5. Discussion
5.4 CHD8-dependent transcription of DNA damage response factors
In the literature, CHD8 is characterized mainly as to its regulation of transcription by various mechanisms (see also 1.8). We therefore grouped the results from microarray analysis
according to cellular functions (Table 4.2). Of those, some candidates appeared promising to us because they are already mechanistically involved in the DDR.
The downregulation of USP11 (Ubiquitin-specific protease 11) following CHD8 knockdown has already been found before (Rodriguez-Paredes et al. 2009) and could be detected and confirmed via qPCR and on a protein basis in our lab (data not shown). USP11 is an established DDR factor acting in the BRCA2-dependent HR pathway. While patients with germline BRCA2 mutations have a high risk to develop pancreatic and other tumors, sporadic BRCA2 mutations in pancreatic cancer samples are comparatively rare.
Pancreatic cancer cells with an intact HR pathway are susceptible to mitoxantrone treatment that is, among its conventional DNA damaging properties, able to potently inhibit USP11 (Burkhart et al. 2013). However, while this study found mitoxantrone being much more effective to reduce pancreatic cancer cell proliferation than is gemcitabine, it did not find a significant cytoreductive effect with RNAi-mediated USP11 depletion to 60 % in untreated cells. Possibly, mitoxantrone exhibits its cytoreductive effect through other mechanisms than via USP11 inhibition. Nonetheless, the RNAi-mediated reduction of cellular USP11 levels did have a sensitizing effect for gemcitabine treatment, making it plausible that the enhanced DDR after CHD8 depletion is a secondary effect mediated by the suppression of USP11. Still, the knockdown of CHD8 in our experiments reduces USP11 levels more than does the knockdown of USP11 itself in the above-mentioned publication, which is to 31 % instead of 60 %. Another study showed that USP11 binds to BRCA2 to ubiquitinate it, and it sensitized cells to mitomycin C treatment only in a BRCA2 wild type condition. However, cellular levels of BRCA 2 were not lowered after USP11 depletion, so that it the question remains whether USP11 specifically promotes HR or whether BRCA2 defective cells exhibit high chemoresistance in general (Schoenfeld et al.
2004). Of note, USP11 stabilizes p53 by reducing its ubiquitination, leading to a pro-apoptotic response of a cell (Ke et al. 2014). In the same way it stabilizes p21 to promote cell cycle arrest and senescence (Deng et al. 2018). Besides the influence on protein turnover, USP11 promotes the transcription of IKKα in response to NF-κB signaling and its depletion leads to reduced p53 expression in p53 wild type conditions (Yamaguchi et al.
2007).
Although we cannot detect a p53-stabilizing effect following CHD8 knockdown, the known features of USP11 depletion would well explain at least some of our observations like cell cycle progression and reduced p53 levels in CHD8-depleted cells. Still, it is not known whether USP11 has any effect on the transcription of mutant p53.
With its DNA repair and p53/p21-promoting properties, USP11 would rather act as a tumor suppressor and its knockdown should lead to an increase in DDR through impaired HR, but
also to apoptosis resistance through reduced p53 levels and cell survival after DNA damage.
The downregulation of the scaffolding protein KSR2 (kinase-suppressor of Ras 2) connects CHD8 to cellular stress and proliferation signaling cascades. KSR2 positively regulates Ras signaling, which is a signaling cascade connecting receptor tyrosine kinase signaling to DNA damage and transcriptional responses of a cell. The most common tumor driving mutation of pancreatic cancer, an activating KRAS mutation, leads to a constant induction of this kinase cascade. While oncogenic KRAS activations cannot be targeted yet, the inhibition of downstream kinases such as ERKs induces DNA damage signaling and apoptosis (Kirkpatrick et al. 2013) as well as p53 phosphorylation and stabilization (Melnikova et al. 2003). KSR2 positively regulates receptor tyrosine kinase-mediated ERK signaling and might thus have tumor-promoting functions, but the KSR2-dependent growth induction of several tumor cell lines is independent of upstream kinase signaling (Fernandez et al. 2012). Based on the above-mentioned, the decrease in KSR2 levels could also be a mediator of the effects of CHD8 knockdown on DNA damage signaling and apoptosis.
When we focused on genes whose transcription is induced upon CHD8 knockdown, two DNA repair factors are noticeable: First, the 1.5-fold induction of the histone acetylase KAT5/TIP60 links CHD8 to the repair of DSBs, too. Following the induction of DSBs, the acetylation of H4 by the histone acetyl transferase TIP60 is required for efficient recruitment of repair proteins like ATM, which itself is acetylated by KAT5 to increase its activity (Murr et al. 2006; Sun et al. 2005). TIP60 further acetylates γH2AX as a prerequisite for H2AX ubiquitination and removal from DNA after damage is repaired (Ikura et al. 2007). If CHD8 acted via KAT5, CHD8-deficient tumor cells would hyper-activate ATM, DNA repair and, if not defective for it, apoptosis.
Second, with MDC1, another DNA repair protein is induced after CHD8 knockdown. MDC1 is a large scaffolding protein which has a central role in the activation of ATM and therefore the spreading of γH2AX away from the actual DNA lesion to promote not only DDR, but also apoptosis (see section 1.3.3). Indeed, high expression levels of MDC1, although a repair protein, are associated with chemoresistance in malignant cells. The cause seems to be a general activation of the DNA repair machinery in response to an amplification of DNA damage signaling after drug-induced damage, leading to high levels of γH2AX on the one hand, but fast DNA repair and the resumption of cell cycle progression and tumor proliferation on the other hand. MDC1 recruits the ubiquitin ligases RNF8 and RNF168 to chromatin, where they ubiquitinate histone H2A as a prerequisite for BRCA1-dependent repair of DSBs (Luijsterburg and van Attikum 2011). MDC1 is increased to 1.6-fold after CHD8 depletion, so to a much lower extent as usually seen for transcriptional responses in
signaling cascades. Still, as MDC1 acts far upstream of the DDR and its abundance delicately influences the intensity of DNA damage signaling, already small changes would have a dramatic effect. Interestingly, MDC1 was among the main candidates whose knockdown robustly lowered γH2AX levels in our siRNA screen showing that it promotes H2AX phosphorylation in response to gemcitabine treatment (Fig. 4.12, table 7.1 and 7.2).