Graduation Year

2013

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Robert J. Deschenes

Keywords

eIF4E, mRNA Translation, mTOR, Radiosensitivity

Abstract

Current approaches aimed at improving the efficacy of radiation as a cancer treatment modality involve the development and application of molecularly targeted radiosensitizers, a strategy that requires a thorough understanding of the fundamental processes comprising the cellular radioresponse. Recent data indicating that radiation modifies gene expression primarily through translational control rather than transcriptional events suggests that mRNA translation contributes to cell survival after irradiation. The overall goal of this project is to determine whether the regulatory/rate-limiting components of the translational machinery provide targets for tumor cell radiosensitization. The majority of translation in mammalian cells occurs in a cap-dependent manner and is highly dependent on eIF4E. As such, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E knockdown enhanced the radiosensitivity of tumor but not normal cells. eIF4E knockdown inhibited the dispersal of radiation-induced γH2AX foci. Furthermore, radiation was found to increase the binding of >1000 unique mRNAs to eIF4E, many involved in DNA replication, recombination, and repair. S6 kinase 1 (S6K1), also an important regulatory component of the translational machinery, enhances the translation of specific mRNA subpopulations, independent from eIF4E, and mediates ribosome biogenesis. The role of S6K1 in determing cell survival after radiation was determined in several tumor cell lines and one normal cell line. S6K1 knockdown enhanced the radiosensitivity of all 3 tumor lines. In contrast S6K1 knockdown had no effect on the cellular radiosensitivity of the one normal line tested. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. Importantly, it plays a major role in regulating eIF4E availability as well as S6K1 activity. The radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the ATP competitive mTOR inhibitor PP242 were compared. Based on immunoblot analyses, whereas rapamycin only partially inhibited mTORC1 activity and had no effect on mTORC2, PP242 inhibited the activity of both mTOR containing complexes. In the two tumor cell lines evaluated, PP242 treatment 1h before irradiation increased radiosensitivity, whereas rapamycin had no effect. PP242 had no effect on the cellular radiosensitivity of a normal lung fibroblast line. PP242 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. A next generation analog of PP242, INK128, is currently undergoing analysis in clinical trials. Given our data showing ATP-competitive mTOR inhibition is a strategy for tumor radiosensitization as well as the fact that radiotherapy is a primary treatment modality for locally advanced pancreatic ductal adenocarcinoma, the effects of INK128 on pancreatic cancer radiosensitivity were determined. In three pancreatic cancer cell lines addition of INK128 immediately after radiation resulted in radiosensitization. Consistent with the effects of PP242 on other cell lines, INK128 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Furthermore, in pancreatic tumor xenografts INK128 inhibits mTOR activity as well as cap-complex formation in a time-dependent manner. Lastly, INK128 treatment significantly prolonged the radiation-induced tumor growth delay of pancreatic tumor xenografts. In summary, the data provided in this thesis have begun to characterize the role of the translational machinery in determining the cellular response to radiation.

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