The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G₂/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G₂/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit; Plk1, polo-like kinase 1; PBD, polo box domain     

Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling

The plasma membrane‐associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up‐regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/β‐catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem‐like characteristics. NEU3 silencing in HT‐29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down‐regulation of stemness and Wnt‐related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently β‐catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3β and Axin, whereas its silencing exerted the opposite effects. NEU3 activity‐null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere‐forming conditions, when stemness genes are up‐regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere‐formation and in vivo tumor incidence in NOD‐SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem‐like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.     

Expression of DDX27 contributes to colony-forming ability of gastric cancer cells and correlates with poor prognosis in gastric cancer

Previously, we have reported that gain at chromosome 20q13 is the most common genomic copy number aberration in gastric cancer (GC) (29/30 cases), and that among the genes located in this region, we have identified DDX27, whose expression level shows the highest correlation with genomic copy number, as a candidate therapeutic target for GC. Here, we analyzed the clinicopathological significance of DDX27 using immunohistochemistry and studied its functions using knockdown assays. We found that DDX27 was frequently upregulated in GC tissues (98 of 140 cases, 70%), and significantly associated with venous invasion and liver metastasis. Furthermore, multivariate analysis of GC patients showed that high expression of DDX27 was independently associated with poorer prognosis. In functional assays, knockdown of DDX27 reduced the ability of GC cells to form colonies both on conventional plates and soft agar, but had little effect on their invasiveness. We also found that knockdown of DDX27 reduced the viability of GC cells through inhibition of cell cycle progression independently of apoptosis. Interestingly, DDX27 depletion induced accumulation of TP53 in a TP53 wild-type cell line, AGS, but not in a TP53-deleted cell line, 44As3, although DDX27 knockdown commonly reduced the viability of both, indicating the TP53-dependent and independent cell cycle control of DDX27. Thus, our results suggest that expression of DDX27 contributes to colony formation by GC cells through cell cycle control and may be a potential therapeutic target for GC patients with chromosome gain at 20q13.     

Predictive validity of weekly monitoring of wound status using DESIGN‐R score change for pressure ulcer healing: A multicenter prospective cohort study

There are few studies on predictive validity of methods to monitor the healing process of pressure ulcers. We evaluated whether the change of DESIGN‐R (rating) score could predict subsequent healing, and determined the optimal cutoff points. In a multicenter prospective cohort study, patients were followed until wound healing or censoring. Wound severity was evaluated by the DESIGN‐R tool every week, and the score change was calculated over 1–4 weeks (n = 411, 286, 224, and 170, respectively). In the multivariate analyses stratified by depth, a one‐point improvement in DESIGN‐R score over any period was positively associated with healing within the next 30 days independent of initial wound severity (hazard ratios over each 1–4 weeks ranging from 1.16 to 1.33 for superficial ulcers and from 1.21 to 1.27 for deep ulcers; all p < 0.05). The optimal cutoff points over 1–4 weeks were set as negative change for superficial ulcers and as positive change of ≥two points for deep ulcers. Nonhealing rate was higher for ulcers with DESIGN‐R score change below the cutoff points than that aforementioned for both depths. Weekly monitoring by the DESIGN‐R tool will be advantageous for evaluating prognosis of pressure ulcers independent of initial wound severity and depth.