Kazi M. Ahmed

Cyclin B1/Cdk1 Coordinates Mitochondrial Respiration for Cell-Cycle G2/M Progression

A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. Here, we show that a fraction of cyclin B1/Cdk1 proteins localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression.

NF-κB regulates radioresistance mediated by β1-integrin in three-dimensional culture of breast cancer cells.

β1-integrin induction enhances breast cancer cell survival after exposure to ionizing radiation (IR), but the mechanisms of this effect remain unclear. Although NF-κB initiates prosurvival signaling pathways post-IR, the molecular function of NF-κB with other key elements in radioresistance, particularly with respect to extracellular matrix-induced signaling, is not known. We discovered a typical NF-κB-binding site in the β1-integrin promoter region, indicating a possible regulatory role for NF-κB. Using three-dimensional laminin-rich extracellular matrix (3D lrECM) culture, we show that NF-κB is required for β1-integrin transactivation in T4-2 breast cancer cells post-IR. Inhibition of NF-κB reduced clonogenic survival and induced apoptosis and cytostasis in formed tumor colonies. In addition, T4-2 tumors with inhibition of NF-κB activity exhibit decreased growth in athymic mice, which was further reduced by IR with downregulated β1-integrin expression. Direct interactions between β1-integrin and NF-κB p65 were induced in nonmalignant breast epithelial cells, but not in malignant cells, indicating context-specific regulation. As β1-integrin also activates NF-κB, our findings reveal a novel forward feedback pathway that could be targeted to enhance therapy.

β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells.MethodsWe measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence.ResultsP-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, α5β1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-κB) to the β1-integrin promoter region, associated with up-regulation of α5β1-integrins. Inhibition of NF-κB or β1-integrin signaling abrogated emergence of the invasive activity.ConclusionsP-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by β1-integrin and NF-κB signaling.

PARP-1 modulates β1-integrin/NF-κB-mediated radioresistance in human breast cancer

Abstract Genomic damage triggers a signal transduction network that modulates gene expression and protein stability to repair DNA and influence cell survival. Inhibition of the DNA damage response protein PARP-1 is the subject of

Abstract C130: Inhibition of NF-κB activity promotes radiosensitivity of breast cancer cells in three-dimensional culture via a β1-integrin-mediated pathway.

Radioresistance markedly impairs the efficacy of tumor radiotherapy and involves prosurvival signal transduction pathways that prevent radiation-induced cell death. Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling in response to ionizing radiation (IR). The cooperative function of NF-κB with other key stress elements in radioresistance has not been clearly defined. We have previously reported that induction of β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to clinically relevant doses of IR. Because a typical NF-κB binding site was identified in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-κB. The aim of the present study was to reveal a connection between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-κB activity was correlated with enhanced clonogenic survival with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-κB activation significantly reduced clonogenic survival with the inhibition of β1-integrin, suggesting that NF-κB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of malignant human breast cell colonies, T4-2 with NF-κB activation inhibitor at Day 4 in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Using an ELISA-based DNA binding assay, we next determined that the binding of NF-κB to its target sequence on β1-integrin promoter region was significantly increased by IR. These data suggest that IR-induced increase in β1-integrin protein expression is mediated by NF-κB activation and subsequent binding of NF-κB to the β1-integrin promoter. Taken together, our results provide the first evidence that NF-κB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-κB/β1-integrin pathway could provide new insight into the development of new therapeutic modalities to re-sensitize radioresistant tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C130.

P3-01-06: NF-kB Inhibition Promotes Radiosensitivity of Breast Cancer Cells in Three-Dimensional Culture through Abating b1-Integrin Expression.

Therapy-associated tumor resistance, giving rise to recurrence and mortality, is a critical issue in cancer therapy. The molecular mechanisms causing tumor resistnace to the therapeutic radiation remain elusive. Nuclear factor-KB (NF-KB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling pathways. The cooperative function of NF-KB with other key stress elements in radioresistance remains to be elucidated. We have previously reported that induction of α5β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to high dose IR (ionizing radiation). Because a typical NF-KB binding site was located in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-KB. The aim of the present study was to reveal a connection between NF-KB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D ***lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-KB activity was correlated with enhanced clonogenic survival, and increased NF-KB heterodimer p50/p65 levels were associated with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-KB activation significantly reduced clonogenic survival with the inhibition of β1-integrin. These results indicate that NF-KB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of T4-2 colonies, formed at day 4, with NF-KB activation inhibitor in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Together, these results provide the first evidence that NF-KB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-KB/β1-integrin pathway may serve as an efficient drug target to re-sensitize radioresistant tumor cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-06.