Gu et al had previously reported that a soluble peptide

Gu et al. had previously reported that a soluble peptide, R9-caPep, derived from the interdomain loop of the replication clamp PCNA (proliferating cell nuclear antigen) selectively inhibited the proliferation of neuroblastoma Cyanine5.5 alkyne Supplier that harbored MYCN amplifications (Gu et al., 2014). However, the underlying mechanism(s) remained elusive. The present study confirms and significantly extends previous results by the Maris laboratory that demonstrated that neuroblastomas in which MYCN was amplified overexpressed and constitutively activated CHK1 (Cole et al., 2011). Gu et al. re-analyzed microarray data from 472 neuroblastoma tumors and examined a total of 36 new patient-derived specimens to validate the notion that high-level MYCN expression strongly correlates with chronic replication stress. In a separate set of experiments, the authors show convincingly that depletion of MYCN in neuroblastoma cell lines reduces the basal burden of genome instability several-fold. At the same time, chronic replication stress confers sensitivity to the PCNA-derived peptide R9-caPep. The peptide interferes with normal PCNA function likely by sequestering binding partners whose actions PCNA coordinates during DNA replication (Stoimenov and Helleday, 2009). Importantly, the inhibitory effect of R9-caPep is diminished when MYCN expression is downregulated, consistent with the original observation that the peptide is highly effective in neuroblastoma cells in which the MYCN locus is amplified. A key experiment in this new study is a single-molecule fiber analysis that utilizes consecutive labeling of nascent DNA by two different thymidine analogs. When the second label was applied in the presence of the R9-caPep peptide, replication fork progression was significantly reduced. It is highly unlikely that the observed effect is not specific to the peptide sequence, as the authors had previously shown that a scrambled control peptide did not have any inhibitory effect on DNA synthesis nor cell growth (Gu et al., 2014). Importantly, the PCNA-derived peptide synergizes with CHK1 inhibitors to increase genome instability, enhance the activation of so-called backup origins – another hallmark of replication stress – and induce apoptosis in MYCN overexpressing neuroblastoma cells. Whether the cells succumb to RPA exhaustion (Toledo et al., 2013), which has been proposed as a molecular mechanism for replication stress-induced cell death, remains to be explored. In the same vein, it remains to be determined whether MYCN drives cells prematurely into S phase, thereby limiting origin licensing and restricting the number of replication forks that can actively contribute to genome duplication. Alternatively, MYC has recently been described to have an active role in replication initiation that is completely independent of its function in transcription (Srinivasan et al., 2013). It is conceivable that MYCN could play a similar role, leading to an overall dysregulation of origin firing that causes replication stress. Both lines of action are not mutually exclusive and could work in concert. Taken together, the work by Gu et al. suggests that the combination of CHK1 inhibitors and soluble, cell penetrable PCNA-peptides that block replication fork progression could be a promising therapeutic tool to aim at the Achilles heel of MYCN-overexpressing neuroblastomas.
Conflict of Interest
The canonical WNT signaling pathway is ultimately involved in the regulation of cytoplasmic levels of free β-catenin. When inactive, the β-catenin not incorporated in adherent junctions is captured by the adenomatous polyposis coli (APC)-based protein complex, phosphorylated and then processed for degradation by the proteasome. Activation by WNT ligands such as those found in the intestinal stem cell niche located in the lower crypts prevents β-catenin ubiquitination allowing its accumulation in the cytoplasm and shuttling to the nucleus where it associates with the DNA-binding proteins of the lymphoid enhancer-binding factor/T-cell factor (TCF) family to transactivate specific gene expression such as , and other genes that drive cell proliferation and stemness (). Colorectal cancer (CRC) cells frequently display a constitutively active WNT–β-catenin signaling pathway as a consequence of mutations in or other genes that encode the APC-based protein destruction complex or β-catenin itself, which allows β-catenin to accumulate in the nucleus and contribute to cellular transformation (). There are many factors that interact and modulate the WNT cascade in both normal and transformed cells. As depicted in seminal reviews (), WNT signaling is strictly controlled at the ligand–receptor level by a series of inhibitors and activators that regulate signal strength. Furthermore, WNT signaling is also modulated at the transcriptional level by a series of β-catenin-interacting co-factors such as cyclic AMP response element-binding protein and B-cell lymphoma 9 (BCL9 and BCL9L) which can strengthen the activity (). While many of these modulators of the WNT pathway represent potential targets for cancer therapeutics, their disruption can also lead to alterations in the WNT signaling pathway of healthy tissues, a difficulty that has to be taken into consideration in the design of pre-clinical and, eventually, clinical studies ().