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Final Report Summary - ANDYHREP (Anatomy and dynamics of the human replisome)

CCDC75 is a previously uncharacterized spliceosome interactor required for normal cell cycle progression

Cancer is one of the most frequent causes of death in the European Union averaging 167 deaths per 100,000 inhabitants (2010) and affecting over 2 million patients newly diagnosed every year. Additionally to the human suffering caused by cancer, the treatment costs for the European population and governments is extremely high, for instance reaching an average of 70,000 € per patient per year in Spain. Accordingly, the European Union, within its 2013 FP7 Cooperation Work Programme for Health, has emphasized cancer research as a major priority, highlighting the utmost importance of cancer research.
Cancer arises when a cell loses control over its normal proliferation. In fact, extremely sophisticated machinery has evolved to ensure the control of cell cycle progression.
Whereas several of the involved molecular factors have been elucidated during the last decades, we are still far away from understanding how this complex machinery works and how it interacts with all other cellular processes, particularly when it comes to mammalian cells.
In this context, the aim of this proposal was to find new factors involved in DNA replication in human cells using different interaction screens. Moreover, I aimed to characterise any novel human factor with regard to its effects on DNA replication and normal cell cycle progression in general.
Using mass spectrometry and yeast-two-hybrid screens, I found the previously characterised human factor CCDC75 or GPATCH11. I have characterised CCDC75 with regards to its cellular localization and stability. Further, I studied the role of CCDC75 in the cell by carefully studying its effects on both cell cycle progression and DNA replication. I have assessed the effects of both depletion and over-expression of CCDC75 and found its direct interactors. Moreover, I have performed a preliminary small-scale screen to assess the levels of CCDC75 in cancer versus non-cancer cell lines.
CCDC75 or GPATCH11 (for Coiled-Coil Domain Containing 75 or G-patch domain-containing protein 11, respectively) is a protein conserved in multicellular organisms and highly conserved in vertebrates. It is expressed in all tissues at medium to high levels. Interestingly, in a small-scale preliminary screen, I found that CCDC75 seems to be over-expressed in tumor versus non-tumor cell lines. Further characterization showed that CCDC75 is a highly stable nuclear protein with constant levels over the cell cycle.
Manipulating the levels of CCDC75 at specific cell cycle stages allowed us discover that normal CCDC75 cellular levels are required for undisturbed cell cycle progression. Indeed, depleting CCDC75 prevents cells from entering S-phase and duplicating their genome normally even if replication origin licensing happens at an undisturbed rate.
Moreover, cells that are not cycling are not capable of re-entering the cell cycle in the absence of CCDC75. On the other hand, mild and short-term overexpression of CCDC75 results in an increased percentage of cells in a population undergoing genome replication. Furthermore, long-term overexpression, also at low levels, is toxic.
Using a yeast-two-hybrid screen and mass spectrometry after pulling down a GFP-tagged over-expressed CCDC75 from human cells I found that CCDC75 directly interacts with Sf3b1 (splicing factor 3b, subunit 1) and several KPNA subunits (karyopherin alpha 1, 2 and 6). Interestingly, both proteins have been implicated in cell cycle progression. Depletion of Sf3b1impairs the proliferative capacity of hematopoietic stem cells (Wang et al. Blood 2014) and KPNA2 promotes cell proliferation and tumorigenicity in epithelial ovarian carcinoma (Huang et al. Cell Death and Disease 2013). We therefore propose that CCDC75 might be an important factor in either or both of these pathways contributing to normal cell cycle progression.
The continuation of this work will allow the elucidation of the mechanisms by which CCDC75 plays a role in normal cell cycle. Particularly two of our main findings make CCDC75 an extremely interesting protein to study further, possibly even exploring a potential role as a therapeutic or diagnostic cancer target: the preliminary evidence of its up-regulation in tumor cell lines, as well as the fact that its depletion results in proliferation defects even in cancer cells. Studying a possible addiction of cancer cells to high levels of CCDC75 will reveal whether CCDC75 could be a target for cancer therapy and thus a contribute to improve the outcome of cancer patients.

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