The differential role of CDC6 in normal and cancer cells

Homeodynamics (Lloyd, Aon et al. 2001) is a fundamental feature of single and multi-cellular organisms that is maintained by convenient cellular counteractions to a plethora of deleterious intrinsic and extrinsic signals. Aberrations in the modulating pathways that govern these responses result in excess of damage, functional defects and disease (Gorgoulis and Halazonetis 2010). Cancer comprises such an example. In cancer cells, one of the most frequently affected cellular functions is the proper execution of the cell cycle that under circumstances can impose genomic instability (Negrini, Gorgoulis et al. 2010). This established hallmark of cancer is closely related to dysfunction of the replication licensing machinery (Petrakis, Komseli et al. 2015).Cell division cycle 6 (CDC6) is a cardinal molecule of this apparatus. Coordinated expression of CDC6, together with ORC and CDT1, facilitate timely loading of MCM2-7 onto the chromatin in G1 phase, forming the pre-replicative complex. This results in licensing of the replication origins to fire once per cell cycle, ensuring that way the accurate duplication of the whole genome before cell division (Blow and Gillespie 2008). Moreover, CDC6 is engaged to the emergence of checkpoints that regulate S phase and mitosis (Borlado and Mendez 2008).On the contrary, accumulating amount of data supports that deregulated expression of CDC6 exerts oncogenic properties. Particularly, it is frequently overexpressed in cancer, usually from its earliest stages and associated with poor prognosis (Williams, Romanowski et al. 1998, Karakaidos, Taraviras et al. 2004, Liontos, Koutsami et al. 2007, Sideridou, Zakopoulou et al. 2011). CDC6 forced expression results in re-replication supplying DNA damage and genomic instability (Vaziri, Saxena et al. 2003, Liontos, Koutsami et al. 2007, Sideridou, Zakopoulou et al. 2011, Walter, Hoffmann et al. 2016). Subsequent activation of DNA damage response checkpoints leads to rise of antitumor barriers of senescence and apoptosis (Bartkova, Horejsi et al. 2005, Bartkova, Rezaei et al. 2006, Petrakis, Komseli et al. 2015), while selective loss of p53 promotes malignant behavior (Karakaidos, Taraviras et al. 2004, Liontos, Koutsami et al. 2007, Halazonetis, Gorgoulis et al. 2008) and acquisition of mesenchymal traits through E-cadherin down-regulation, a hallmark of epithelial-to-mesenchymal transition (EMT) (Liontos, Koutsami et al. 2007, Sideridou, Zakopoulou et al. 2011). This down-regulation involves transcriptional repression of the CDH1 gene (coding E-cadherin), as it has also been shown for the INK4/ARF locus coding three important tumor suppressors (Gonzalez, Klatt et al. 2006). More recently, CDC6 has been involved in regulation of initiation of the transcription of rRNA (Huang, Xu et al. 2016)and CXCL12 (Petrakis, Komseli et al. 2015).To investigate the role of CDC6 in carcinogenesis we developed a prototypical non-malignant epithelial cellular system overexpressing CDC6 in an inducible manner utilizing immortalized Human Bronchial Epithelial Cells (HBEC) as a platform. These cells, immortalized with a combined expression of human telomerase reverse transcriptase (hTERT) and ectopic mutant cyclin dependent kinase (CDK) 4, maintain their epithelial phenotype and have intact the p53 checkpoint pathway (Ramirez, R.D., et al., 2004). Thus, they represent an almost normal epithelial environment being a valuable tool for studying carcinogenesis, since the majority of malignancies have epithelial origin.To examine the role of CDC6 in tumorigenesis, we applied our newly developed system in an Epithelial Cancer Evolution Experiment (ECEE), which means constitutive overexpression of CDC6 in the benign environment of HBECs and monitor of the cellular behavior over time without genetic manipulations. In that way, we allowed the action of natural selection and managed to recapitulate in vitro the events that take place during the carcinogenetic process in vivo.Analyzing the results of ECEE, we found that CDC6 induction resulted acutely in diminished cell proliferation rate and rise of the tumor suppressive mechanism of senescence. Although this seemed intriguing at first, given that CDC6 overexpression was functional leading to a substantial increase in chromatin-loaded MCM2-7 helicase complex, meaning more licensed replication origins (Moreno, Carrington et al. 2016), it strongly supported CDC6 role as an oncogene (Prieur and Peeper 2008). According to the oncogene-induced DNA damage model for cancer development, the tumor-suppressive mechanism of senescence is overcome at some point giving rise to cancerous cells with aggressive features (Halazonetis, Gorgoulis et al. 2008). In alliance, downregulation of p16INK4A, necessary for irreversible senescence (Beausejour, Krtolica et al. 2003), due to CDC6-dependent repression of INK4/ARF locus (Gonzalez, Klatt et al. 2006, Petrakis, Komseli et al. 2015), queried the endurance of CDC6-mediated senescence in HBECs. Indeed, after protracted CDC6 overexpression and an initial phase of stalled growth, a fraction of proliferating cells emerged generating cellular clones that escaped from senescence. Interestingly, these cells have acquired spindle morphology, accompanied by loss of E-cadherin and increase of vimentin, implying an EMT, and have been more efficient in migration in vitro. Consequently, different time points of ECEE reflect the whole spectrum of epithelial carcinogenesis; normal epithelial, precancerous-senescent and cancerous cells with mesenchymal features.From a mechanistic point of view, CDC6-dependent senescence must be owed to DNA damage response (DDR) activation (d'Adda di Fagagna 2008, Halazonetis, Gorgoulis et al. 2008). As expected (Bartkova, Rezaei et al. 2006, Liontos, Koutsami et al. 2007, Walter, Hoffmann et al. 2016), CDC6 induction resulted in aberrant DNA replication, origin over-usage and/or re-firing of the same origin, known as re-replication, feeding DNA damage (shown by alkaline comet assay) and triggering DDR activation. Since most of the DDR pathways converge on p53 stabilization leading to p21WAF/CIP1 upregulation and cell cycle arrest (Deng, Zhang et al. 1995), both p53 and p21WAF/CIP1 protein levels were found increased. However, contrary to the existing literature where inactivation of DDR key gene products leads to senescence avoidance (d'Adda di Fagagna, Reaper et al. 2003, Herbig, Jobling et al. 2004, Bartkova, Rezaei et al. 2006, Di Micco, Fumagalli et al. 2006, Mallette, Gaumont-Leclerc et al. 2007, Borodkina, Shatrova et al. 2016), neither p53 depletion nor ATM kinase inhibition resulted in bypass of CDC6-driven senescence. On the contrary, silencing of ATM resulted in massive cell death (Petrakis, Komseli et al. 2015). One potential explanation is that CDC6-induced senescence is p53 independent and owed to p73 (Liontos, Niforou et al. 2009), which protein levels have been found increased, or p38 (Bulavin and Fornace 2004). Moreover, it has been proposed that robust DDR activation and foci formation itself, or even the interaction of DDR factors alone, are able to promote cellular senescence (Bonilla, Melo et al. 2008, d'Adda di Fagagna 2008).Apart from the well-established replication stress, here we report for the first time R loop formation as another source of DNA damage (Skourti-Stathaki and Proudfoot 2014) deriving from CDC6 overexpression. As already mentioned, CDC6 regulates rDNA transcription initiation (Huang, Xu et al. 2016), while it has been reported that actively transcribed rDNA repeats form R loops in yeast (El Hage, French et al. 2010). As a result, identification of R loops within the nucleoli in CDC6-induced cells did not come as a surprise to us. Double immunofluorescent analysis of nucleophosmin (NPM) or upstream binding factor (UBF), bound on rDNA array (Grob, Colleran et al. 2014), and 53BP1 revealed: a) nucleolar reorganization, known as segregation, which has been found to be correlated with DSBs introduced into rDNA, b) UBF relocalization from the interior of nucleoli to the periphery, and c) association of CDC6-mediated DSBs with UBF forming structures that resemble nucleolar caps (van Sluis and McStay 2015, van Sluis and McStay 2017). One possible explanation for the recession of R loops formation in 6-day induced cells is that DSBs introduced into rDNA due CDC6 overexpression result in inhibition of RNA Polymerase I transcription (necessary for R loop formation) until damage is repaired. Re-appearance of R loops in the escaped cells implies DNA recovery, as well as diverse functions in this new context or in the presence of the R loop-affecting factors. It should be pointed out, though, that it is not the majority of DSBs related to R loops and nucleoli, which means that other threats to DNA integrity also exist.No matter the provenance of DNA damage, it rabidly induces DDR activation and senescence. This stage of ECEE resembles pre-neoplastic lesions, as identification of DDR and senescent markers peaks in the dysplastic precancerous phase and progressively recesses during cancer progression in vivo (Bartkova, Horejsi et al. 2005, Gorgoulis, Vassiliou et al. 2005, Bartkova, Rezaei et al. 2006, Nuciforo, Luise et al. 2007). In accordance, HBEC CDC6 overexpressing cells that have escaped from senescence have reduced DSBs, as well as p53, p21WAF/CIP1 and p73 protein levels. A similar phenomenon has been observed in p21WAF/CIP1-constitutive expression, which results in activation of a Rad52-dependent repair process and the emergence of cellular “offspring” which are more aggressive and chemoresistant (Galanos, Vougas et al. 2016). It is possible that senescence ensures that there is enough time so that cells repair their damages enabling cell transformation. In the future, it will be interesting to find out the activated repair mechanism(s) upon CDC6 induction in HBECs. Except for the morphological differences, high-throughput RNA and micro-RNA sequencing analysis of the escaped cells was indicative of extensive alterations in their transcriptome landscape compared to the non-induced and senescent cells. Adapting Gene Ontology analysis to the hallmarks of cancer, we found that these cells share most of the cancerous characteristics. In alliance, cytogenetic analysis of HBEC CDC6 Tet-ON cells revealed that they are near diploid, even though they obtain a few chromosomal rearrangements typical of clonal expansion, whereas the escaped cells showed novel and random chromosomal rearrangements, which characterize chromosomal instability, typical of cancer cells. Another interesting finding is that the escaped cells are more sensitive to common chemotherapeutics, doxorubicin and cisplatin, compared to near normal non-induced cells, further supporting malignant transformation, as it has long been established that normal cells are more resistant than cancer ones to therapeutics (Sonneveld, Mulder et al. 1981, Ajani, Blaauw et al. 1985, Yalowich, Zucali et al. 1985, Lampidis, Krishan et al. 1986, Matthews, Kutlaca et al. 1987, Borenfreund, Babich et al. 1990, Nygren and Larsson 1991, Bennani-Baiti, Lafarge-Frayssinet et al. 1993, Kuhl, Duran et al. 1993).Overall, our results indicate that deregulated expression of CDC6 which is part of replication licensing machinery, the first that responds to mitogenic stimuli, is able to drive carcinogenesis. However, other oncogenic stimuli are also necessary. Morover, HBEC CDC6 Tet-ON system is optimal for studying a) the molecular basis of epithelial carcinogenesis, b) genome dynamics given the role of CDC6 in replication and transcription, and c) repair mechanisms in both euchromatic and heterochromatic regions.Η ομοιόσταση συνιστά θεμελιώδη ιδιότητα των οργανισμών και εξασφαλίζεται μέσω πληθώρας κυτταρικών αποκρίσεων έναντι ενδογενών και εξωγενών παραγόντων. Η διαταραχή των μηχανισμών που ελέγχουν τις διαδικασίες αυτές οδηγεί στη συσσώρευση βλαβών και γενικά στην απορρύθμιση της κυτταρικής λειτουργίας με επακόλουθο την εμφάνιση νόσων, όπως ο καρκίνος. Στους όγκους παρατηρείται συχνά διατάραξη της ομαλής εξέλιξης του κυτταρικού κύκλου, που υπό προϋποθέσεις, μπορεί να οδηγήσει στην πρόκληση γενωμικής αστάθειας. Η τελευταία έχει καθιερωθεί τα τελευταία χρόνια ως χαρακτηριστικό των καρκινικών κυττάρων και σχετίζεται στενά με την διαταραχή του μηχανισμού αδειοδότησης της αντιγραφής του DNA.Ο παράγοντας CDC6 αποτελεί κύριο συστατικό αυτής της λειτουργίας. Το γονίδιο CDC6 εδράζεται στην περιοχή 17q21.3 και κωδικοποιεί μια πρωτεΐνη με μοριακό βάρος ~60 KDa που ανήκει στην οικογένεια των AAA+ ATPασών. Το CDC6 μαζί με τις πρωτεΐνες ORC και CDT1 διευκολύνουν τη στρατολόγηση των παραγόντων MCM2-7 στη χρωματίνη, προκειμένου να σχηματιστεί το προ-αντιγραφικό σύμπλοκο. Έτσι εξασφαλίζεται η έναρξη της αντιγραφής από ένα καθορισμένο σημείο μία φορά ανά κυτταρικό κύκλο, με αποτέλεσμα τον ακριβή διπλασιασμό ολόκληρου του γονιδιώματος πριν από κάθε κυτταρική διαίρεση. Επιπλέον, το CDC6 εμπλέκεται στην ορθή λειτουργεία των σημείων ελέγχου του κυτταρικού κύκλου G1/S και G2/M.Απο την άλλη, η απορρυθμισμένη έκφραση του CDC6 ενέχει ογκογόνες ιδιότητες. Συγκεκριμένα ο παράγοντας CDC6 υπερεκφράζεται από τα αρχικά στάδια της καρκινογένεσης, και σχετίζεται με κακή πρόγνωση. Σύμφωνα με το μοντέλο για την επαγωγή της καρκινογένεσης μέσω των προκαλούμενων από τα ογκογονίδια βλαβών του DNA, η υπερέκφραση ογκογονιδίων, όπως το CDC6, οδηγεί σε γενωμική αστάθεια και στην ενεργοποίηση των αντικαρκινικών φραγμών της απόπτωσης ή της κυτταρικής γήρανσης, ενεργοποιώντας τον μηχανισμό απόκρισης σε βλάβες του DNA (DDR). H επακόλουθη απώλεια ογκοκατασταλτικών γονιδίων, όπως του TP53, οδηγεί στην παράκαμψη των αντικαρκινικών φραγμών και στην προαγωγή του καρκίνου καθώς και στην απόκτηση γνωρισμάτων μεσεγχυματικών κυττάρων (ΕΜΤ), με παράλληλη απώλεια έκφρασης της πρωτεΐνης Ε-καδερίνης, που είναι βασικό χαρακτηριστικό της ΕΜΤ. Η απώλεια έκφρασης της Ε-καδερίνης οφείλεται στη μεταγραφική καταστολή του γονιδίου CDH1 (που κωδικοποιεί την Ε-καδερίνη) από το CDC6. Εκτός από το CDH1, το CDC6 καταστέλλει μεταγραφικά και τα γονίδια του γενετικού τόπου INK4/ARF που κωδικοποιούν τρεις ογκοκατασταλτικές πρωτεΐνες, ενώ πιο πρόσφατα δεδομένα υποστηρίζουν ότι το CDC6 ενεργοποιεί τη μεταγραφή του rDNA και του γονιδίου CXCL12.Για να διερευνηθεί ο ρόλος του CDC6 στην καρκινογένεση αναπτύχθηκε ένα πρωτότυπο μη κακόηθες επιθηλιακό κυτταρικό σύστημα υπερέκφρασης του CDC6 με επαγώγιμο τρόπο χρησιμοποιώντας ανθρώπινα κύτταρα βρογχικού επιθηλίου (HBEC), αθανατοποιημένα με συνδυαστική υπερέκφραση τελομεράσης (hTERT) και πρωτεΐνης CDK4. Τα κύτταρα αυτά διατηρούν τον επιθηλιακό φαινότυπό τους και έχουν άθικτo τo μονοπάτι ελέγχου μέσω του p53. Έτσι, αντιπροσωπεύουν ένα σχεδόν φυσιολογικό επιθηλιακό περιβάλλον και αποτελούν ένα πολύτιμο εργαλείο για τη μελέτη της καρκινογένεσης, καθώς η πλειοψηφία των κακοηθειών έχει επιθηλιακή προέλευση.Η επαγόμενη υπερέκφραση του CDC6 σε αυτό το σχεδόν φυσιολογικό γενετικό περιβάλλον προκαλεί μείωση του πολλαπλασιασμού των κυττάρων και πυροδοτεί τον αντικαρκινικό φραγμό της γήρανσης. Ακολουθεί διαφυγή από αυτή και εμφάνιση επιθετικότερων κυτταρικών κλώνων με χαρακτηριστικά EMT. Ως εκ τούτου, το πρότυπο αυτό κυτταρικό σύστημα συνοψίζει ολόκληρο το φάσμα της επιθηλιακής καρκινογένεσης από το μη κακόηθες στάδιο, στην ενεργοποίηση του αντικαρκινικού φραγμού της γήρανσης (προκαρκινικό στάδιο) και, εν τέλει, στη διαφυγή από αυτή και στην προαγωγή του καρκίνου