In this report, we demonstrate that sodium hydrosulfide (NaHS), a hydrogen sulfide (H(2)S) donor, promoted adventitious root formation mediated by auxin and nitric oxide (NO). Application of the H(2)S donor to seedling cuttings of sweet potato (Ipomoea batatas L.) promoted the number and length of adventitious roots in a dose-dependent manner. It was also verified that H(2)S or HS(-) rather than other sulfur-containing components derived from NaHS could be attributed to the stimulation of adventitious root formation. A rapid increase in endogenous H(2)S, indole acetic acid (IAA) and NO were sequentially observed in shoot tips of sweet potato seedlings treated with HaHS. Further investigation showed that H(2)S-mediated root formation was alleviated by N-1-naphthylphthalamic acid (NPA), an IAA transport inhibitor, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO scavenger. Similar phenomena in H(2)S donor-dependent root organogenesis were observed in both excised willow (Salix matsudana var. tortuosa Vilm) shoots and soybean (Glycine max L.) seedlings. These results indicated that the process of H(2)S-induced adventitious root formation was likely mediated by IAA and NO, and that H(2)S acts upstream of IAA and NO signal transduction pathways.

No abstract

IntroductionAberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.MethodsWe examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.ResultsWe identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.ConclusionsOur study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

No abstract

Dickkopf-related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour-specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial-mesenchymal transition (EMT) and down-regulating stem cell markers. DKK3 inhibited canonical Wnt/β-catenin signalling through mediating β-catenin translocation from nucleus to cytoplasm and membrane, along with reduced active-β-catenin, further activating non-canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.

Rationale: Breast cancer (BrCa) is the most common cancer worldwide, and the 5-year relative survival rate has declined in patients diagnosed at stage IV. Advanced BrCa is considered as incurable, which still lack effective treatment strategies. Identifying and characterizing new tumor suppression genes is important to establish effective prognostic biomarkers or therapeutic targets for late-stage BrCa. Methods: RNA-seq was applied in BrCa tissues and normal breast tissues. Through analyzing differentially expressed genes, DRD2 was selected for further analysis. And expression and promoter methylation status of DRD2 were also determined. DRD2 functions were analyzed by various cell biology assays in vitro . Subcutaneous tumor model was used to explore DRD2 effects in vivo . A co-cultivated system was constructed to investigate interactions of DRD2 and macrophages in vitro . WB, IHC, IF, TUNEL, qRT-PCR, Co-IP, Antibody Array, and Mass Spectrum analysis were further applied to determine the detailed mechanism. Results: In BrCa, DRD2 was found to be downregulated due to promoter methylation. Higher expression of DRD2 positively correlated with longer survival times especially in HER2-positive patients. DRD2 also promoted BrCa cells sensitivity to Paclitaxel. Ectopic expression of DRD2 significantly inhibited BrCa tumorigenesis. DRD2 also induced apoptosis as well as necroptosis in vitro and in vivo . DRD2 restricted NF-κB signaling pathway activation through interacting with β-arrestin2, DDX5 and eEF1A2. Interestingly, DRD2 also regulated microenvironment as it facilitated M1 polarization of macrophages, and triggered GSDME-executed pyroptosis. Conclusion: Collectively, this study novelly manifests the role of DRD2 in suppressing BrCa tumorigenesis, predicting prognosis and treatment response. And this study further reveals the critical role of DRD2 in educating M1 macrophages, restricting NF-κB signaling pathway and triggering different processes of programmed cell death in BrCa. Taking together, those findings represent a predictive and therapeutic target for BrCa.

Our results show that the antitumor effect of DADS on TNBC cells is mediated by the β-catenin pathway, suggesting that DADS could be used as a potential therapeutic agent for treating or preventing breast cancer.

Zinc-finger and BTB/POZ domain-containing family proteins (ZBTB) are important transcription factors functioning as tumor suppressors or oncogenes, such as BCL6/ZBTB27 as a key oncoprotein for anti-cancer therapy. Through epigenome study, we identified ZBTB28/BCL6B/BAZF, a BTB/POZ domain protein highly homologous to BCL6, as a methylated target in multiple tumors. However, the functions and mechanism of ZBTB28 in carcinogenesis remain unclear. Methods: ZBTB28 expression and methylation were examined by reverse-transcription PCR and methylation-specific PCR. The effects and mechanisms of ectopic ZBTB28 expression on tumor cells were assessed with molecular biological and cellular approaches in vitro and in vivo. Results: Albeit broadly expressed in multiple normal tissues, ZBTB28 is frequently downregulated in aero- and digestive carcinoma cell lines and primary tumors, and correlated with its promoter CpG methylation status. Further gain-of-function study showed that ZBTB28 functions as a tumor suppressor inhibiting carcinoma cell growth in vitro and in vivo, through inducing cell cycle arrest and apoptosis of tumor cells. ZBTB28 suppresses cell migration and invasion by reversing EMT and cell stemness. ZBTB28 transactivates TP53 expression, through binding to the p53 promoter in competition with BCL6, while BCL6 itself was also found to be a direct target repressed by ZBTB28. Conclusion: Our results demonstrate that ZBTB28 functions as a tumor suppressor through competing with BCL6 for targeting p53 regulation. This newly identified ZBTB28/BCL6/p53 regulatory axis provides further molecular insight into carcinogenesis mechanisms and has implications in further improving BCL6-based anticancer therapy.