Kazi Mokim Ahmed

Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells

Mechanisms governing inducible resistance to ionizing radiation in untransformed epithelial cells pre-exposed to low-dose ionizing radiation (LDIR; </=10 cGy) are not well understood. The present study provides evidence that pre-exposure to 10 cGy X-rays increases clonogenic survival of mouse skin JB6P+ epithelial cells subsequently exposed to 2 Gy doses of gamma-rays. To elucidate the molecular pathways of LDIR-induced adaptive radioresistance, the transcription factor nuclear factor-kappaB (NF-kappaB) and a group of NF-kappaB-related proteins [i.e., p65, manganese superoxide dismutase (MnSOD), phosphorylated extracellular signal-regulated kinase, cyclin B1, and 14-3-3zeta] were identified to be activated as early as 15 min after LDIR. Further analysis revealed that a substantial amount of both 14-3-3zeta and cyclin B1 accumulated in the cytoplasm at 4 to 8 h when cell survival was enhanced. The nuclear 14-3-3zeta and cyclin B1 were reduced and increased at 4 and 24 h, respectively, after LDIR. Using YFP-fusion gene expression vectors, interaction between 14-3-3zeta and cyclin B1 was visualized in living cells, and LDIR enhanced the nuclear translocation of the 14-3-3zeta/cyclin B1 complex. Treatment of JB6P+ cells with the NF-kappaB inhibitor IMD-0354 suppressed LDIR-induced expression of MnSOD, 14-3-3zeta, and cyclin B1 and diminished the adaptive radioresistance. In addition, treatment with small interfering RNA against mouse MnSOD was shown to inhibit the development of LDIR-induced radioresistance. Together, these results show that NF-kappaB, MnSOD, 14-3-3zeta, and cyclin B1 contribute to LDIR-induced adaptive radioresistance in mouse skin epithelial cells.

NF-κB-Mediated HER2 Overexpression in Radiation-Adaptive Resistance

Abstract Cao, N., Li, S., Wang, Z., Ahmed, K. M., Degnan, M. E., Fan, M., Dynlacht, J. R. and Li, J. J. NF-κB-Mediated HER2 Overexpression in Radiation-Adaptive Resistance. Radiat. Res. 171, 9–21 (2009). The molecular mechanisms governing acquired tumor resistance during radiotherapy remain to be elucidated. In breast cancer patients, overexpression of HER2 (human epidermal growth factor receptor 2) is correlated with aggressive tumor growth and increased recurrence. In the present study, we demonstrate that HER2 expression can be induced by radiation in breast cancer cells with a low basal level of HER2. Furthermore, HER2-postive tumors occur at a much higher frequency in recurrent invasive breast cancer (59%) compared to the primary tumors (41%). Interestingly, NF-κB is required for radiation-induced HER2 transactivation. HER2 was found to be co-activated with basal and radiation-induced NF-κB activity in radioresistant but not radiosensitive breast cancer cell lines after long-term radiation exposure, indicating that NF-κB-mediated HER2 overexpression is involved in radiation-induced repopulation in heterogeneous tumors. Finally, we found that inhibition of HER2 resensitizes the resistant cell lines to radiation. Since HER2 is shown to activate NF-κB, our data suggest a loop-like HER2-NF-κB-HER2 pathway in radiation-induced adaptive resistance in breast cancer cells.

NF-κB-Mediated HER2 Overexpression inRadiation-Adaptive Resistance

Abstract Cao, N., Li, S., Wang, Z., Ahmed, K. M., Degnan, M. E., Fan, M., Dynlacht, J. R. and Li, J. J. NF-κB-Mediated HER2 Overexpression in Radiation-Adaptive Resistance. Radiat. Res. 171, 9–21 (2009). The molecular mechanisms governing acquired tumor resistance during radiotherapy remain to be elucidated. In breast cancer patients, overexpression of HER2 (human epidermal growth factor receptor 2) is correlated with aggressive tumor growth and increased recurrence. In the present study, we demonstrate that HER2 expression can be induced by radiation in breast cancer cells with a low basal level of HER2. Furthermore, HER2-postive tumors occur at a much higher frequency in recurrent invasive breast cancer (59%) compared to the primary tumors (41%). Interestingly, NF-κB is required for radiation-induced HER2 transactivation. HER2 was found to be co-activated with basal and radiation-induced NF-κB activity in radioresistant but not radiosensitive breast cancer cell lines after long-term radiation exposure, indicating that NF-κB-mediated HER2 overexpression is involved in radiation-induced repopulation in heterogeneous tumors. Finally, we found that inhibition of HER2 resensitizes the resistant cell lines to radiation. Since HER2 is shown to activate NF-κB, our data suggest a loop-like HER2-NF-κB-HER2 pathway in radiation-induced adaptive resistance in breast cancer cells.

Nuclear Factor-κB p65 Inhibits Mitogen-Activated Protein Kinase Signaling Pathway in Radioresistant Breast Cancer Cells

The molecular mechanism by which tumor cells increase their resistance to therapeutic radiation remains to be elucidated. We have previously reported that activation of nuclear factor-κB (NF-κB) is causally associated with the enhanced cell survival of MCF+FIR cells derived from breast cancer MCF-7 cells after chronic exposure to fractionated ionizing radiation. The aim of the present study was to reveal the context of NF-κB pathways in the adaptive radioresistance. Using cell lines isolated from MCF+FIR populations, we found that the elevated NF-κB activity was correlated with enhanced clonogenic survival, and increased NF-κB subunit p65 levels were associated with a decrease in phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK in all radioresistant MCF+FIR cell lines. Further irradiation with 30 fractions of radiation also inhibited MEK/ERK phosphorylation in paired cell lines of MCF+FIR and parental MCF-7 cells. Activation of ataxia-telangiectasia mutated (ATM) protein, a sensor to radiation-induced DNA damage, was elevated with increased interaction with NF-κB subunits p65 and p50. The interaction between p65 and MEK was also enhanced in the presence of activated ATM. In contrast, both interaction and nuclear translocation of p65/ERK were reduced. Inhibition of NF-κB by overexpression of mutant IκB increased ERK phosphorylation. In addition, MEK/ERK inhibitor (PD98059) reduced the interaction between p65 and ERK. Taken together, these results suggest that NF-κB inhibits ERK activation to enhance cell survival during the development of tumor adaptive radioresistance. (Mol Cancer Res 2006;4(12):945–55)

c-MYC Suppresses BIN1 to Release Poly(ADP-Ribose) Polymerase 1: A Mechanism by Which Cancer Cells Acquire Cisplatin Resistance

c-MYC promotes cisplatin resistance by enabling the increased activity of a DNA repair enzyme. Losing Your Inhibitions The antineoplastic drug cisplatin binds to actively replicating DNA, eliciting DNA lesions and, eventually, apoptosis. However, overexpression of the oncoprotein c-MYC can enable cancer cells to become resistant to cisplatin’s effects. Pyndiah et al. found that the c-MYC inhibitor BIN1 sensitized cells to DNA damage through a direct interaction with and inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1). c-MYC, when overexpressed, inhibited expression of BIN1, thereby setting up a positive feedback loop for increased c-MYC activity and promoting cancer cell resistance to DNA-damaging agents like cisplatin. Cancer cells acquire resistance to DNA-damaging therapeutic agents, such as cisplatin, but the genetic mechanisms through which this occurs remain unclear. We show that the c-MYC oncoprotein increases cisplatin resistance by decreasing production of the c-MYC inhibitor BIN1 (bridging integrator 1). The sensitivity of cancer cells to cisplatin depended on BIN1 abundance, regardless of the p53 gene status. BIN1 bound to the automodification domain of and suppressed the catalytic activity of poly(ADP-ribose) polymerase 1 (PARP1, EC 2.4.2.30), an enzyme essential for DNA repair, thereby reducing the stability of the genome. The inhibition of PARP1 activity was sufficient for BIN1 to suppress c-MYC–mediated transactivation, the G2-M transition, and cisplatin resistance. Conversely, overexpressed c-MYC repressed BIN1 expression by blocking its activation by the MYC-interacting zinc finger transcription factor 1 (MIZ1) and thereby released PARP1 activity. Thus, a c-MYC–mediated positive feedback loop may contribute to cancer cell resistance to cisplatin.

ATM-NF-κB Connection as a Target for Tumor Radiosensitization

Ionizing radiation (IR) plays a key role in both areas of carcinogenesis and anticancer radiotherapy. The ATM (ataxia-telangiectasia mutated) protein, a sensor to IR and other DNA-damaging agents, activates a wide variety of effectors involved in multiple signaling pathways, cell cycle checkpoints, DNA repair and apoptosis. Accumulated evidence also indicates that the transcription factor NF-κB (nuclear factor-kappaB) plays a critical role in cellular protection against a variety of genotoxic agents including IR, and inhibition of NF-κB leads to radiosensitization in radioresistant cancer cells. NF-κB was found to be defective in cells from patients with A-T (ataxia-telangiectasia) who are highly sensitive to DNA damage induced by IR and UV lights. Cells derived from A-T individuals are hypersensitive to killing by IR. Both ATM and NF-κB deficiencies result in increased sensitivity to DNA double strand breaks. Therefore, identification of the molecular linkage between the kinase ATM and NF-κB signaling in tumor response to therapeutic IR will lead to a better understanding of cellular response to IR, and will promise novel molecular targets for therapy-associated tumor resistance. This review article focuses on recent findings related to the relationship between ATM and NF-κB in response to IR. Also, the association of ATM with the NF-κB subunit p65 in adaptive radiation response, recently observed in our lab, is also discussed.

Cyclin D1 in low-dose radiation-induced adaptive resistance

Cyclin D1 is involved in cell-cycle arrest in DNA-damage response. This study tested the hypothesis that cyclin D1 regulates mitochondrial apoptosis. Cyclin D1 was induced by low-dose ionizing radiation (LDIR; 10-cGy X-ray) in human keratinocytes with an adaptive radioresistance that can be inhibited by short interfering RNA (siRNA)-mediated cyclin D1 inhibition. Cyclin D1 was found to form complex with chaperon 14-3-3ζ in unstressed cells and mutation of 14-3-3ζ Ser-58 to Asp (S58D) significantly impaired 14-3-3ζ binding to cyclin D1. The formation of cyclin D1/14-3-3ζ complex was differently regulated by exposure to low (10-cGy X-ray) versus high (5-Gy γ-ray) doses of radiation. Unlike exposure to 5-Gy that predominantly enhanced cyclin D1 nuclear accumulation, LDIR induced the dissociation of the cyclin D1/14-3-3ζ complex without nuclear translocation, indicating that cytosolic accumulation of cyclin D1 was required for LDIR-induced adaptive response. Further studies revealed a direct interaction of cyclin D1 with proapoptotic Bax and an improved mitochondrial membrane potential (Δψm) in LDIR-treated cells. Consistently, blocking cyclin D1/Bax formation by cyclin D1 siRNA reversed Δψm and inhibited the LDIR-associated antiapoptotic response. These results demonstrate the evidence that cytosolic cyclin D1 is able to regulate apoptosis by interaction with Bax in LDIR-induced adaptive resistance.

Identification of a novel effector domain of BIN1 for cancer suppression

Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein with tumor suppressor properties. The protein interacts with and inhibits the c‐MYC transcription factor through the BIN1 MYC‐binding domain (MBD). However, in vitro colony formation assays have clearly demonstrated that the MBD is not essential for BIN1‐mediated growth arrest. We hypothesized that BIN1 contains a MYC‐independent effector domain (MID) for cancer suppression. Because a functionally unique domain frequently contains a distinct structure, the human full‐length BIN1 protein was subjected to limited trypsin digestion and the digested peptides were analyzed with Edman sequencing and mass spectrometry. We identified a trypsin‐resistant peptide that corresponds to amino acids 146–268 of BIN1. It encompassed part of the BAR region, a putative effector region of BIN1. Computational analysis predicted that the peptide is very likely to exhibit coiled‐coil motifs, implying a potential role for this region in sustaining the BIN1 structure and function. Like MBD‐deleted BIN1, the trypsin‐resistant peptide of BIN1 was predominantly present in the cytoplasm and was sufficient to inhibit cancer growth, regardless of dysregulated c‐MYC activity. Our results suggest that the coiled‐coil BIN1 BAR peptide encodes a novel BIN1 MID domain, through which BIN1 acts as a MYC‐independent cancer suppressor. J. Cell. Biochem. 112: 2992–3001, 2011.