Constantinos G. Broustas

DNA Damage Response Genes and the Development of Cancer Metastasis

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-)factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genome-wide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo.

Contributions of Rad9 to tumorigenesis.

Rad9 plays a crucial role in maintaining genomic stability by regulating cell cycle checkpoints, DNA repair, telomere stability, and apoptosis. Rad9 controls these processes mainly as part of the heterotrimeric 9‐1‐1 (Rad9‐Hus1‐Rad1) complex. However, in recent years it has been demonstrated that Rad9 can also act independently of the 9‐1‐1 complex as a transcriptional factor, participate in immunoglobulin class switch recombination, and show 3′‐5′ exonuclease activity. Aberrant Rad9 expression has been associated with prostate, breast, lung, skin, thyroid, and gastric cancers. High expression of Rad9 is causally related to, at least, human prostate cancer growth. On the other hand, deletion of Mrad9, the mouse homolog, is responsible for increased skin cancer incidence. These results reveal that Rad9 can act as an oncogene or tumor suppressor. Which of the many functions of Rad9 are causally related to initiation and progression of tumorigenesis and the mechanistic details by which Rad9 induces or suppresses tumorigenesis are presently not known, but are crucial for the development of targeted therapeutic interventions. J. Cell. Biochem. 113: 742–751, 2012.

Rad9 Protein Contributes to Prostate Tumor Progression by Promoting Cell Migration and Anoikis Resistance

Background: Rad9, a cell cycle checkpoint and DNA repair protein, is functionally related to human prostate tumorigenesis. Results: Rad9 deletion in prostate cancer cells impairs migration and invasion, sensitizes to anoikis, and down-regulates integrin β1. Conclusion: Rad9 controls ITGB1 expression, migration, invasion, and anoikis resistance of prostate cancer cells. Significance: This study reveals the significance of Rad9 in prostate tumor migration and invasion. Rad9 as part of the Rad9-Hus1-Rad1 complex is known to participate in cell cycle checkpoint activation and DNA repair. However, Rad9 can act as a sequence-specific transcription factor, modulating expression of a number of genes. Importantly, Rad9 is up-regulated in prostate cancer cell lines and clinical specimens. Its expression correlates positively with advanced stage tumors and its down-regulation reduces tumor burden in mice. We show here that transient down-regulation of Rad9 by RNA interference reduces DU145 and PC3 prostate cancer cell proliferation and survival in vitro. In addition, transient or stable down-regulation of Rad9 impairs migration and invasion of the cells. Moreover, stable reduction of Rad9 renders DU145 cell growth anchorage-dependent. It also decreases expression of integrin β1 protein and sensitizes DU145 and LNCaP cells to anoikis and impairs Akt activation. On the other hand, stable expression of Mrad9, the mouse homolog, in DU145/shRNA Rad9 cells restores migration, invasion, anchorage-independent growth, integrin β1 expression, and anoikis resistance with a concomitant elevation of Akt activation. We thus demonstrate for the first time that Rad9 contributes to prostate tumorigenesis by increasing not only tumor proliferation and survival but also tumor migration and invasion, anoikis resistance, and anchorage-independent growth.

p53 and RAD9, the DNA Damage Response, and Regulation of Transcription Networks

The way cells respond to DNA damage is important since inefficient repair or misrepair of lesions can have deleterious consequences, including mutation, genomic instability, neurodegenerative disorders, premature aging, cancer or death. Whether damage occurs spontaneously as a byproduct of normal metabolic processes, or after exposure to exogenous agents, cells muster a coordinated, complex DNA damage response (DDR) to mitigate potential harmful effects. A variety of activities are involved to promote cell survival, and include DNA repair, DNA damage tolerance, as well as transient cell cycle arrest to provide time for repair before entry into critical cell cycle phases, an event that could be lethal if traversal occurs while damage is present. When such damage is prolonged or not repairable, senescence, apoptosis or autophagy is induced. One major level of DDR regulation occurs via the orchestrated transcriptional control of select sets of genes encoding proteins that mediate the response. p53 is a transcription factor that transactivates specific DDR downstream genes through binding DNA consensus sequences usually in or near target gene promoter regions. The profile of p53-regulated genes activated at any given time varies, and is dependent upon type of DNA damage or stress experienced, exact composition of the consensus DNA binding sequence, presence of other DNA binding proteins, as well as cell context. RAD9 is another protein critical for the response of cells to DNA damage, and can also selectively regulate gene transcription. The limited studies addressing the role of RAD9 in transcription regulation indicate that the protein transactivates at least one of its target genes, p21/waf1/cip1, by binding to DNA sequences demonstrated to be a p53 response element. NEIL1 is also regulated by RAD9 through a similar DNA sequence, though not yet directly verified as a bonafide p53 response element. These findings suggest a novel pathway whereby p53 and RAD9 control the DDR through a shared mechanism involving an overlapping network of downstream target genes. Details and unresolved questions about how these proteins coordinate or compete to execute the DDR through transcriptional reprogramming, as well as biological implications, are discussed.

Comparison of gene expression response to neutron and x-ray irradiation using mouse blood.

BackgroundIn the event of an improvised nuclear device detonation, the prompt radiation exposure would consist of photons plus a neutron component that would contribute to the total dose. As neutrons cause more complex and difficult to repair damage to cells that would result in a more severe health burden to affected individuals, it is paramount to be able to estimate the contribution of neutrons to an estimated dose, to provide information for those making treatment decisions.ResultsMice exposed to either 0.25 or 1 Gy of neutron or 1 or 4 Gy x-ray radiation were sacrificed at 1 or 7 days after exposure. Whole genome microarray analysis identified 7285 and 5045 differentially expressed genes in the blood of mice exposed to neutron or x-ray radiation, respectively. Neutron exposure resulted in mostly downregulated genes, whereas x-rays showed both down- and up-regulated genes. A total of 34 differentially expressed genes were regulated in response to all ≥1 Gy exposures at both times. Of these, 25 genes were consistently downregulated at days 1 and 7, whereas 9 genes, including the transcription factor E2f2, showed bi-directional regulation; being downregulated at day 1, while upregulated at day 7. Gene ontology analysis revealed that genes involved in nucleic acid metabolism processes were persistently downregulated in neutron irradiated mice, whereas genes involved in lipid metabolism were upregulated in x-ray irradiated animals. Most biological processes significantly enriched at both timepoints were consistently represented by either under- or over-expressed genes. In contrast, cell cycle processes were significant among down-regulated genes at day 1, but among up-regulated genes at day 7 after exposure to either neutron or x-rays. Cell cycle genes downregulated at day 1 were mostly distinct from the cell cycle genes upregulated at day 7. However, five cell cycle genes, Fzr1, Ube2c, Ccna2, Nusap1, and Cdc25b, were both downregulated at day 1 and upregulated at day 7.ConclusionsWe describe, for the first time, the gene expression profile of mouse blood cells following exposure to neutrons. We have found that neutron radiation results in both distinct and common gene expression patterns compared with x-ray radiation.

RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels

Mouse embryonic stem cells null for Rad9 are sensitive to deleterious effects of ionizing radiation exposure. Likewise, integrin β1 is a known radioprotective factor. Previously, we showed that RAD9 downregulation in human prostate cancer cells reduces integrin β1 protein levels and ectopic expression of Mrad9 restores inherent high levels.

Impact of Neutron Exposure on Global Gene Expression in a Human Peripheral Blood Model

The detonation of an improvised nuclear device would produce prompt radiation consisting of both photons (gamma rays) and neutrons. While much effort in recent years has gone into the development of radiation biodosimetry methods suitable for mass triage, the possible effect of neutrons on the endpoints studied has remained largely uninvestigated. We have used a novel neutron irradiator with an energy spectrum based on that 1–1.5 km from the epicenter of the Hiroshima blast to begin examining the effect of neutrons on global gene expression, and the impact this may have on the development of gene expression signatures for radiation biodosimetry. We have exposed peripheral blood from healthy human donors to 0.1, 0.3, 0.5 or 1 Gy of neutrons ex vivo using our neutron irradiator, and compared the transcriptomic response 24 h later to that resulting from sham exposure or exposure to 0.1, 0.3, 0.5, 1, 2 or 4 Gy of photons (X rays). We identified 125 genes that responded significantly to both radiation qualities as a function of dose, with the magnitude of response to neutrons generally being greater than that seen after X-ray exposure. Gene ontology analysis suggested broad involvement of the p53 signaling pathway and general DNA damage response functions across all doses of both radiation qualities. Regulation of immune response and chromatin-related functions were implicated only following the highest doses of neutrons, suggesting a physiological impact of greater DNA damage. We also identified several genes that seem to respond primarily as a function of dose, with less effect of radiation quality. We confirmed this pattern of response by quantitative real-time RT-PCR for BAX, TNFRSF10B, ITLN2 and AEN and suggest that gene expression may provide a means to differentiate between total dose and a neutron component.

Abstract B030: MEK5 downregulation enhances radiosensitization of human prostate cancer cells by inhibiting DNA repair

Radiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure almost always ensues due to tumor intrinsic or acquired radioresistance. Mitogen-activated protein kinase kinase 5 (MAP2K5 or MEK5), belongs to the family of MAP kinases. It is activated by the upstream kinases MEKK2 and MEKK3 at Ser311/Thr315. MEK5, in turn, phosphorylates and activates extracellular signal-regulated kinase 5 (ERK5 or BMK1) at Thr218/Tyr220. MEK5/ERK5 pathway plays a pivotal role in tumor initiation and progression. MEK5 protein is overexpressed in prostate cancer cells compared with normal prostate epithelial cells, and MEK5 levels are correlated with prostate cancer metastasis. High expression of ERK5 in prostate cancer is also found to correlate with poor disease-specific survival and can serve as an independent prognostic factor. To determine whether the MEK5/ERK5 pathway is activated in response to ionizing radiation (IR), RNA interference was used to deplete MEK5 from PC3 and DU145 cells. Western blot analysis demonstrated that control cells with normal levels of MEK5 exposed to 3-Gy γ-rays had an increase in phospho-ERK5 levels at 5 and 15 min post-IR, diminishing at later time points. No activated ERK5 was detected in MEK5-depleted cells. Downregulation of MEK5 did not impact on cell cycle checkpoint activation in irradiated cells. In contrast, depletion of MEK5 markedly impaired phosphorylation of DNA-PKcs at Ser2056 in response to IR treatment. Furthermore, MEK5 knockdown did not influence the initial appearance of γH2AX or 53BP1 foci after irradiation, but significantly delayed the resolution of radiation-induced γH2AX and 53BP1 foci, detectable even 48 h post-irradiation, indicating a DNA repair defect. Cell based assay showed that nonhomologous end-joining is compromised in PC3 cells with ablated MEK5 protein expression. Finally, long-term clonogenic survival analyses and short-term cell growth assays indicated that MEK5 knockdown sensitized PC3 and DU145 prostate cancer cell lines to IR. Likewise, the topoisomerase II inhibitor etoposide that causes double-strand breaks also sensitized MEK5-depleted cells. These data indicate that MEK5 influences the response of prostate cancer cells to radiation and MEK5 downregulation is associated with delayed double-strand break repair kinetics. Inhibition of MEK5 in combination with radiation may provide a strategy to improve survival of prostate cancer patients. Citation Format: Constantinos G. Broustas. MEK5 downregulation enhances radiosensitization of human prostate cancer cells by inhibiting DNA repair [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B030.