Caspase-2 is most well-known for initiating the intrinsic apoptosis pathway [1] and as a tumor suppressor [2]. Despite its pro-apoptotic role, caspase-2-deficient tumors have comparable basal apoptosis levels to wild-type tumors [3, 4]. This suggests a non-apoptotic role for caspase-2 could drive its tumor suppression function. It has long been noted that caspase-2-deficient cells proliferate faster [2], and elegant studies have shown that caspase-2 induces cell cycle arrest following cytokinesis failure [5]. However, whether caspase-2 plays an active role in regulating the cell cycle has up until now been unclear. In our paper published in Oncogene [6], we show that caspase-2 plays an essential role in regulating normal cell division by protecting DNA replication forks from replication stress. 

During normal cell division, we show that caspase-2 is activated in G1 in dividing cells [6]. These cells do not undergo apoptosis, and the primary outcome of caspase-2 activation is continued, cell division. This indicates a role distinct from caspase-2’s reported role in inducing apoptosis to remove damaged or aneuploid cells [7][8]. The inhibitor of the intrinsic apoptotic pathway, Bcl-X_L, potently inhibits caspase-2-induced apoptosis [9]; therefore, we overexpressed Bcl-X_L to distinguish caspase-2’s role in cell division and DNA protection from its role in apoptosis. If caspase-2 regulates cell division through the same pathway, it would be expected to phenocopy the cell cycle results of caspase-2 null cells. In contrast, while caspase-2-deficiency led to an increased proportion of S-phase cells and a progressive decrease in the proportion of G1-phase cells following treatment with arresting concentrations of the DNA damaging agent camptothecin and increased DNA damage following S-phase arrest, overexpression of Bcl-X_L did not change the cell cycle profiles or the amount of DNA damage. While other groups have demonstrated non-apoptotic roles for caspase-2 in DNA repair, cell cycle arrest [2], this is the first demonstration that these roles are regulated by a distinct pathway to apoptosis.

If apoptosis is not an essential mechanism for its tumor suppression function, then a role in regulating the cell cycle may be. We show that caspase-2 protects cells from a range of S-phase-related defects, including delayed exit from S-phase, increased stalled replication forks, increased new replication origins, and decreased repair of S-phase-associated chromosomal aberrations [6]. All of these defects are associated with impaired activation of the ATR/CHK1 pathway. However, in the absence of caspase-2, we observed some impairment of ATR autophosphorylation, but we did not detect any differences in CHK1 activation. Similarly, p53 activity was minimally impacted by the absence of caspase-2. In response to cytokinesis failure, caspase-2-mediated cleavage of MDM2 stabilizes p53, leading to p21-induced G1 arrest [5]. However, we did not observe any MDM2 cleavage or caspase-2-dependent p21 expression following exposure to DNA damage or replication stress. Thus, caspase-2 activation during replication stress engages a pathway distinct from that engaged by cytokinesis failure. This may suggest that caspase-2 activates a non-canonical replication checkpoint to facilitate DNA repair. Consistent with this, we noted a caspase-2-dependent slowdown of the cell cycle following mild replication stress. This is likely to allow for DNA repair.

How exactly caspase-2 contributes to DNA repair is unclear. Caspase-2-deficient cells show a significantly higher percentage of S-phase-associated chromosomal aberrations. This suggests that caspase-2 plays a role in ensuring correct homologous recombination.  Consistent with our evidence that caspase-2 regulates a replication checkpoint, the chromosomal aberrations detected in caspase-2-deficient cells are not repaired before metaphase. Homologous recombination is the main mechanism to repair DNA lesions that block replication forks and fill ssDNA gaps left behind the fork [11]. Caspase-2 may play a role in this process, leading to restart of stalled replication forks and suppression of new origins of replication. It will be interesting to examine the homologous recombination machinery to see if caspase-2 plays a direct or indirect role in the repair process. 

Caspase-2 is activated by proximity-induced activation upon recruitment to its activation platform. The known activation platform for caspase-2 is the PIDDosome, comprising of  p53-induced protein with a death domain (PIDD) and RIP-associated ICH-2/CAD-3 homologous protein with a death domain (RAIDD), but studies show that PIDD-independent caspase-2 activation complexes exist [2].  In contrast to our data, PIDD silencing in UV-challenged cells reduced stalled replication forks [12]. PIDD^-/- and RAIDD^-/- mice do not phenocopy Casp2^-/- mice with respect to accelerated Eμ-Myc lymphomagenesis. In fact, PIDD-deficiency actually delays the onset of disease [13, 14]. These data call into question the necessity of PIDD for the tumor suppression function of caspase-2, and caspase-2’s functions in protecting from replication stress may be PIDD-independent. We used a bimolecular fluorescence complementation (BiFC)-based probe for caspase-2 activation that measures proximity-induced dimerization [9]. This allowed us to distinctly visualize when and where caspase-2 was activated relative to the cell cycle. Caspase-2 activation occurred shortly after mitosis, and when we applied mild replication stress, the caspase-2 reporter lit up earlier than in untreated cells. This effect dissipated when we knocked out caspase-2, indicating that endogenous caspase-2 is required to assemble the platform. This kinetic change could mean a change in the activation platform for caspase-2 or even that caspase-2 can form an alternative complex independent of other factors. A similar complex called the FADDosome has been reported where caspase-8 forms a scaffold for recruitment of FADD and RIPK1 in a manner that does not require its catalytic activity [15]. Non-catalytic functions have been reported for caspase-2 in modulating p21 expression [16] and activation of NFκB [17], so it is tempting to speculate that caspase-2 could work in a similar fashion to protect from replication stress. Investigation into both the necessity of catalytic activity and the required components of the activation platform is warranted to establish the mechanisms of caspase-2 activation in the DNA damage response.

Altogether, our data show that caspase-2 has an integral role in cell division and DNA repair, separate from its role in apoptosis. With the necessity of the pro-apoptotic pathway of caspase-2 in tumor suppression called into question [3, 4], further investigation into the non-apoptotic mechanisms, activation platforms, and targets of caspase-2 is essential to determine if this is a druggable pathway that will benefit future cancer research.

References

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