When cells are exposed to physical or chemical agents that damage DNA, or develop damage during normal metabolic processes, deleterious effects can ensue, including mutation, cancer or death. However, mechanisms are available to repair the damage, stabilize the genome and neutralize harmful effects. RAD9 plays a prominent role in these processes that promote survival and genomic integrity. Our studies indicate that this evolutionarily conserved gene has multiple functions needed for the cellular response to DNA damage, including cell cycle checkpoints, DNA repair and pro-apoptotic activities. In addition, RAD9 can, like p53, act as a sequence specific transcription factor. RAD9 can bind p53 consensus sequences in the p21 promoter and cause transcription when overexpressed. There is strong evidence that an optimum level of RAD9 is needed for proper cell functioning, and too much or too little can be detrimental. For examples, too much protein can cause apoptosis or lead to prostate cancer. Too little can cause cellular sensitivity to DNA damage, defects in DNA repair and cell cycle checkpoints, as well as lead to the development of cataracts or skin cancer. Therefore, it is important to understand mechanisms that regulate RAD9 levels. Recently, we discovered a very novel regulatory pathway that causes aberrant up regulation of Rad9 in prostate cancer. We found that DNA methyltransferases DNMT1 and DNMT3b levels are aberrantly high in several prostate cancer cell lines, and can methylate a """"""""suppressor of transcription"""""""" region within Rad9 intron 2 in DU145, AVLA31 and AVLA41 cells in particular, causing """"""""suppression"""""""" of the """"""""transcription suppressor"""""""", upregulation of the gene and subsequent transactivation of at least three downstream targets, Cox-2, Flt-1 and p21. We also provide evidence of a new carcinogenesis related function for Rad9, in metastasis. We propose to address and extend the novel Rad9 regulatory mechanism in greater detail, as well as define at the cellular and molecular levels the role of Rad9 in metastasis, both of which are important for prostate carcinogenesis. We will focus on three hypotheses: 1) DNMT1 and DNMT3b are highly expressed in certain prostate cancer cell lines because of chromatin structure, promoter methylation abnormalities or mutation, and bind more tightly to Rad9 only in DU145, AVLA31 and AVLA41 versus other cancer cell lines due to altered Rad9 chromatin;2) Rad9 upregulation induces transcription of multiple downstream genes and pathways critical for genomic integrity, growth control, prostate carcinogenesis and metastasis;and 3) Knock down of Rad9 expression will manifest as reduced intensity of phenotypes that reflect metastasis, including migration towards fibronectin, invasion, cell adhesion to various extracellular matrix proteins, and metastatic behavior in an in vivo model. The results of this study will elucidate a very novel Rad9 regulatory network we found active in multiple prostate cancer cell lines, and provide evidence for in primary prostate tumors, and expand our understanding of the biological function of the protein with respect to prostate carcinogenesis. As such, the findings should impact on basic research and public health where DNA damaging agents are prevalent environmental carcinogens that can induce genomic instability and cancer, and at the same time the work should reveal novel targets for anti-cancer agents.

Public Health Relevance

Rad9 is an evolutionarily conserved gene that participates in numerous, rudimentary biological processes important for influencing genomic integrity. There is strong evidence indicating that an optimum level of Rad9 is needed for proper cell functioning, and too much or too little can have deleterious consequences, including susceptibility to exogenous environmental DNA damaging agents and carcinogenesis. We describe experiments to define a very novel mechanism in prostate cancer cells for regulation of Rad9 level in greater detail, as well as a newly discovered role in metastasis, which is important since the results could impact on public health and lead to the development of new strategies for cancer therapy or to reduce risks associated with unwanted exposure.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-OTC-C (04))
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Pelroy, Richard
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Columbia University (N.Y.)
Schools of Medicine
New York
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Lieberman, Howard B; Panigrahi, Sunil K; Hopkins, Kevin M et al. (2017) p53 and RAD9, the DNA Damage Response, and Regulation of Transcription Networks. Radiat Res 187:424-432
Panigrahi, Sunil K; Hopkins, Kevin M; Lieberman, Howard B (2015) Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair. Nucleic Acids Res 43:4531-46
Broustas, Constantinos G; Lieberman, Howard B (2014) RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels. Prostate 74:1359-70
Ghandhi, Shanaz A; Ponnaiya, Brian; Panigrahi, Sunil K et al. (2014) RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response. Radiat Oncol 9:206
Broustas, Constantinos G; Lieberman, Howard B (2014) DNA damage response genes and the development of cancer metastasis. Radiat Res 181:111-30
Vasileva, Ana; Hopkins, Kevin M; Wang, Xiangyuan et al. (2013) The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse. J Cell Sci 126:3927-38
Cheng, Haiying; Zhang, Zhenfeng; Borczuk, Alain et al. (2013) PARP inhibition selectively increases sensitivity to cisplatin in ERCC1-low non-small cell lung cancer cells. Carcinogenesis 34:739-49
Broustas, Constantinos G; Zhu, Aiping; Lieberman, Howard B (2012) Rad9 protein contributes to prostate tumor progression by promoting cell migration and anoikis resistance. J Biol Chem 287:41324-33
Young, Erik F; Smilenov, Lubomir B; Lieberman, Howard B et al. (2012) Combined haploinsufficiency and genetic control of the G2/M checkpoint in irradiated cells. Radiat Res 177:743-50
Broustas, Constantinos G; Lieberman, Howard B (2012) Contributions of Rad9 to tumorigenesis. J Cell Biochem 113:742-51

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