ARISTEIA II

“Transcriptional reprogramming features triggered by oncogenic Cdc6” Funded by the GSRT with 200.000 Euros, “ARISTEIA II” program, project code 3020, 2014-2015

Cdc6 is a pivotal replication licensing factor. Together with the origin recognition complex (ORC) and Cdt1 they facilitate the loading of the minichromosome maintenance proteins 2-7 (MCM) on the chromatin forming the pre-Replication Complex (pre-RC). Pre-RCs bound to the replication origins, license them to fire once per cell cycle in normal cells. We and others have shown that deranged expression of Cdc6 and Cdt1 leads to re-replication, replication stress and aberrant recombination, which drive genomic instability and promote malignant behavior.

We demonstrated that Cdc6 is overexpressed in cancer from its earliest stages. This overexpression in epithelial cells is accompanied by appearance of mesenchymal features (epithelial to mesenchymal transition - EMT) and loss of E-cadherin expression. Furthermore, we showed that Cdc6 repression of E-cadherin (CDH1) is mediated by a transcriptional mechanism involving its binding to the E-boxes of CDH1 promoter, dissociation of the chromosomal insulator CTCF, and displacement of the histone variant H2A.Z and promoter heterochromatinization. This transcriptional suppressive mechanism is also linked with the activation of an adjacent replication origin.

This new and unique ability of Cdc6 to function as a molecular switch, linking transcriptional reprogramming to activation of replication, remains largely unexplored in cancer development. Therefore in the current proposal we aim to address the following: Which other genes are directly regulated by Cdc6? Which other factors are involved in the transcriptional reprogramming function of Cdc6? Does Cdc6 function as a molecular switch at these new genes; repressing transcription and co-currently triggering replication? Are the Cdc6 induced mesenchymal traits accompanied by emergence of cancer stem cell like features? Can Cdc6 trigger cancer development per se or does it co-operate with other tumor-promoting pathways?

Objectives:

  1. Identification of genes that are transcriptionally regulated by by Cdc6 / Gene expression pattern / ChIP analysis / Sequencing of cells that overexpress Cdc6.
  2. Functional characterization of proteins that interact with Cdc6.
  3. The role of Cdc6 as a molecular switch. Detection of "hidden" spots which initiate the transcription process to genes that are regulated by Cdc6 and effect on derregulation of miRNAs.
  4. Detection of the potential role of prolonged Cdc6 overexpression in cancer stem cells induction.
  5. Creation of transgenic mice which overexpress Cdc6 protein in several tissues / Study of cooperativeness with the mutant gene ras in cancer development.

Description of Work Packages (WPs):

  • WP1 - Investigation of expression profile and high-throughput ChIP / sequencing of human and mice cells which overexpress Cdc6

Deliverables of WP1:

1.1: Study of the expression profile regarding cell arrays with continuous Cdc6 overexpression, through high-throughput analysis systems

1.2: Study of the expression profile regarding cell arrays with induced Cdc6 overexpression, through high-throughput analysis systems

1.3: Revealing of the Cdc6 role regarding the derregulation of gene expression

  • WP2 - Biochemical and cellular characterization of proteins interacting with Cdc6

Deliverables of WP2:

2.1: Confirmation of the interaction between Cdc6 and BMI in the cellular systems available

2.2: Identification of new proteins interacting with Cdc6

2.3: Determination of the Cdc6 partners biological role in transcriptional regulation

  • WP3 - Determination of the presence of (re)activated sites concerning the initiation of the transcription process in the Cdc6-regulated genes instigators / Avocation with the induced by Cdc6 genomic instability which is caused during the activation of common fragile sites (CFCs) and the miRNA derregulation

Deliverables of WP3:

3.1: List of genes regulated by Cdc6 in combination with activated sites of the initiation of the transcription process

3.2: Cdc6-influenced CFCs models

3.3: miRNAs models which are affected because of the CFCs' changes that are induced by Cdc6

3.4: Cellular pathways / networks affected by the previously mentioned miRNAs

  • WP4 - Cdc6 role regarding the inducement of stem cell features in microculture systems

Deliverables of WP4:

4.1: Isolation and characterization of cancer stem cells after prolonged Cdc6 overexpression

4.2: Cdc6-dependent oncogene activity of cells which overexpress Cdc6

  • WP5 - Creation and characterization of mice models which overexpress Cdc6 under controlled conditions / Genetic interaction with ras mutation

Deliverables of WP5:

5.1: Mice model overexpressing Cdc6 in the lung

5.2: Mice model overexpressing Cdc6 in the breats

5.3: Mice model overexpressing Cdc6 in the intestine

5.4: Pathologoanatomical report on mice models

5.5: Cellular and molecular characterization of tumors induced by Cdc6

5.6: Cross-fertilization of Cdc6 transgenic mice with other carrying the mutant ras: Study of the synergistic effect

  • WP6 - Publicity and dissemination actions

Deliverables of WP6:

6.1: 1 Scientific publication

6.2: Creation of a subspace in the main researcher's official website, regarding the programme ARISTEIA

6.3: Press releases and advertisements

 

VG New

Prof. Vassilis G. Gorgoulis

Laboratory of Histology-Embryology
Molecular Carcinogenesis Group
Medical School
National and Kapodistrian University of Athens

 

Biomedical Research Foundation of the Academy of Athens

 

Faculty Institute for Cancer Sciences, University of Manchester,
Manchester Academic Health Science Centre, Manchester, UK

Manchester Centre for Cellular Metabolism,
University of Manchester, Manchester Academic Health Science Centre, Manchester

 

EMBO member

 

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