The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.

OBJECTIVE

Gene promoter region hypermethylation is a significant event in primary breast cancer. However, its impact on tumor progression and potential predictive implications remain relatively unknown.

METHODS

We conducted hypermethylation profiling of 151 primary breast tumors with association to known prognostic factors in breast cancer using methylation-specific PCR for six known tumor suppressor and related genes: RASSF1A, APC, TWIST, CDH1, GSTP1, and RAR-beta2. Furthermore, correlation with sentinel lymph node (SLN) tumor status was assessed as it represents the earliest stage of metastasis that is readily detected. Hypermethylation for any one gene was identified in 147 (97%) of 151 primary breast tumors. The most frequently hypermethylated gene was RASSF1A (81%).

RESULTS

Hypermethylation of the CDH1 was significantly associated with primary breast tumors demonstrating lymphovascular invasion (P = 0.008), infiltrating ductal histology (P = 0.03), and negative for the estrogen receptor (P = 0.005), whereas RASSF1A and RAR-beta2 gene hypermethylation were significantly more common in estrogen receptor-positive (P < 0.001) and human epidermal growth factor receptor 2-positive (P < 0.001) tumors, respectively. In multivariate analysis, hypermethylation of GSTP1 and/or RAR-beta2 was significantly associated with patients having macroscopic SLN metastasis compared with those with microscopic or no sentinel node metastasis (odds ratio, 4.59; 95% confidence interval, 2.02-10.4; P < 0.001). In paired SLN metastasis, CDH1 was the most frequently methylated gene (90%) and provides evidence in patients corroborating its role in the clinical development of metastasis.

CONCLUSIONS

Hypermethylation profiling of primary breast tumors is significantly associated with known pathologic prognostic factors and may have additional clinical and pathologic utility for assessing patient prognosis and predicting early regional metastasis.

BACKGROUND

Cervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood.

RESULTS

We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.

CONCLUSIONS

These results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment.

Genome-wide association studies (GWAS) have identified more than a dozen loci associated with colorectal cancer (CRC) risk. Here, we examined potential effect-modification between single-nucleotide polymorphisms (SNP) at 10 of these loci and probable or established environmental risk factors for CRC in 7,016 CRC cases and 9,723 controls from nine cohort and case-control studies. We used meta-analysis of an efficient empirical-Bayes estimator to detect potential multiplicative interactions between each of the SNPs [rs16892766 at 8q23.3 (EIF3H/UTP23), rs6983267 at 8q24 (MYC), rs10795668 at 10p14 (FLJ3802842), rs3802842 at 11q23 (LOC120376), rs4444235 at 14q22.2 (BMP4), rs4779584 at 15q13 (GREM1), rs9929218 at 16q22.1 (CDH1), rs4939827 at 18q21 (SMAD7), rs10411210 at 19q13.1 (RHPN2), and rs961253 at 20p12.3 (BMP2)] and select major CRC risk factors (sex, body mass index, height, smoking status, aspirin/nonsteroidal anti-inflammatory drug use, alcohol use, and dietary intake of calcium, folate, red meat, processed meat, vegetables, fruit, and fiber). The strongest statistical evidence for a gene-environment interaction across studies was for vegetable consumption and rs16892766, located on chromosome 8q23.3, near the EIF3H and UTP23 genes (nominal P(interaction) = 1.3 × 10(-4); adjusted P = 0.02). The magnitude of the main effect of the SNP increased with increasing levels of vegetable consumption. No other interactions were statistically significant after adjusting for multiple comparisons. Overall, the association of most CRC susceptibility loci identified in initial GWAS seems to be invariant to the other risk factors considered; however, our results suggest potential modification of the rs16892766 effect by vegetable consumption.

In diffuse gastric carcinoma, despite common E-cadherin gene (CDH1) mutations, tumors show absence of CDH1 loss of heterozigosity (LOH) in most cases. This observation challenges the classical two-hit model of tumor suppressor gene inactivation. In order to investigate whether or not CDH1 promoter methylation may function as the second hit we analysed a series of 23 sporadic gastric carcinomas for the presence of CDH1 mutations, CDH1 promoter methylation, LOH and E-cadherin expression. CDH1 mutations were detected in nine of the 16 (56.3%) diffuse gastric carcinomas and in none of the seven intestinal gastric carcinomas. In diffuse gastric carcinomas harboring CDH1 mutations, LOH was observed in a single case. Loss of plasma membrane E-cadherin expression was consistently found in all nine cases with CDH1 mutation, suggesting that tumors inactivated the remaining CDH1 allele via a different mechanism. CDH1 promoter methylation was observed in nine of the 16 (56.3%) diffuse-type gastric carcinoma cases, including six of the nine cases (66.7%) harboring CDH1 mutations. CDH1 promoter methylation was also seen in two (28.6%) intestinal-type cases. Our results show that CDH1 promoter methylation is the second hit in more than half of the sporadic diffuse gastric carcinoma cases harboring CDH1 mutations.

By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCF(Skp2) and APC(Cdh1) complexes, respectively, Fucci technique labels mammalian cell nuclei in G(1) and S/G(2)/M phases with different colors. Transgenic mice expressing these G(1) and S/G(2)/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G(1) marker based on the SCF(Skp2)-mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-to-posterior waves of cell-cycle transitions, G(1)/S and M/G(1), in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4(Ddb1)-mediated Cdt1 degradation pathway common to all metazoans for the development of a G(1) marker that works in the nonmammalian animal model.

Aurora B kinase, a subunit of the chromosomal passenger protein complex, plays essential roles in spindle assembly, chromosome bi-orientation, and cytokinesis. The kinase activity of Aurora B, which peaks in mitosis, is tightly controlled in the cell cycle. Modulation of Aurora B protein levels could partly account for the regulation of its kinase activity in the cell cycle. However, little is known on the molecular mechanism of regulation of Aurora B levels. Here, we examined Aurora B protein levels and confirmed that they fluctuate during the cell cycle, peaking in mitosis and dropping drastically in G1. This profile for Aurora B in the cell cycle is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase essential for mitotic progression. Indeed, Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/C-Cdh1 is specific as it requires the presence of a conserved D-box at the COOH terminus of Aurora B. Furthermore, endogenous Aurora B and Cdh1 form a complex exclusively in mitotic cells. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as a reduction of the Cdh1 level by RNA interference stabilizes the Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is regulated both by its activation during early mitosis and by its destruction by APC/C-Cdh1 in late mitosis and in G1.

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5' differences and in support of this we report that a 5' isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5' isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

E-cadherin (CDH1) gene expression is strictly regulated. The transcriptional factors SNAIL and ZEB1 are involved in its repression, whereas activation of vitamin D receptor (VDR) by vitamin D induces its transcription. We study the expression and functional correlation of SNAIL, CDH1, VDR and ZEB1 genes and examine their possible involvement in colon cancer. The expression of these four genes was measured by real time-PCR in 114 patients with colorectal cancer, and tumor characteristics were analyzed in each patient. SNAIL expression was associated with downregulation of CDH1 (P < 0.001) and VDR (P < 0.001) gene products. We also found a positive correlation between CDH1 and VDR expressions. However, the association between SNAIL and CDH1 was not found in patients with high expression of ZEB1. We observed a correlation between downregulation of: a) ZEB1 and presence of polyps in surgical resections; b) VDR and poor differentiation and c) CDH1 and poor differentiation, vascular invasion, presence of lymph node metastases and advanced stages; as well as a trend toward a correlation between SNAIL expression in tumors and vascular invasion. The correlations between SNAIL, CDH1, VDR and ZEB1 and the association between reduced expression of CDH1 and VDR and aggressive tumor characteristics emphasize the value of analyzing these genes in colon cancer patients for prognostic purposes and for predicting response to possible therapies with vitamin D or its analogs.

Aberrant promoter hypermethylation is frequently observed in cancer. The potential for this mechanism to contribute to tumor development depends on whether the genes affected are repressed because of their methylation. Many aberrantly methylated genes play important roles in development and are bivalently marked in ES cells, suggesting that their aberrant methylation may reflect developmental processes. We investigated this possibility by analyzing promoter methylation in 19 breast cancer cell lines and 47 primary breast tumors. In cell lines, we defined 120 genes that were significantly repressed in association with methylation (SRAM). These genes allowed the unsupervised segregation of cell lines into epithelial (EPCAM+ve) and mesenchymal (EPCAM-ve) lineages. However, the methylated genes were already repressed in normal cells of the same lineage, and >90% could not be derepressed by treatment with 5-aza-2'-deoxycytidine. The tumor suppressor genes APC and CDH1 were among those methylated in a lineage-specific fashion. As predicted by the epithelial nature of most breast tumors, SRAM genes that were methylated in epithelial cell lines were frequently aberrantly methylated in primary tumors, as were genes specifically repressed in normal epithelial cells. An SRAM gene expression signature also correctly identified the rare claudin-low and metaplastic tumors as having mesenchymal characteristics. Our findings implicate aberrant DNA methylation as a marker of cell lineage rather than tumor progression and suggest that, in most cases, it does not cause the repression with which it is associated.

Familial gastric cancer comprises at least three major syndromes: hereditary diffuse gastric cancer, gastric adenocarcinoma and proximal polyposis of the stomach, and familial intestinal gastric cancer. The risk of development of gastric cancer is high in families affected b-y these syndromes, but only hereditary diffuse gastric cancer is genetically explained (caused by germline alterations of CDH1, which encodes E-cadherin). Gastric cancer is also associated with a range of several cancer-associated syndromes with known genetic causes, such as Lynch, Li-Fraumeni, Peutz-Jeghers, hereditary breast-ovarian cancer syndromes, familial adenomatous polyposis, and juvenile polyposis. We present contemporary knowledge on the genetics, pathogenesis, and clinical features of familial gastric cancer, and discuss research and technological developments, which together are expected to open avenues for new genetic testing approaches and novel therapeutic strategies.

We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans. Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23. We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels.

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

BACKGROUND

Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition.

RESULTS

To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates.

CONCLUSIONS

Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

BACKGROUND

Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A alpha-transcript), p14ARF (CDKN2A beta-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables.

RESULTS

The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling.

CONCLUSIONS

The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.

We have identified the zinc-finger transcription factor Kruppel-like factor 4 (Klf4) among the transcription factors that are significantly downregulated in their expression during epithelial-mesenchymal transition (EMT) in mammary epithelial cells and in breast cancer cells. Loss and gain of function experiments demonstrate that the down-regulation of Klf4 expression is required for the induction of EMT in vitro and for metastasis in vivo. In addition, reduced Klf4 expression correlates with shorter disease-free survival of subsets of breast cancer patients. Yet, reduced expression of Klf4 also induces apoptosis in cells undergoing TGFβ-induced EMT. Chromatin immunoprecipitation/deep-sequencing in combination with gene expression profiling reveals direct Klf4 target genes, including E-cadherin (Cdh1), N-cadherin (Cdh2), vimentin (Vim), β-catenin (Ctnnb1), VEGF-A (Vegfa), endothelin-1 (Edn1) and Jnk1 (Mapk8). Thereby, Klf4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. Specifically, increased expression of Jnk1 (Mapk8) upon down-regulation of its transcriptional repressor Klf4 is required for EMT cell migration and for the induction of apoptosis. The data demonstrate a central role of Klf4 in the maintenance of epithelial cell differentiation and the prevention of EMT and metastasis.

BACKGROUND

Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes.

METHODS

We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors.

RESULTS

While the following sixteen genes remained the unmethylated in all tumor and normal tissues: <em>CDH1</em>, APAF1, hMLH1, BRCA1, hTERC, VHL, RARbeta, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and <em>CDH1</em>3 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%).

CONCLUSIONS

Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the <em>CDH1</em>3 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.

OBJECTIVE

To establish the methylation profile of the promoter CpG islands of 31 genes that might play etiological roles in colon carcinogenesis.

METHODS

The methylation specific PCR in conjunction of sequencing verification was used to establish the methylation-profile of the promoter CpG islands of 31 genes in colorectal cancer (n = 65), the neighboring non-cancerous tissues (n = 5), colorectal adenoma (n = 8), and normal mucosa (n = 1). Immunohistochemically, expression of 10 genes was assessed on the home-made tissue microarrays of tissues from 58 patients. The correlation of tumor specific changes with each of clinical-pathologic features was scrutinized with relevant statistic tools.

RESULTS

In comparison with the normal mucosa of the non-cancer patients, the following 14 genes displayed no tumor associated changes: breast cancer 1, early onset (BRCA1), cadherin 1, type 1, E-cadherin (epithelial) (<em>CDH1</em>), death-associated protein kinase 1 (DAPK1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), melanoma antigen, family A, 1 (directs expression of antigen MZ2-E) (MAGEA1), tumor suppressor candidate 3 (N33), cyclin-dependent kinase inhibitor 1A (p21, Cip1) (p21(WAF1)), cyclin-dependent kinase inhibitor 1B (p27, Kip1) (p27(KIP1)), phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN), retinoic acid receptor, beta (RAR- , Ras association (RalGDS/AF-6) domain family 1 C (RASSF1C), secreted frizzled-related protein 1 (SFRP1), tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) (TIMP3), and von Hippel-Lindau syndrome (VHL). The rest 17 targets exhibited to various extents the tumor associated changes. As changes in methylation of the following genes occurred marginally, their impact on the formation of colorectal cancer were trivial: adenomatous polyposis coli (APC) (8%, 5/65), Ras association (RalGDS/AF-6) domain family 1A (RASSF1A) (3%, 2/65) and cyclin-dependent kinase inhibitor 2A, alternated reading frame (p14(ARF)) (6%, 4/65). The following genes exhibited moderate changes in methylation: O-6-methylguanine-DNA methyltransferase (MGMT) (20%, 13/65), mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) (hMLH1) (18%, 12/65), cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (p16(INK4a)) (10%, 10/65), methylated in tumor 1 (MINT1) (15%, 10/65), methylated in tumor 31 (MINT31) (11%, 7/65). The rest changed greatly in the methylation pattern in colorectal cancer (CRC): cyclin A1 (cyclin a1) (100%, 65/65), caudal type homeobox transcription factor 1 (CDX1) (100%, 65/65), RAR- (85%, 55/65), myogenic factor 3 (MYOD1) (69%, 45/65), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (p15(INK4b)) (68%, 44/65), prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) (COX2) (72%, 47/65), cadherin 13, H-cadherin (heart) (<em>CDH1</em>3) (65%, 42/65), CAAX box 1 (CXX1) (58%, 38/65), tumor protein p73 (p73) (63%, 41/65) and Wilms tumor 1 (WT1) (58%, 38/65). However, no significant correlation of changes in methylation with any given clinical-pathological features was detected. Furthermore, the frequent changes in methylation appeared to be an early phase event of colon carcinogenesis. The in situ expression of 10 genes was assessed by the immunohistochemical approach at the protein level: <em>CDH1</em>, <em>CDH1</em>3, COX2, cyclin A1, hMLH1, MGMT, p14(ARF), p73, RAR- , and TIMP3 genes in the context of the methylation status in colorectal cancer. No clear correlation between the hypermethylation of the promoter CpG islands and the negative expression of the genes was established.

CONCLUSIONS

The methylation profile of 31 genes was established in patients with colon cancer and colorectal adenomas, which provides new insights into the DNA methylation mediated mechanisms underlying the carcinogenesis of colorectal cancer and may be of prognostic values for colorectal cancer.

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

lncRNAs play important roles in the epigenetic regulation of carcinogenesis and progression. Previous studies suggest that HOTAIR contributes to gastric cancer (GC) development, and the overexpression of HOTAIR predicts a poor prognosis. In this study, we found that HOTAIR was more highly expressed in diffuse-type GC than in intestinal type (P=0.048). In the diffuse type, there is significant relationship between HOTAIR expression and DFS (P<0.001). CDH1 was downregulated in diffuse-type GC tissues (P=0.0007) and showed a negative relationship with HOTAIR (r(2)=0.154, P=0.0354). In addition, HOTAIR knockdown significantly repressed migration, invasion and metastasis both in vitro and vivo and reversed the epithelial-to-mesenchymal transition in GC cells. We also showed that HOTAIR recruiting and binding to PRC2 epigenetically represses miR34a, which controls the targets C-Met (HGF/C-Met/Snail pathway) and Snail, thus contributing to GC cell-EMT process and accelerating tumor metastasis. Moreover, it is demonstrated that HOTAIR crosstalk with microRNAs during epigenetic regulation. Our results suggest that HOTAIR acts as an EMT regulator and may be a candidate prognostic biomarker and a target for new therapies in GC patients.

Epithelial-mesenchymal transition (EMT) in cancer cells plays a pivotal role in determining metastatic prowess, but knowledge of EMT regulation remains incomplete. In this study, we defined a critical functional role for the Forkhead transcription factor FOXQ1 in regulating EMT in breast cancer cells. FOXQ1 expression was correlated with high-grade basal-like breast cancers and was associated with poor clinical outcomes. RNAi-mediated suppression of FOXQ1 expression in highly invasive human breast cancer cells reversed EMT, reduced invasive ability, and alleviated other aggressive cancer phenotypes manifested in 3-dimensional Matrigel (BD Biosciences) culture. Conversely, enforced expression of FOXQ1 in differentiated human mammary epithelial cells (HMLER) or epithelial cancer cell lines provoked an epithelial to mesenchymal morphologic change, gain of stem cell-like properties, and acquisition of resistance to chemotherapy-induced apoptosis. Mechanistic investigations revealed that FOXQ1-induced EMT was associated with transcriptional inactivation of the epithelial regulator E-cadherin (CDH1). Our findings define a key role for FOXQ1 in regulating EMT and aggressiveness in human cancer.