Identification and analysis of the signalling networks that regulate Ciz1 levels in normal and cancer cell lines

Ciz1 is a nuclear protein that associates with cyclin A – cyclin dependent kinase 2 (CDK2) and facilitates the initiation of DNA replication. Ciz1 overexpression has been linked to common cancer types, including breast, colon, prostate, lung, and liver cancers. This suggests that identification of mechanisms that regulate Ciz1 levels may represent potential drug targets in cancer. This work identifies that CDK2 and DDK activity are required to maintain Ciz1 levels. Chemical or genetic inhibition of CDK2 or DDK (Cdc7-Dbf4) activity in murine fibroblasts reduced Ciz1 levels. Further analysis demonstrated that CDK and DDK activity promotes Ciz1 accumulation in G1 phase by reducing ubiquitin proteasome system (UPS) mediated degradation. Furthermore, Ciz1 levels are actively controlled by the proteasome, as inhibition of protein translation rapidly reduced Ciz1 levels, and this is reversed by proteasomal inhibition. The data suggest a model where Ciz1 is regulated by opposing kinase and UPS activities, leading to Ciz1 accumulation in response to rising kinase activity in G1 phase, and its degradation later in the cell cycle. Significantly, human prostate adenocarcinoma (PC3) and oestrogen receptor positive breast cancer (MCF7) cell lines require Ciz1 for efficient proliferation. The data demonstrate that Ciz1 levels can be reduced with CDK2/ DDK inhibitors via proteasomally mediated degradation in human cancer cell lines similarly to normal fibroblasts. In PC3 and MCF7 cell lines, repurposing small molecule CDK2 inhibitors efficiently reduce Ciz1 levels, decrease E2F mediated transcription and proliferation. The targeted depletion of Ciz1 via CDK2/ DDK inhibition and UPS mediated degradation requires a functional E3 ligase to be effective. As a first step towards identifying the regulatory E3 ligase(s), a biochemical fractionation and mass spectrometry approach revealed three putative E3 ligases: UBR5, FBXO8 and UBE2O, which require further characterisation. Taken together, this work suggests that deregulation of CDK activity or inactivation of UPS signalling may promote Ciz1 overexpression in specific cancers. Importantly, Ciz1 is required for proliferation of some cancer cell lines, suggesting that approaches, which reduce Ciz1 levels may be of clinical benefit. Therefore, the identification of the regulatory mechanisms that control Ciz1 levels, represent potential targets in Ciz1 dependent cancers

Emerging Roles for Ciz1 in Cell Cycle Regulation and as a Driver of Tumorigenesis

Precise duplication of the genome is a prerequisite for the health and longevity of multicellular organisms. The temporal regulation of origin specification, replication licensing, and firing at replication origins is mediated by the cyclin-dependent kinases. Here the role of Cip1 interacting Zinc finger protein 1 (Ciz1) in regulation of cell cycle progression is discussed. Ciz1 contributes to regulation of the G1/S transition in mammalian cells. Ciz1 contacts the pre-replication complex (pre-RC) through cell division cycle 6 (Cdc6) interactions and aids localization of cyclin A- cyclin-dependent kinase 2 (CDK2) activity to chromatin and the nuclear matrix during initiation of DNA replication. We discuss evidence that Ciz1 serves as a kinase sensor that regulates both initiation of DNA replication and prevention of re-replication. Finally, the emerging role for Ciz1 in cancer biology is discussed. Ciz1 is overexpressed in common tumors and tumor growth is dependent on Ciz1 expression, suggesting that Ciz1 is a driver of tumor growth. We present evidence that Ciz1 may contribute to deregulation of the cell cycle due to its ability to alter the CDK activity thresholds that are permissive for initiation of DNA replication. We propose that Ciz1 may contribute to oncogenesis by induction of DNA replication stress and that Ciz1 may be a multifaceted target in cancer therapy

Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor.

The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5'-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.Pfizer ITEN award, Lister Prize Fellowshi

Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor

Hardy, John George/0000-0003-0655-2167; Kivrak, Ezgi/0000-0001-8552-3473; FIRLAK, Melike/0000-0003-1674-9086; ozsoz, mehmet/0000-0003-4037-3445; Copeland, Nikki/0000-0002-6204-4717; Yilmaz, Selahattin/0000-0003-2853-0751; Palaz, Fahreddin/0000-0002-9514-3172WOS:000609857900001PubMed: 33429883The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. in this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5 '-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.Royal SocietyRoyal Society of LondonEuropean Commission [NF151479, RG160449]; Lancaster University Faculty of Science and Technology for a Distinguished Visitor Program Grant; North West Cancer Research NWCR [CR1071, CR879]We thank the Royal Society for a Newton International Fellowship (NF151479) for Melike Firlak, and a Research Grant (RG160449) for John Hardy. We thank the Lancaster University Faculty of Science and Technology for a Distinguished Visitor Program Grant to support collaboration with Mehmet Ozsoz. We thank North West Cancer Research NWCR for project funding for Tekle Pauzaite (CR1071) and an independent research fellowship for Nikki Copeland (CR879)

Dbf4-Cdc7 (DDK) Inhibitor PHA-767491 Displays Potent Anti-Proliferative Effects via Crosstalk with the CDK2-RB-E2F Pathway

Precise regulation of DNA replication complex assembly requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities to activate the replicative helicase complex and initiate DNA replication. Chemical probes have been essential in the molecular analysis of DDK-mediated regulation of MCM2-7 activation and the initiation phase of DNA replication. Here, the inhibitory activity of two distinct DDK inhibitor chemotypes, PHA-767491 and XL-413, were assessed in cell-free and cell-based proliferation assays. PHA-767491 and XL-413 show distinct effects at the level of cellular proliferation, initiation of DNA replication and replisome activity. XL-413 and PHA-767491 both reduce DDK-specific phosphorylation of MCM2 but show differential potency in prevention of S-phase entry. DNA combing and DNA replication assays show that PHA-767491 is a potent inhibitor of the initiation phase of DNA replication but XL413 has weak activity. Importantly, PHA-767491 decreased E2F-mediated transcription of the G1/S regulators cyclin A2, cyclin E1 and cyclin E2, and this effect was independent of CDK9 inhibition. Significantly, the enhanced inhibitory profile of PHA-767491 is mediated by potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network, that provides the molecular basis for its increased anti-proliferative effects in RB+ cancer cell lines

A HIF independent oxygen-sensitive pathway for controlling cholesterol synthesis.

Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.Lister Institute of Preventative Medicine National Institutes of Health (NIH) grant Pfizer ITEN Awar