RESEARCH DIRECTIONS (Kolettas lab)
I. Functional roles and mechanisms of action of IKKα- and IKKβ-mediated NF-κB-dependent or -independent signalling pathways, and IKK/NF-κB-miRNA regulatory network in DNA damage and inflammation impacting on senescence and cancer
NF-κB transcription factors (TFs) are critical regulators of pro-inflammatory/stress-like responses, and their immediate upstream signaling components are aberrantly expressed and/or activated in pulmonary diseases and in non-small cell lung cancer (NSCLC), with an unfavourable prognosis for patient survival. NF-κB TFs bind to DNA as hetero- or homodimers of 5 subunits: RelA/p65, c-Rel, RelB, p50 and p52. These TFs are activated by two upstream activating serine/threonine kinases IKKα and IKKβ. Activation of NF-κB is achieved by two main signalling pathways: An IKKβ-mediated canonical NF-κB pathway involving the nuclear translocation of c-Rel/p50 or p65/50 heterodimes and target gene regulation, and an IKKα-mediated non-canonical or alternative NF-κB pathway involving the nuclear translocation of p52/RelB heterodimers to regulate the expression of a distinct set of NF-κB target genes. NF-κB TFs can either activate or repress target gene transcription in different physiological contexts.
|Mechanism of action of ΙΚΚ/NF-κB in lung cancer. IKKα acts as tumour suppressor by influencing hypoxia-inducible pathways required for the enhanced tumour growth in vivo. IKKβ/canonical NF-κB promotes human NSCLC growth in vivo by inhibiting the expression of tumour metastasis cell surface suppressors and promoting cell motility, EMT and invasion.|
IKKβ/canonical NF-κB signalling and non-canonical heterodimers have been implicated in the onset, development and progression of NSCLC in the context of oncogene activation. Canonical NF-κB-induced genes promote cancer cell survival, cell cycle progression, metastasis and angiogenesis, and a cancer cell metabolic switch from oxidative phosphorylation to glycolysis (Warburg effect). Canonical NF-κB signalling functions as tumour promoting within transformed cells, but also influence the host’s innate immune response to cancer cells by regulating functions of infiltrating lymphocytes and macrophages and promoting inflammation in the tumour microenvironment. In contrast to the involvement of IKKβ/NF-κB in inflammation-linked NSCLC, the functional roles of IKKα in NSCLC remain unclear, as it can act as a tumour promoter or suppressor in different contexts.
Because each IKK functions differently, with IKKα acting as a growth suppressor and IKKβ as a promoter, we aim to understand the molecular mechanisms of action of each IKK in DNA damage and inflammation impacting on senescence and cancer:
1. Mechanism of action of canonical IKKβ/NF-κB in NSCLC: While somatic oncogenic mutations and the chemical carcinogen urethane lead to activation of canonical NF-κB, the mechanism(s) by which it contributes to NSCLC is still under investigation. Specifically, we would like to investigate the:
(a) Impact of canonical NF-κB in mutant EGFR-mediated NSCLC to identify novel NF-κB-regulated cancer biomarkers and potential therapy targets.
(b) Inflammatory cytokine-induced DNA damage, and vice versa, leading to genomic instability and cancer
(In collaboration with Dr. A. Klinakis and Dr. Z. Kanaki, Biomedical Research Foundation, Academy of Athens/BRFAA, Greece; Prof. A. Goussia, UoI Medical School; Prof. K. B. Marcu, Stony Brook University NY, USA; Dr. M. & K. Kriegsmann, University Hospital Heidelberg, Germany; Dr. E. Karteris, Brunel University London, UK)
2. NF-κB-miRNA network in NSCLC: We employed Nanostring miR analysis to identify IKKα or IKKβ-regulated miRNAs involved in DNA damage responses and NSCLC growth acting either as NSCLC tumour promoters or suppressors by influencing the expression of specific transcription factors and chromatin remodellers
(In collaboration with Dr. C. Polytarchou and Dr. M. Hatziapostolou, Nottingham Trent University, UK; Prof. K. B. Marcu, Stony Brook University NY, USA Prof. R. Sandaltzopoulos, Democritus University of Thrace, Greece; Dr. A. Klinakis & Dr. Z. Kanaki, BRFAA)
3. Mechanism of action of IKKα in NSCLC: RNA-seq and bioinformatics analysis identified genes including TFs and chromatin remodelers involved in human NSCLC growth. In addition, one of the IKKα-regulated miRNAs appears to affect chromatin remodeling. We aim at elucidating the role of IKKα-TF-miRNA in NSCLC.
(In collaboration with Prof. Emeritus K. B. Marcu, Stony Brook University, NY, USA)
• Markopoulos et al. (2017). Senescence-associated microRNAs target cell cycle regulation genes in human lung fibroblasts. Exp. Gerontol. 96:110-122.
• Markopoulos et al. (2017). A step-by-step microRNA guide to cancer development and metastasis. Cellular Oncol. 40(4):303-339.
• Markopoulos et al. (2018). Roles of NF-κB signalling in the regulation of miRNAs impacting on inflammation in cancer. Biomedicines 6(2):40, 198-216
• Markopoulos et al. (2019). Epigenetic regulation of inflammatory cytokine-induced epithelial-to-mesenchymal cell transition and in the generation of cancer stem cells. Cells 8(10), Sep 25, pii:E1143
• Chavdoula et al. (2019). CHUK/IKKα loss in lung epithelial cells enhances non-small cell lung cancer (NSCLC) growth associated with HIF up-regulation. Life Science Alliance 2(6):e201900460
• Roupakia et al. (2021). Genes and pathways involved in senescence bypass identified by functional genetic screens. Mech Ageing Dev 194:111432
(B) DNA Damage and Senescence
1. Involvement of the canonical NF-κB in the molecular crosstalk between the DNA damage response (DDR) and the mitotic Spindle Assembly Checkpoint (SAC) impacting on senescence and cancer, in response to chemotherapeutic drugs: We aim at identifying (a) Molecular connections between SAC and DDR, and (b) If NF-κΒ regulates SAC and connects DDR and SAC impacting on these surveillance mechanisms, using molecular cell biology and genetics, and functional assays for SAC activation. We will assay for SAC activation in response to several drugs such as VP16, nocodazole and paclitaxel in human cancer epithelial cells. Specifically, we will investigate (i) the impact of DDR checkpoint proteins in the activation of SAC in drug-treated human cancer cells, and (ii) Impact of canonical NF-κB on the activation and regulation of DDR, mitotic SAC and genomic instability.
2. IKK-regulated miRNAs in senescence: We have identified, Nanostring miRNA analysis, IKKα or IKKβ-regulated miRNAs involved in DDR and NSCLC growth, acting either as NSCLC tumour promoters or suppressors. We would investigate the impact of these miRNAs on normal human fibroblast senescence.
(In collaboration with Dr. C. Polytarchou & Dr. M. Hatziapostolou, Nottingham Trent University, UK; Prof. K. B. Marcu, Stony Brook University NY, USA Dr. A. Klinakis & Dr. Z. Kanaki, BRFAA, Greece)
3. Mechanism of action of IKKα in oncogene-induced senescence: Because IKKα acts as a growth suppressor, we aim to define its functional role in K-Ras expressing human fibroblast cell cycle progression, DDR and SASP, during OIS, by developing inducible cell systems.
(In collaboration with Dr. Charalampos Rallis, University of Essex, UK; Prof. K. Marcu, Stony Brook University NY, USA)
II. CRISPR/Cas9 screening to identify novel regulators of DNA damage, inflammation and cancer
1. Identifying novel regulators of cell signalling involved in NSCLC growth: We will employ a functional human kinase-specific CRISPR/Cas9 knockout lentiviral library to ablate 482 protein kinase (PKs) genes and identify novel regulators of cell signalling involved in NSCLC growth and in the responses of NSCLC cells to anticancer therapy. NGS will be used to identify the specific kinase involved.
2. Identifying novel regulators involved in DNA damage and cancer: We also want to employ a DNA binding domain-focused CRISPR knockout lentiviral library to ablate 1427 transcription factor (TFs), and identify TFs regulating DNA damage responses and certain aspects during the multistep process of carcinogenesis such as cell motility and invasion including EMT. NGS will be used to identify the specific TF involved.
• Giamas G, et al (2011) Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer. Nat Med 17(6):715-9.
• Shalem O, et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84-87.
• Shalem O, et al (2015) High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 16:299-311
• Shi J, et al (2015). Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol 33:661-67
• Tarumoto T, et al. (2018). LKB1, salt-inducible kinases, and MEF2C are linked dependencies in acute myeloid leukemia. Mol. Cell 69:1017-1027.
(In collaboration with Professor Georgios Giamas, Professor of Cell Signalling/Biochemistry, Head of the Department of Biochemistry & Biomedicine, School of Life Sciences. University of Sussex, UK)