The faithful genome duplication and segregation of the entire genome to progeny cells are the most fundamental elements of the continuity of life. Aberrant events promote genomic instability, a characteristic of cancer development. One well-studied example for regulating key mitotic events is a set of proteins called the chromosomal passenger complex (CPC), which includes the Aurora B kinase, Survivin, Borealin and INCENP. All CPC members are overexpressed in a multitude of cancers. They regulate key mitotic events, including correction of chromosome-microtubule attachment errors, activation of the spindle assembly checkpoint and construction and regulation of the contractile apparatus that drives cytokinesis. Besides its nuclear function during mitosis, a cytoplasmic fraction of Survivin, without CPC association, exhibits anti-apoptotic functions as a member of the inhibitor of apoptosis protein (IAP) family. In addition, recent studies have also shown an involvement of the CPC in the DNA damage response after irradiation. Overexpression of at least one CPC member is associated with high rates of tumor recurrence, abbreviated patient survival and resistance to chemo- and radiotherapy. Besides its function as a molecular marker for malignancies, this designates the CPC as a potential target for cancer therapy. In contrast to the localization and according functions of the CPC during mitosis, its function during interphase is still unknown. It is important to understand the underlying mechanism to improve cancer therapies. Thus, the present project aims to dissect the functional role of the CPC during interphase, especially during replication and after damage response.

 

Survivin was already expressed in early S phase and formed nuclear foci. Some of these foci were in close proximity or co-localized with PCNA, especially during late S phase. Similarly, Aurora B also accumulated in nuclear foci during S phase, co-localizing with PCNA. In order to track the formation of CPC foci, time-lapse studies were performed but need to be improved to obtain further evidence regarding the spatial and temporal regulation of CPC foci formation throughout the cell cycle. Since the CPC co-localized with PCNA especially during late S phase, when the constitutive heterochromatin is replicated, the localization referring to heterochromatin protein 1 (HP1) was investigated in more detail. Both overexpressed and endogenous CPC proteins co-localized with heterochromatin in interphase cells. The CPC co-localized at replication sites defined by PCNA not only with HP1, but also to centromeric chromatin defined by a staining with a specific centromere antiserum (CREST).

 

In order to address the question if the CPC is bound to the replisome via PCNA or to heterochromatin, an iPOND assay was performed. While Survivin seems to be bound to the newly synthesized DNA strand in the vicinity of the replisome, it is still unknown whether this link is provided through an interaction with chromatin or with replication proteins, respectively. PLA analysis demonstrated a close proximity of all CPC members to PCNA, indicative for an interaction. Indeed, also in co-IP experiments Aurora B-HA, Borealin-HA and myc-INCENP were detected in immunoprecipitates of PCNA. A conserved PCNA-interacting protein (PIP)-box motif was found in the human protein INCENP and comfirmed in PLA and co-IP experiments either by blocking the interaction via inhibitor treatment or by mutation of the PIP box. A disruption of the interaction by mutating the respective binding site did not result in localization changes of INCENP, thus indicating that the PIP box motif in INCENP is necessary for interaction with PCNA but does not play a role in foci formation. Since the CPC proteins co-localized to the heterochromatin marker HP1 co-IPs were conducted and revealed a potential binding of HP1 and Borealin, but still need to be confirmed by further studies.

 

To evaluate whether the CPC could be functionally implicated in processes during replication, flow cytometry was performed and has demonstrated that cells in G2/M phase increased dramatically while the amount of S and G1 phase cells decreased upon depletion of Survivin. However, in this case the mitotic and a potential replicative dysfunction can not be unambiguously distinguished from one another. Further functional analysis of replication fork velocities via fiber assay analysis revealed a reduced replication fork speed in Survivin-depleted cells but not after Aurora B inhibition.

 

Since Survivin depletion decelerated the replication fork, thus causing replication stress, the question arises whether induced replication stress reversely affects the CPC. After induction of replication stress by different chemicals and irradiation, all CPC proteins showed similar expression levels in all samples of whole cell lysates, indicating that the total protein quantity remains the same. Aurora B and Survivin were slightly increased only in the cytoplasmic fraction after CPT and APH treatment as well as after irradiation, which still needs to be elucidated in more detail. Both soluble nuclear and chromatin bound fractions showed similar levels independently of the type of treatment in contrast to previous results after irradiation. Likewise, quantitative analysis of immunostaining revealed only slight or no alterations in nuclear protein expression of Survivin and Aurora B after induction of replication stress. In addition to replication fork stalling, replication stress can also lead to fork collapse. In this case, replication stress leads to functional uncoupling of the helicase complex from the replicative polymerase resulting in dissociation of elongating factors from the replication fork. While PCNA dissociated from forks, CPC proteins still showed an accumulation after fork collapse. This leads to the assumption that even if the CPC can bind to PCNA, this interaction seems not to be sufficient for targeting the CPC to replication sites. Furthermore, recent studies have shown that in cases of replication stress without checkpoint activation, DNA synthesis takes place even during mitosis. However, INCENP was not detected on newly synthesized DNA in mitotic cells, indicating that CPC members conduct their mitotic function without a relocation to replication sites.

 

Taken together, these results indicate a novel role of the CPC in DNA replication or replicationassociated processes as well as after damage response. The CPC accumulates in nuclear foci, especially during late S phase, and these proteins interact via a PIP box motif in INCENP with PCNA. In addition, these foci are also located at heterochromatin, more precisely at centromeres. Furthermore, Survivin-depletion affected replication fork velocities thus causing replication stress. Overall, these results emphasize the concept of the multifunctional nature of the CPC. This kind of multitasking accomplished by a single protein complex could be on the one hand much more economical for a cell than producing many different proteins or protein complexes to regulate different tasks during the cell cycle. On the other hand, this vast array of tasks in maintaining genomic stability also support the CPC’s importance for cancer therapy as treatment against one single protein complex can multiply its effect by targeting a multitude of cellular processes.