrators: Michelle Cooley and Sue Yager

The Administrative Core (Core A) provides overall management of the NSCOR award by ensuring that projects make satisfactory progress. During the second year of funding, the Administrative Core has monitored project progress by conducting biweekly Duke NSCOR meetings, an annual Internal Advisory Committee Meeting, and multiple teleconferences with NASA. Minutes were recorded at these meetings in order to ensure that tasks were completed in a timely manner. Core A made travel arrangements for the Duke NSCOR team to travel to Brookhaven National Laboratory in Spring and Fall of 2012 in order to expose mice to high energy ionizing radiation. Travel arrangements were also made for all of the meetings described above to facilitate communication between the Duke NSCOR and other NASA investigators. Furthermore, Core A organized the visit by the UTSW NSCOR on March 7, 2012, which was held at the R. David Thomas Center at Duke. Core A also provided administrative support for credentialing Duke NSCOR investigators to work at BNL and for submitting and renewing the animal protocols at Duke and BNL. Core A provided budget oversight for the Duke NSCOR. Project expenditures were monitored by Erin Dillard. Ms. Dillard met monthly with David Kirsch M.D., Ph.D. to review spending and fiscal matters for each NSCOR project and Core. Marcia Painter assisted with ordering supplies and financial accounting for the Duke NSCOR.

Core B: Physics Core. Terry Yoshizumi, Ph.D., Lead

The Physics Core (Core B) provides comprehensive measurements of radiation dose (dosimetry) and oversees the radiation safety of experiments performed by investigators in the Duke NSCOR for experiments with X-rays. By performing routine dosimetry measurements on the standard small animal X-Ray irradiator, the Physics Core provides quality control for radiation exposure experiments. In addition, members of the physics core participate and present physics reports in the monthly NSCOR meeting.

Core C: Education Core. Rochelle Schwartz-Bloom, Ph.D., Lead

The Education Core (Core C) is developing an online problem-based unit to teach high school students about radiation in space by incorporating principles of physics, chemistry, and biology. The unit contains a hypothetical scenario in which a group of young astronauts are selected to travel to Mars in the year of 2040. The astronauts must learn about the types of radiation they will encounter in space (compared to on earth), the damage these high energy particles and cosmic rays can cause to their DNA molecules, how their bodies can deal with the damage using a protein called p53, and what would happen if their p53 gene has a mutation. They also learn how mutations in p53 genes can increase the risk of cancer, especially of the lung. The astronauts will meet some “virtual” scientists (the PIs of projects 1-3) who study these topics and whose research findings are crucial to the development of a successful space program that includes a trip to Mars. The educational unit will be field-tested in local high schools for impact on content knowledge and interests in science.

Lung cancer can be divided into two major forms: small-cell lung cancer and non-small cell lung cancer. Both non-small cell lung cancers and small cell lung cancers have developed in survivors of the atomic bombs in Japan. Similarly, both types of lung cancer arise in smokers. Cancers arising in never smokers preferentially develop in the distal airways and are of the adenocarcinoma histological subtype, which is a type of non-small cell lung cancer. Recently, genomic sequencing technology has been utilized to identify the most commonly mutated genes in adenocarcinomas. Based on this analysis, the two most commonly mutated genes in adenocarcinomas are Trp53, which encodes the tumor suppressor p53, and the oncogene Kras. The Duke NSCOR is utilizing sophisticated genetically engineered mouse models to study the role of p53 and Kras in non-small cell lung cancer. For example, we are studying how mutations in Kras in different kinds of cells in the lung affect lung cancer development with exposure to space radiation. We are also studying mice with an additional copy of p53 or inducible p53 suppression to investigate the timing and mechanism by which p53 suppresses Kras-driven lung adenocarcinoma progression after space radiation exposure. In addition, we are developing a mouse model of radiation-induced small-cell lung cancer. Together these studies will provide new insights into how lung cancer forms, where lung cancers develop, and how Kras and p53 mutation promote lung cancers. As we answer these questions using experiments with space radiation, we expect that our results will not only help us understand how lung cancer develops on earth, but will also provide new insights into preventing and treating lung cancer.

In addition to studying lung cancer development, the Duke NSCOR is also studying lung progenitor cell injury and repair after exposure to either terrestrial or space radiation. Injury and inflammation of the lung are key components of many diseases in people including emphysema, asthma, and lung fibrosis. Furthermore, patients receiving radiotherapy for either primary lung cancer or other neoplasms of the thoracic region (e.g. breast cancer) undergo lung tissue remodeling and declining lung function that is directly related to the dose and location of radiation exposure. By exploring which lung cells are injured by space radiation and how these injured lung cells are repaired, we anticipate that this knowledge may also lead to a better understanding of how lung diseases besides cancer develop and strategies that may be employed to moderate the effects of radiotherapy on lung tissue remodeling. This information may ultimately be used to develop novel approaches for the prevention and treatment of these lung diseases, and the improvement of public health.

Through these activities, members of the Duke NSCOR learned about space radiation and established collaborations with NASA investigators in other NSCORs. We are now applying this knowledge in our projects described below, which focus on 2 genes that are frequently mutated in human lung cancer: the tumor suppressor p53 and the oncogene K-ras.

Project 1. The role of the tumor suppressor p53 in space radiation-induced lung cancer. David Kirsch, M.D., Ph.D., Lead

We have proposed to study the role and timing of p53 in radiation-induced lung cancer using mice with an extra copy of p53 (Aim 1) and reversible knockdown of p53 (Aim 2). In addition, we are developing a model of radiation-induced small cell lung cancer (Aim 3).

For Aim 1, we have performed experiments to confirm increased induction of p53 transcriptional targets in mice with an additional copy of p53 (super p53 mice) after x-ray radiation. We have also established breeding cages to produce mice predisposed to lung cancer in mice with 2 or 3 copies of p53. Thirteen of these mice were sent to Brookhaven National Lab (BNL) and exposed to space radiation with iron. These mice are currently being monitored for lung tumor development. An additional 40 mice will be shipped to BNL for iron exposure in November. In parallel, we have irradiated two cohorts of mice with x-rays for comparison to the mice exposed to space radiation.

For Aim 2, we have performed experiments to quantify p53 knockdown in lung epithelial cells isolated from mice. We have observed p53 knockdown using a systemically inducible p53 shRNA, and we are currently breeding mice with a lung-targeted p53 shRNA. These mice will be used for pilot experiments to determine the best system to investigate the temporal role of p53 in high LET radiation-induced lung cancer.

For Aim 3, we have bred mice to investigate initiation of small cell lung cancer by space radiation. We have performed pilot experiments to determine the best fluorescent reporter to confirm deletion of these conditional genes in lung tissue. Twenty mice will be sent to BNL in November for exposure to space radiation. In addition, we have irradiated two cohorts of mice with x-rays and are monitoring them for the development of lung tumors.

Project 2. The role of cell of origin in space radiation-induced lung cancer. Mark Onaitis, M.D., Lead

We have proposed to study the cell of origin of K-RasG12D-induced lung cancer in response to high-LET radiation. Our aims include studying the effects of high LET radiation on mice in which K-rasG12D is inducibly expressed in different cell types of the lung: Clara cells (Aim 1), basal cells (Aim 2), and Type II cells (Aim 3).

For Aim 1, we have set up breeding cages to obtain many mice with the necessary group of genes. Five mice were sent to Brookhaven, exposed to space radiation with iron particles, and returned to Duke. These mice were sacrificed seven weeks after exposure to space radiation and analyzed for tumor formation. Additional mice will be exposed to space radiation in November at BNL.

In the next year, we plan to expose many, many more of these mice to space radiation. We also plan to isolate lung cells from these mice for in vitro colony-forming assays and to transplant these cells into the lungs of mice lacking an immune system.

For Aim 2, we have set up breeding cages and plan to irradiate many of these basal cell-specific K-rasG12D mice over the next year.

For Aim 3, we have set up multiple breeding cages and generated multiple litters with Type II cell specific K-rasG12D that will be ready for exposure to space radiation in the future. All of the assays described above will be performed.

Project 3. Effects of space radiation and p53 signaling on lung progenitor cells. Barry Stripp, Ph.D., Lead

We have proposed to determine how space radiation impacts epithelial cells that line the airways of the lung. Many different progenitor cell types reside in conducting airways. These progenitor cells normally contribute to replacement of specialized epithelial cell types that function in gas exchange and defense against environmental insults. However, the relative sensitivity of these progenitor cells to radiation effects is not known. We will determine the responses of progenitor cells to radiation injury and repair using in vivo and in vitro assays in normal mice and mice lacking the tumor suppressor p53.

For Aim 1, we have established a method to introduce genetic tags in airway progenitor cells to follow their expansion in vivo following radiation exposure. Our experiments indicate that mice exposed to radiation show evidence of increased clonal expansion of epithelial progenitor cells in vivo. Interestingly, in vitro studies indicate that the number of progenitor cells decreases following radiation exposure. We are applying histopathological methods to quantify these changes and determine the relative effects of X-rays (“terrestrial radiation”) and space radiation. Initial experiments suggest that different epithelial progenitor cells have different sensitivity/resistance to radiation exposure.

For Aim 2, we will genetically tag progenitor cells in mice that are deficient in the p53 tumor suppressor gene. These mice are currently being bred with the expectation that experiments with these mice lines will begin next year. Another goal of experiments in this aim is to generate new genetically engineered “reporter” mice to allow us to identify individual cells that have breaks in their DNA after space radiation exposure. In order to generate these mice, we are first testing the reporter gene constructs in vitro in model epithelial cell culture systems.

Core A: Administrative Core. David Kirsch, M.D., Ph.D., Lead. Duke NSCOR Administrator: Michelle Cooley

The Administrative Core (Core A) provides overall management of the NSCOR award by ensuring that projects make satisfactory progress. During the first year of funding, the Administrative Core has monitored project progress by conducting biweekly Duke NSCOR meetings, an annual Internal Advisory Committee Meeting, and multiple teleconferences with NASA. Minutes were recorded at these meetings in order to ensure that tasks were completed in a timely manner. Core A made travel arrangements for the Duke NSCOR team to travel to Brookhaven National Laboratory in June and November 2011 in order to expose mice to space radiation. Travel arrangements were also made for all of the meetings described above to facilitate communication between the Duke NSCOR and other NASA investigators. Core A also provided administrative support for credentialing Duke NSCOR investigators to work at BNL and for submitting and renewing the animal protocols at Duke and BNL. Core A provided budget oversight for the Duke NSCOR. Project expenditures were monitored by Erin Dillard. Ms. Dillard met monthly with David Kirsch M.D., Ph.D. to review spending and fiscal matters for each NSCOR project and Core. Marcia Painter assisted with ordering supplies and financial accounting for the Duke NSCOR.

Core B: Physics Core. Terry Yoshizumi, Ph.D., Lead

The Physics Core (Core B) provides comprehensive measurements of radiation dose (dosimetry) and oversees the radiation safety of experiments performed by investigators in the Duke NSCOR for experiments with X-rays. By performing routine dosimetry measurements on the standard small animal X-Ray irradiator, the Physics Core provides quality control for radiation exposure experiments. In addition, members of the physics core commissioned a new small animal irradiator (XRAD C225 Cx from Precision X-Ray), which has the capability to perform CT scans on mice and to deliver radiation not only to the entire mouse, but also to volumes as small as 1 cubic millimeter.

Core C: Education Core. Shelly Schwartz-Bloom, Ph.D., Lead

We are developing an online problem-based unit to teach high school students about radiation in space by incorporating principles of physics, chemistry, and biology. The unit will contain a hypothetical scenario in which a group of young astronauts are selected to travel to Mars in the year of 2040. The astronauts must learn about the types of radiation they will encounter in space (compared to on earth), the damage these high energy particles and cosmic rays can cause to their DNA molecules, how their bodies can deal with the damage using a protein called p53, and what would happen if their p53 gene has a mutation. They will also learn how mutations in p53 genes can increase the risk of cancer, especially of the lung. The astronauts will meet some “virtual” scientists who study these topics and whose research findings are crucial to the development of a successful space program that includes a trip to Mars.