Project description:We report that NEK2 protein level is overexpressed and correlated with the tumor stage and lymph node metastasis in cervical cancer. Furthermore, we provided evidence that NEK2-depleted cervical cancer cells exhibit impaired oncogenesis and enhanced radiosensitivity. Using RNA sequencing, we identify Wnt1 as a key downstream effector of NEK2. Knockdown of NEK2 downregulates the mRNA and protein levels of Wnt1, thereby inhibiting the activation of the Wnt/β-catenin signaling pathway. More importantly, the observed consequences induced by NEK2 depletion in cervical cancer cells can be partially rescued by Wnt1 overexpression. Taken together, these results demonstrate that NEK2 activates the Wnt/β-catenin signaling pathway via Wnt1 to drive oncogenesis and radioresistance in cervical cancer, indicating that NEK2 may be a promising target for the radiosensitization of cervical cancer.
Project description:Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early stage cervical cancer. Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early stage cervical cancer patients was performed for representatives of the identified pathways. Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P<0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3 and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR:0.20, 95%CI:0.06-0.66) and the β-catenin pathway (p120 positivity) to N+ (OR:1.79, 95%CI:1.05-3.05). Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated non-canonical β-catenin pathways are important in pelvic lymph node metastasis in early stage cervical cancer. For the microarray experiment, we selected fresh frozen primary cervical cancer tissue, containing at least 80% tumor cells, of patients with histologically confirmed N0 (n=20) and of patients with N+ (n=19). The N0 and N+ groups were matched for age, FIGO stage and histology (all squamous cell carcinoma).
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin-driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. Here, we combined RNA sequencing with CUT&RUN-LoV-U against β-catenin to assess the correlation between β-catenin recruitment to target loci and its effect on target gene expression. To this end, we performed a bulk RNA sequencing analysis on human embryonic stem cells (hESCs) treated with the the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days, and compared them to untreated hESCs. We then correlated the observed gene expression changes with β-catenin binding events identified from a separate experiment (see “Related Accession Number”). We observed that β-catenin binding is associated with both activation and repression of cell-specific gene expression programs, underscoring how Wnt/b-catenin drives complex cell behaviors.
Project description:Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early stage cervical cancer. Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early stage cervical cancer patients was performed for representatives of the identified pathways. Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P<0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3 and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR:0.20, 95%CI:0.06-0.66) and the β-catenin pathway (p120 positivity) to N+ (OR:1.79, 95%CI:1.05-3.05). Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated non-canonical β-catenin pathways are important in pelvic lymph node metastasis in early stage cervical cancer.
Project description:Using the pools of DNA from squamous cervical carcinomas (SCC) and DNA from normal scraping cells discovered the abnormal changes of genomic wide methylation by methylation BeadChip. The 61 genes were selected to validate by quantitative methylation specific PCR (qMSP) and bisulfite pyrosequencing in further processes. A CpG islands methylator phenotype (CIMP) was confirmed at 14 candidates in an independent set contained a spectrum of scraping cells form abnormal cervical lesions. In addition, we found among of 7 genes were announced to imply to potentially biological functions of β-catenin signal.
Project description:Background: Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/beta-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/beta-catenin -driven transcription. Despite the passage of a decade after the discovery of TCF4 and beta-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Methodology/Principal Findings: Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l, as Tcf4/beta-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a beta-catenin -dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine, in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators dedicated to Wnt target gene regulation. In contrast, previously published b-catenin coactivators p300 and beta-catenin displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10 and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. Conclusions/Significance: We conclude that Mllt10/Af10-Dot1l are essential, dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase Dot1l may present an attractive candidate for drug targeting in colorectal cancer. 6 samples for Ls174T cells: si-b-catenin against si-control and dyeswap of it, si-control, si-MLLT10, si-BRG1 and si-P300 are hybridized against common reference RNA; 6 samples of HEK293T cells: Wnt3A or control medium (CM) induction for 9 hours, si-MLLT10, si-DOT1L, si-BRG1 and si-P300 upon 9 hour Wnt3A induction are all hybridized against common reference RNA
Project description:Wnt/beta-catenin signaling is essential for stem cell regulation and cancer formation by activation of target genes transcription. For transcriptional activation, the histone around promoters needs to be modified to remove transcriptional repressors; however, the underlying mechanisms remain largely unknown. Here, we report that Wnt signal erases TCF4-associated H3K9me2/me3 by recruitment of KDM4C through β-catenin to activate gene transcription. In the absence of Wnt3a, PKR phosphorylates KDM4C which induces its ubiquitination and degradation. Wnt3a stabilizes KDM4C through inhibition of GSK3-dependent PKR kinase activity. Stabilized KDM4C accumulates in nucleus. Through interaction with β-catenin, KDM4C binds to and demethylates TCF4-associate Histone H3K9 which leads to HP1 removal and transcription activation. KDM4C-dependent H3K9 demethylation is essential for Wnt-induced gene expression and tumorigenesis. Importantly, KDM4C levels directly correlate with Wnt signaling activation in human glioblastomas. These findings demonstrate a pivotal epigenetic regulation mechanism for Wnt/β-catenin signaling activation in tumorigenesis.
Project description:Medulloblastoma is the most frequent malignant pediatric brain tumor. Considerable efforts are dedicated to identify markers that help to refine treatment strategies. The activation of the Wnt/beta-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favorable patient outcome. We report a series of 72 pediatric medulloblastomas evaluated for beta-catenin immunostaining, CTNNB1 mutations, and studied by comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of beta-catenin showed extensive nuclear staining (>50% of the tumor cells) in 6 cases and focal nuclear staining (<10% of cells) in 3 cases. The other cases exhibited either a signal strictly limited to the cytoplasm (58 cases) or were negative (5 cases). CTNNB1 mutations were detected in all beta-catenin extensively nucleopositive cases. The expression profiles of these cases documented a strong activation of the Wnt/beta-catenin pathway. Remarkably, 5 out of these 6 tumors showed a complete loss of chromosome 6. In contrast, cases with focal nuclear beta-catenin staining, as well as tumors with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/beta-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5-121.2) from diagnosis. All three patients with a focal nuclear staining of beta-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1-mutated tumors represent a distinct molecular subgroup of medulloblastomas with favorable outcome, indicating that therapy de-escalation should be considered. Yet, international consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved. A series of 72 pediatric medulloblastoma tumors has been studied at the genomic level (array-CGH), screened for CTNNB1 mutations and beta-catenin expression (immunohistochemistry). A subset of 40 tumor samples has been analyzed at the RNA expression level (Affymetrix HG U133 Plus 2.0). Correlations between the genomic data, the expression data, the mutational screening, the pathological classification and clinical data is presented in the study. note: aCGH data not submitted to GEO
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin -driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. Among these, one regards how the Wnt/β-catenin cascade modulates the chromatin behavior: to date, there exists no comprehensive genome-wide annotation of changing chromatin patterns upon Wnt pathway activation. This is important, as shifts in chromatin patterns might underlie how different cells promote diverging gene expression programs in response to Wnt. To address this question, we characterized how Wnt/β-catenin signaling shapes the genome-wide chromatin accessibility landscape in two human cell types, human embryonic kidney cells 293T (HEK293T) and human embryonic stem cells (hESCs), over time. To this end, we treated HEK293T and hESCs with the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days and assessed chromatin accessibility via ATAC-sequencing 4 hours, 24 hours and 3 days after the onset of the stimulation. We found that hESCs respond to Wnt/β-catenin activation by progressively shaping their chromatin accessibility profile in a manner that is consistent with their gradual acquisition of a mesodermal identity: differentiation genes loci open over time, while pluripotency ones close. We refer to this genomic response as plastic. On the other hand, HEK293T, which are known to be highly responsive to Wnt activation, appear more resistant to a long-term Wnt/β-catenin-driven change in cell identity. In this context, the chromatin displays a temporary opening of relevant regions at 4 hours after stimulation, followed by a re-establishment of its pre-stimulation state: we define this transient response as elastic.