Opposing biological functions of the cytoplasm and nucleus DAXX modified by SUMO-2/3 in gastric cancer

Abstract

Death domain-associated protein (DAXX) is a complex biological multifunctional protein and is involved in the tumorigenesis and progression of multiple cancers. The accumulation of DAXX in the nucleus is a common phenomenon in tumor cells. However, altering the subcellular localizations of DAXX results in different biological functions, and we also found that its nuclear/cytoplasmic ratio (NCR) was associated with poor prognosis in gastric cancer (GC). In this study, we investigated the effect of cytoplasmic and nuclear DAXX (cDAXX and nDAXX) in GC and the underlying mechanisms. Immunohistochemical detection performed in 323 GC tissues reveled that cDAXX was associated with a better survival, while high nDAXX expression suggested a poorer prognosis outcome. Upregulation of DAXX in the cytoplasm inhibited cell proliferation and promoted apoptosis, whereas downregulation of DAXX in the nucleus displayed opposite effects. Moreover, Transwell assays revealed that DAXX enhanced GC cell migration and invasion. Analysis from the Gene Expression Profile Interactive Analysis (GEPIA) database showed that the expression of DAXX was significantly associated with SUMO-2/3 in GC tissues. Co-immunoprecipitation combined with immunofluorescence analysis indicated that DAXX interacted directly with SUMO-2/3. Subsequently, down-regulating the expression of SUMO-2/3 resulted in altered subcellular localization of DAXX. Bioinformatics analysis showed that RanBP2 may act as SUMO E3 ligase to promote nuclear-plasma transport via combining with RanGAP1. Taken together, our results indicated that DAXX plays opposing roles in GC and suggest a new model whereby cDAXX, nDAXX, and SUMO-2/3 form a molecular network that regulates the subcellular localization of DAXX and thereby modulates its opposing biological effects. Thus, our findings provide a foundation for future studies of DAXX as a novel therapeutic target for patients with GC.

NSUN2 modified by SUMO-2/3 promotes gastric cancer progression and regulates mRNA m5C methylation

“…The expression level of NSUN2 in tumor and normal tissues was determined using IHC on a tissue microarray (TMA), constructed as previously described [ 20 ]. Briefly, sections were incubated with anti-NSUN2 (1:800 dilution, 20854-1-AP, Proteintech, Wuhan, China), followed by incubation with an HRP-conjugated secondary antibody, visualized with DAB (Dako, Cytomation, CA, USA), and counterstained with hematoxylin.…”

The Human Cytomegalovirus US31 Gene Predicts Favorable Survival and Regulates the Tumor Microenvironment in Gastric Cancer

“…Total proteins were harvested, and western blots were performed as described previously (23). Briefly, proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes.…”

“…The tissue microarray (TMA) was constructed and immunohistochemistry (IHC) performed as described previously (23). Briefly, TMA slides were dewaxed in xylene, incubated in a 0.5% hydrogen peroxide bath for 10 min, blocked in sheep serum for 30 min, and then incubated with a US31 polyclonal rabbit antibody (diluted 1:500), or CD4, CD8, CD66b, and CD163 polyclonal rabbit antibodies (diluted 1:1000) at 24°C for 2 h. After incubation with secondary antibodies at 24°C, and staining with 3,3′-diaminobenzidine (Dako) and counterstaining with hematoxylin, the TMA slides were analyzed using an Easyscan6 digital slice scanner (MOTIC Medical Diagnostic Systems).…”

“…Transwell migration and invasion assays were performed as described previously (23). Briefly, transfected cells were collected and counted.…”

Three-Dimensional Ex Vivo Culture for Drug Responses of Patient-Derived Gastric Cancer Tissue

“…Cell type and distribution were determined by H&E staining and immunohistochemical staining. The specific experimental steps refer to the previous literature of our team ( 28 ). Primary and secondary antibodies are listed in Table S2 .…”