The p21-Dependent Radiosensitization of Human Breast Cancer Cells by MLN4924, an Investigational Inhibitor of NEDD8 Activating Enzyme

Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS ONE (Impact Factor: 3.23). 03/2012; 7(3):e34079. DOI: 10.1371/journal.pone.0034079
Source: PubMed


Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells, respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However, blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924 could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.

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    • "Induction of DNA re-replication and p21-mediated cell cycle arrest has been primarily attributed to growth suppression [33], [34]. Suppressing the overall neddylation affects cellular response to conventional DNA damaging agents, shown by increased sensitivity of cancer cells to DNA damaging agents [33], [35]–[38]. Disrupting the normal DNA damage response has been proposed as a module for increasing drug sensitivity in cancer cells. "
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    ABSTRACT: Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.
    PLoS ONE 07/2014; 9(7):e101844. DOI:10.1371/journal.pone.0101844 · 3.23 Impact Factor
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    • "Chk2 phosphorylation at threonine 68 occurs following exposure to radiation as part of the DNA damage response [28], [29]. Several studies show the involvement of p21 in radiosensitization in several types of cancer [19], [20], [30]. Moreover, studies show that the phosphorylation of Chk2 at threonine 68 and increased p21 expression reduces cell proliferation by inducing senescence [31]–[33]. "
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    ABSTRACT: Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.
    PLoS ONE 02/2014; 9(2):e88322. DOI:10.1371/journal.pone.0088322 · 3.23 Impact Factor
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    • "Its dysfunction associates with tumor development and progression, suggesting that CRL could be a potential anticancer target. MLN4924, a small molecule inhibitor to inactivated CRL by inhibiting cullins activity, could effectively inhibit growth of various cancer cells [7], further suggesting the importance of CRL in maintaining tumor growth [8], [9]. However, Regulator of Cullins-1 (ROC1), another key subunit of CRL that heterodimerizes with distinct cullins to constitute the catalytic cores, whose function associated with cancer is poorly understand [5]. "
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    ABSTRACT: Regulator of Cullins-1 (ROC1) is a key subunit in the Cullin-RING ligase (CRL) protein complex. Overexpression of ROC1 protein is associated with tumor progression and poor prognosis of non-muscle invasive bladder transitional cell carcinoma (NMIBC). This study was designed to assess the effects of ROC1 knockdown in bladder cancer cells and to determine the potential mechanisms involved. A total of 112 bladder cancer tissue specimens were recruited for immunohistochemical analyses of ROC1 overexpression. Bladder cancer cell lines were used to knockdown ROC1 expression using ROC1 siRNA. Our data showed that ROC1 knockdown remarkably inhibited bladder cancer cell growth, arrested cells at the G2 phase of the cell cycle, and induced the p53-dependent cell senescence. Molecularly, G2 arrest was associated with upregulation of p21, p27, cyclin B1, and Cdc2 proteins. ROC1 knockdown induced-senescence functioned through p53/p21 pathway. Knockdown of p21 expression partially rescued ROC1 knockdown-induced growth inhibition in cancer cells. Furthermore, nude mouse xenograft analyses confirmed these in vitro data. In conclusion, data from the current study indicate that ROC1 plays an essential role in bladder cancer progression and could serve as a novel anticancer target for bladder transitional cell carcinoma (BTCC).
    PLoS ONE 05/2013; 8(5):e62734. DOI:10.1371/journal.pone.0062734 · 3.23 Impact Factor
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