Yuanzhong Wu

HSSB1 binds and protects p21 from ubiquitin-mediated degradation and positively correlates with p21 in human hepatocellular carcinomas

Downregulation of hSSB1, a single-stranded DNA-binding protein, causes increased radiosensitivity, defective checkpoint activation and genomic instability. However, the mechanisms of hSSB1 function in these responses remain to be uncovered. Here, we present evidence that hSSB1 directly binds p21 and this interaction may prevent p21 from ubiquitin-mediated degradation. Furthermore, both promotion of the G1/S transition and abrogation of the G2/M checkpoints induced by hSSB1 knockdown are partially dependent on p21. Most importantly, hSSB1 and p21 levels are positively correlated in human hepatocellular carcinomas (HCC), as determined by immunostaining. Therefore, hSSB1 may positively modulate p21 to regulate cell cycle progression and DNA damage response, implicating hSSB1 as a novel, promising therapeutic target for cancers such as HCC.

hSSB1 regulates both the stability and the transcriptional activity of p53

The tumor suppressor p53 is essential for several cellular processes that are involved in the response to diverse genotoxic stress, including cell cycle arrest, DNA repair, apoptosis and senescence. Studies of the regulation of p53 have mostly focused on its stability and transactivation; however, new regulatory molecules for p53 have also been frequently identified. Here, we report that human ssDNA binding protein SSB1 (hSSB1), a novel DNA damage-associated protein, can interact with p53 and protect p53 from ubiquitin-mediated degradation. Furthermore, hSSB1 also associates with the acetyltransferase p300 and is required for efficient transcriptional activation of the p53 target gene p21 by affecting the acetylation of p53 at lysine382. Functionally, the hSSB1 knockdown-induced abrogation of the G2/M checkpoint is partially dependent on p53 or p300. Collectively, our results indicate that hSSB1 may regulate DNA damage checkpoints by positively modulating p53 and its downstream target p21.

Damaged DNA-binding Protein 1 (DDB1) Interacts with Cdh1 and Modulates the Function of APC/CCdh1

APC/CCdh1 plays a key role in mitotic exit and has essential targets in the G1 phase; however, these mechanisms are poorly understood. In this report, we provide evidence that damaged DNA-binding protein 1 (DDB1) is capable of binding the WD40 domains of Cdh1, but not of Cdc20, through its BPA and BPC domains. Moreover, cells lacking DDB1 exhibit markedly elevated levels of the protein substrates of APC/CCdh1. Depletion of DDB1 in mitotic cells significantly delays mitotic exit, which demonstrates that the interaction between DDB1 and Cdh1 plays a critical role in regulating APC/CCdh1 activity. However, cells depleted of Cdh1 demonstrated no change in the UV-induced degradation of Cdt1, the main function of DDB1 as an E3 ligase. Strikingly, the APC/CCdh1 substrate levels are normal in cell knockdowns of Cul4A and Cul4B, which, along with DDB1, form an E3 ligase complex. This finding indicates that DDB1 modulates the function of APC/CCdh1 in a manner independent on the Cul4-DDB1 complex. Our results suggest that DDB1 may functionally regulate mitotic exit by modulating APC/CCdh1 activity. This study reveals that there may be cross-talk among DDB1, Cdh1, and Skp2 in the control of cell cycle division.

KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway

Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a spheroid formation assay and a xenograft tumor model. Mechanistically, KCTD12 suppresses CRC cell stemness markers, such as CD44, CD133 and CD29, by inhibiting the ERK pathway, as the ERK1/2 inhibitor U0126 abolishes the increase in expression of CRC cell stemness markers induced by the down-regulation of KCTD12. Indeed, a decreased level of KCTD12 is detected in CRC tissues compared with their adjacent normal tissues and is an independent prognostic factor for poor overall and disease free survival in patients with CRC (p = 0.007). Taken together, this report reveals that KCTD12 is a novel regulator of CRC cell stemness and may serve as a novel prognostic marker and therapeutic target for patients with CRC.

CBX4 Suppresses Metastasis via Recruitment of HDAC3 to the Runx2 Promoter in Colorectal Carcinoma

Polycomb chromobox (CBX) proteins participate in the polycomb repressive complex (PRC1) that mediates epigenetic gene silencing and endows PRC1 with distinct oncogenic or tumor suppressor functions in a cell-type-dependent manner. In this study, we report that inhibition of cell migration, invasion, and metastasis in colorectal carcinoma requires CBX4-mediated repression of Runx2, a key transcription factor that promotes colorectal carcinoma metastasis. CBX4 inversely correlated with Runx2 expression in colorectal carcinoma tissues, and the combination of high CBX4 expression and low Runx2 expression significantly correlated with overall survival, more so than either CBX4 or Runx2 expression alone. Mechanistically, CBX4 maintained recruited histone deacetylase 3 (HDAC3) to the Runx2 promoter, which maintained a deacetylated histone H3K27 state to suppress Runx2 expression. This function of CBX4 was dependent on its interaction with HDAC3, but not on its SUMO E3 ligase, its chromodomain, or the PRC1 complex. Disrupting the CBX4-HDAC3 interaction abolished Runx2 inhibition as well as the inhibition of cell migration and invasion. Collectively, our data show that CBX4 may act as a tumor suppressor in colorectal carcinoma, and strategies that stabilize the interaction of CBX4 with HDAC3 may benefit the colorectal carcinoma patients with metastases. Cancer Res; 76(24); 7277-89. ©2016 AACR.

Acetylation-dependent function of human single-stranded DNA binding protein 1

Human single-stranded DNA binding protein 1 (hSSB1) plays a critical role in responding to DNA damage and maintaining genome stability. However, the regulation of hSSB1 remains poorly studied. Here, we determined that hSSB1 acetylation at K94 mediated by the acetyltransferase p300 and the deacetylases SIRT4 and HDAC10 impaired its ubiquitin-mediated degradation by proteasomes. Moreover, we demonstrated that the hSSB1-K94R mutant had reduced cell survival in response to DNA damage by radiation or chemotherapy drugs. Furthermore, the p300/CBP inhibitor C646 significantly enhanced the sensitivity of cancer cells to chemotherapy drugs, and a positive correlation between hSSB1 and p300 level was observed in clinical colorectal cancer samples. Acetylation, a novel regulatory modification of hSSB1, is crucial for its function under both physiological and pathological conditions. This finding supports the notion that the combination of chemotherapy drugs with acetylation inhibitors may benefit cancer patients.

A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1DCAF8 was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.

Human KIAA1018/FAN1 nuclease is a new mitotic substrate of APC/CCdh1

A recently identified protein, FAN1 (FANCD2-associated nuclease 1, previously known as KIAA1018), is a novel nuclease associated with monoubiquitinated FANCD2 that is required for cellular resistance against DNA interstrand crosslinking (ICL) agents. The mechanisms of FAN1 regulation have not yet been explored. Here, we provide evidence that FAN1 is degraded during mitotic exit, suggesting that FAN1 may be a mitotic substrate of the anaphase-promoting cyclosome complex (APC/C). Indeed, Cdh1, but not Cdc20, was capable of regulating the protein level of FAN1 through the KEN box and the D-box. Moreover, the up- and down-regulation of FAN1 affected the progression to mitotic exit. Collectively, these data suggest that FAN1 may be a new mitotic substrate of APC/CCdh1 that plays a key role during mitotic exit.

CBX8 suppresses tumor metastasis via repressing snail in esophageal squamous cell carcinoma

The poor clinical outcome and prognosis of esophageal squamous cell carcinoma (ESCC) is mainly attributed to its highly invasive and metastatic nature, making it urgent to further elicit the molecular mechanisms of the metastasis of ESCC. The function of each polycomb chromobox (CBX) protein in cancer is cell-type-dependent. Although CBX8 has been reported to promote the esophageal squamous cell carcinoma (ESCC) tumorigenesis, its role in ESCC metastasis has not been explored yet. In this study, we report that the inhibition of cell migration, invasion, and metastasis in ESCC requires CBX8-mediated repression of Snail, a key transcription factor that induces epithelial-to-mesenchymal transition (EMT), and that CBX8 inversely correlated with Snail in the ESCC tissues. Moreover, this novel function of CBX8 is dependent on its binding with the Snail promoter, which in turn suppresses the transcription of Snail. Collectively, CBX8 may play paradoxical roles in ESCC, inhibiting metastasis while promoting cell proliferation.

Twist promotes tumor metastasis in basal-like breast cancer by transcriptionally upregulating ROR1

Rationale: Twist is a key transcription factor for induction of epithelial-mesenchymal transition (EMT), which promotes cell migration, invasion, and cancer metastasis, confers cancer cells with stem cell-like characteristics, and provides therapeutic resistance. However, the functional roles and targeted genes of Twist in EMT and cancer progression remain elusive. Methods: The potential targeted genes of Twist were identified from the global transcriptomes of T47D/Twist cells by microarray analysis. EMT phenotype was detected by western blotting and immunofluorescence of marker proteins. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional induction of ROR1 by Twist. A lung metastasis model was used to study the pro-metastatic role of Twist and ROR1 by injecting MDA-MB-231 cells into tail vein of nude mice. Bio-informatics analysis was utilized to measure the metastasis-free survival of breast cancer patients. Results: Twist protein was proved to directly activate the transcription of ROR1 gene, a receptor of Wnt5a in non-canonical WNT signaling pathway. Silencing of ROR1 inhibited EMT process, cell migration, invasion, and cancer metastasis of basal-like breast cancer (BLBC) cells. Knockdown of ROR1 also ameliorated the pro-metastatic effect of Twist. Furthermore, analyses of clinical specimens indicated that high expression of both ROR1 and Twist tightly correlates with poor metastasis-free survival of breast cancer patients. Conclusion: ROR1 is a targeted gene of Twist. Twist/ROR1 signaling is critical for invasion and metastasis of BLBC cells.