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Sick sinus syndrome (SSS) is a term used for a variety of disorders defined by abnormal cardiac impulse formation and by abnormal propagation from the heart’s sinoatrial node. In this study, we present a case from a Chinese family in which two closely related individuals had the symptoms and electrocardiographic evidence of SSS. We hypothesized that multiple individuals affected by the disease in the family was an indication of its genetic predisposition, and thus performed high-throughput sequencing for the participants from the family to detect potential disease-associated variants. One of the potential variants that was identified was a KCNG2 gene variant (NC_000018.9: g.77624068_77624079del). Further bioinformatic analysis showed that the observed variant may be a pathogenic mutation. The results of protein-protein docking and whole cell patch-clamp measurements implied that the deletion variant in KCNG2 could affect its binding the KV2.1 protein, and finally affect the function of Kv channel, which is an important determinant in regulation of heartbeat. Therefore, we inferred that the variable KCNG2 gene may affect the function of Kv channel by changing the binding conformation of KCNG2 and KV2.1 proteins and then adversely affect propagation from the sinoatrial node and cardiac impulse formation by changing the action potential repolarization of heart cells. In summary, our findings suggested that the dominant KCNG2 deletion variant in the examined Chinese family with SSS may be a potential disease-associated variant.

DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G 1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G 1 arrest. The mechanism of UV-induced G 1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C Cdh1 ), a critical ubiquitin ligase in G 1 phase, thereby stabilizing Cyclin A and promoting G 1 –S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C Cdh1 -mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G 1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G 1 –S block. These results establish CUEDC2 as an APC/C Cdh1 inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G 1 arrest.

Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.

Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C-Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results suggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability.

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil-containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras-induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.

In order to capture the mechanism of roadway instability in deep mines, a new approach of Physically Finite Elemental Slab Assemblage (PFESA) is proposed in order to construct a large-scale physical model simulating the geologically horizontal strata. We carried out physical modeling on the deformation and failure processes of roadways subjected to a plane loading scheme. Our laboratory tests were based on work which incorporated infrared (IR) detection, IR radiation temperature (IRT) statistics, image feature extraction and 2D Fourier transformation, from resulting thermographies. The IRT characterizes the mechanical responses from the roadway after loading with two stages, i.e., IRT evolving at higher levels corresponded to shallow mining (fl500 m) during which the roadway deformed gradually (referred to as the “steady deformation stage”); IRT evolving in a quasi-cyclical manner with multiple peaks corresponded to deep mining (800–2600 m), in which the failure mode for the roadway are dominated by breakage and collapse (called the “unsteady deformation stage”). The IR images and 2D Fourier spectra illustrate detailed information in terms of initiation, nucleation and coalescence of the damage to rock masses and the eventual failure of roadways subject to external loading.

Early detection of breast cancer is paramount to successful clinical therapy. Yet, early-stage breast cancer lacks specific symptoms or biomarkers. With the emerging of the mass spectrometric (MS)–based signatures as biomarkers, we investigated breast cancer-related serum profile pattern through class prediction and independent validation, and used Fourier transfer MS to identify breast cancer signature. We now show a distinctive serum peptide pattern that discriminates breast cancer from healthy controls with 93.2% sensitivity and 95.4% specificity. m/z 5901.70 and 4465.74 of ion fragment of FPA and alpha1-antichymotrypsin are found in the signatures that predominantly discriminate breast cancer from healthy individuals. These novel findings identify an MS-based serum peptide pattern of breast cancer that may have direct clinical utility in future.

A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significance of ubiquitination. There are, however, a few effective screening methods for rapid analysis of ubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combined with cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulate to form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consisted of 112 cDNAs encoding key components of major cellular pathways and potential targets of polyubiquitination. Among them, 40 proteins formed accumulation dots in response to proteasome inhibitor, MG-132, treatment. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK, and RhoGDI2, are previously not known as the targets of ubiquitin. We further validated our findings by examining the endogenous counterparts of some of these proteins and found that those endogenous proteins form a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.

SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) that interacted with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localized with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically repressed SOX4 transcriptional activity in 293T cells. We further demonstrated that Ubc9 could functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, did not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity.

The response rules of pressing pain on the back section in the Governor Vessel in patients with gastro-esophageal reflux disease (GERD) were studied to provide references for the diagnosis and treatment of GERD. Seventy-six cases of GERD were included into an observation group while 30 healthy volunteers were recruited into a control group. A mechanical measurement device of pressing pain that could measure the pain threshold was adapted to observe the pressing pain on the back section in the Governor Vessel in GERD patients and healthy volunteers. The test area is from spinous process of the 1st thoracic vertebra to that of the 12th thoracic vertebra (T1 -T12), including acupoints and non-acupoints on the Governor Vessel. As a result, in the observation group the pain threshold of T5-T7 spinous process clearance, which was the location of Shendao (GV 11), Lingtai (GV 10) and Zhiyang (GV 9), was lower than that in the control group (all P0.05). This result indicated that there was significant pressing pain in T5-T7 spinous process clearance in patients with GERD, which could be taken as an important auxiliary diagnosis and a new thinking method in the treatment of GERD with acupuncture.

To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis.4T1-luc cells carrying shGankyrin construct were established by lentivirus infection and antibiotic screening. Western blotting and real-time PCR were used to check the expression levels of Gankyrin. In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice.The cell expression decreased at the protein and mRNA levels. Gankyrin mRNA expression in different shGankyrin 4T1-luc cells was respectively 4.9%, 25.1% and 69.8% versus the control cells. ShGankyrin#2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers. The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4T1-luc cell was 3.02 × 10(6), while that of lung metastasis induced by control cells was 10.9 × 10(6). The differences between two groups were significant. In pathology, Gankyrin was detected positive in lung metastasis tumors induced by control group. However, Gankyrin was negative in the Gankyrin knockdown group.Lentivirus infection may be effectively used to establish Gankyrin knocking down 4T1-luc cell model. Because of its involvement in the in vivo pulmonary metastasis of breast cancers, Gankyrin should be a novel target for tumor therapy.

Hepatic metastasis is responsible for the majority of colorectal cancer (CRC)-related mortalities. Although Gankyrin (PSMD10) has been implicated in cancer metastasis, its exact role and underlying mechanisms of CRC hepatic metastasis remain largely unknown. Herein, we showed that the expression of Gankyrin was higher in primary CRC with hepatic metastasis compared with CRC without metastasis. RNAi-mediated silencing of Gankyrin expression in highly metastatic human CRC cells impaired their migratory and metastatic capacity in vivo. Genome-wide transcriptome profiling revealed activation of the interleukin (IL)-8 signaling pathway by Gankyrin. Protein levels of IL-8 and cyclin D1 (CCND1), the two important molecules in the IL-8 pathway, were positively correlated with Gankyrin expression in human CRC specimens. Furthermore, genetic deletion of cyclin D1 showed its requirement in Gankyrin-mediated cell migration. Finally, administration of recombinant IL-8 rescued the migratory defect of CRC cells where Gankyrin expression was silenced. Together, our findings highlight the importance of Gankyrin in hepatic metastasis of CRC and point out its candidature as a potential prognostic marker and therapeutic target to improve the clinical management of metastatic CRC. Cancer Res; 73(14); 4548–58. ©2013 AACR.

RIG-I/MDA5 plays a pivotal role in innate immunity by detecting intracellular double-stranded RNA (dsRNA) and activating the transcription of type I interferons and proinflammatory factors, but the exactly regulating mechanism of RIG-I/MDA5 signaling remains elusive. In this study, UbL-UBA domain containing protein RAD23A was identified as a negative regulator of RIG-I/MDA5-mediated signaling activation through a small interfering RNA (siRNA)-based screening. Knockdown of RAD23A augmented the expression of RIG-I/MDA5-mediated expression of proinflammatory cytokines and IFN-β whereas ectopic expression of RAD23A showed the converse effect. Moreover, we confirmed the interaction between RAD23A and tumor necrosis factor receptor-associated factor 2 (TRAF2), an essential mediator of RIG-I/MDA5 signaling, and found that RAD23A down-regulated TRAF2 protein level through ubiquitin–proteasome system. Therefore, this study identified RAD23A as a novel negative regulator of RIG-I/MDA5 mediated anti-virus response.

Tumor metastasis is responsible for most cancer patients' deaths. Understanding the mechanism of metastasis is crucial for improving the cure rate for cancer. Here, we report that Gankyrin, a chaperone of ubiquitin-proteasome, has an essential role in breast cancer metastasis. We find that Gankyrin is highly overexpressed in human breast cancers and the expression correlates strongly with lymph node metastasis. Knocking down Gankyrin expression in highly metastatic human breast cancer cells significantly decreases cancer cell migration and invasion. Furthermore, we demonstrate that depletion of Gankyrin inhibits intrinsic Rac1 activity and induces large focal adhesions. Overexpression of Gankyrin accelerates focal adhesion turnover and increases cell migration. Notably, reduction of Gankyrin expression in mouse mammary tumor cell significantly decreases tumor metastasis to lung in animal models. Therefore, our findings suggest that Gankyrin is crucial for breast cancer metastasis and highlight the potential of Gankyrin as a therapeutic target for tumor metastasis.

DNA damage response (DDR) acts as a tumorigenesis barrier, and any defects in the DDR machinery may lead to cancer. SOX4 expression is elevated in many types of tumors; however, its role in DDR is still largely unknown. Here, we show that SOX4, a new DNA damage sensor, is required for the activation of p53 tumor suppressor in response to DNA damage. Notably, SOX4 interacts with and stabilizes p53 protein by blocking Mdm2-mediated p53 ubiquitination and degradation. Furthermore, SOX4 enhances p53 acetylation by interacting with p300/CBP and facilitating p300/CBP/p53 complex formation. In concert with these results, SOX4 promotes cell cycle arrest and apoptosis, and it inhibits tumorigenesis in a p53-dependent manner. Therefore, these findings highlight SOX4 as a potential key factor in regulating DDR-associated cancer.

Despite rapid progress in elucidating the molecular mechanisms of activation of the kinase IKK, the processes that regulate IKK deactivation are still unknown. Here we demonstrate that CUE domain-containing 2 (CUEDC2) interacted with IKKalpha and IKKbeta and repressed activation of the transcription factor NF-kappaB by decreasing phosphorylation and activation of IKK. Notably, CUEDC2 also interacted with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). We found that IKK, CUEDC2 and PP1 existed in a complex and that IKK was released from the complex in response to inflammatory stimuli such as tumor necrosis factor. CUEDC2 deactivated IKK by recruiting PP1 to the complex. Therefore, CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting PP1.

Accumulated evidence indicates that progesterone receptors (PR) are involved in proliferation of breast cancer cells and are implicated in the development of breast cancer. In this paper, a yeast two-hybrid screen for PR led to the identification of CUE domain containing 2 (CUEDC2), whose function is unknown. Our results demonstrate that CUEDC2 interacts with PR and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. The inhibition of endogenous CUEDC2 by siRNA nearly abrogated the progesterone-induced degradation of PR, suggesting that CUEDC2 is involved in progesterone-induced PR ubiquitination and degradation. Moreover, we identify the sumoylation site Lys-388 of PR as the target of CUEDC2-promoted ubiquitination. CUEDC2 decreases the sumoylation while promoting ubiquitination on Lys-388 of PRB. We also show that CUEDC2 represses PR transactivation, inhibits the ability of PR to stimulate rapid MAPK activity, and impairs the effect of progesterone on breast cancer cell growth. Therefore, our results identify a key post-translational mechanism that controls PR protein levels and for the first time provide an important insight into the function of CUEDC2 in breast cancer proliferation.

We implemented a proteomics approach to the systematical analysis of the alterations in the proteome of NIH3T3 cells transformed by oncogenic H-RasV12. Forty-four proteins associated with Ras-mediated transformation have been identified, and 28 proteins were not previously reported. RT-PCR analysis showed that approximately 44% of target proteins identified showed concomitant changes in mRNA abundance. A principal finding was the up-regulation of gankyrin, which was the first evidence to show that gankyrin pathway was implicated in Ras-activated transformation.

Progesterone receptor (PR) plays a critical role in cell proliferation and differentiation, and its transcriptional activity is known to be modulated by cofactor proteins. In the present study, we demonstrated that in the presence of progesterone, protein inhibitor of activated STAT-3 (PIAS3) significantly inhibited the PR transcriptional activity and the expression of progesterone-responsive genes. Reduction of endogenous PIAS3 by PIAS3 small-interfering RNA enhanced PR transactivation in a ligand-dependent manner. PIAS3 interacted with PR both in vitro and in vivo and the interaction was enhanced by progesterone. Furthermore, our findings suggested that PIAS3 strongly induced PRB sumoylation at three sites, Lys-7, Lys-388 and Lys-531. In addition, novel roles in PRB nuclear retention and transactivation were identified for these sites. Our data also suggested that PIAS3 was recruited in a largely hormone-dependent manner in response to a progesterone-responsive promoter. Finally, we demonstrated that PIAS3 inhibited the DNA-binding activity of PR and influenced its nuclear export as well as PR transactivation. Taken together, these data strongly suggested that PIAS3 played an important physiological role in PR function.

Recent reports have shown that MDM2 may attenuate hypertrophy of cardiac myocytes. However, mechanism of MDM2 involving in this process is unclear. In this study, we identified a novel specific MDM2-binding protein TCAP by the yeast two-hybrid screen. It was validated by GST pull-down and co-immunoprecipitation assays. Confocal analysis showed that MDM2 and TCAP co-localized in the nucleus, and elevated MDM2 expression could alter the subcellular localization of TCAP. Notably, MDM2 downregulated the protein level of TCAP through the proteasomal pathway, and this downregulation was inhibited by p14ARF. In addition, our results suggested that the degradation of TCAP by MDM2 was through the ubiquitin-independent pathway. Given that TCAP is a key component involving in the cardiac hypertrophy, the degradation of TCAP by MDM2 might be connected with the roles of MDM2 in cardiac hypertrophy. Further investigation will focus on the biological significance of MDM2–TCAP interaction in cardiac hypertrophy.

Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy.

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1–6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.