Metastatic disease accounts for more than 90% of cancer-related deaths, but the development of effective antimetastatic agents has been hampered by the paucity of clinically relevant preclinical models of human metastatic disease. Here, we report the development of a mouse model of spontaneous breast cancer metastasis, which recapitulates key events in its formation and clinical course. Specifically, using the conditional K14cre;Cdh1(F/F);Trp53(F/F) model of de novo mammary tumor formation, we orthotopically transplanted invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once primary tumors were established in recipient mice, we mimicked the clinical course of treatment by conducting a mastectomy. After surgery, recipient mice succumbed to widespread overt metastatic disease in lymph nodes, lungs, and gastrointestinal tract. Genomic profiling of paired mammary tumors and distant metastases showed that our model provides a unique tool to further explore the biology of metastatic disease. Neoadjuvant and adjuvant intervention studies using standard-of-care chemotherapeutics showed the value of this model in determining therapeutic agents that can target early- and late-stage metastatic disease. In obtaining a more accurate preclinical model of metastatic lobular breast cancer, our work offers advances supporting the development of more effective treatment strategies for metastatic disease.

BACKGROUND

Hepatitis C virus infects ~3% of the population and it is a risk factor for hepatocarcinogenesis. The epigenetic mechanisms of HCV-induced hepatocyte transformation towards malignancy in this context are unclear.

OBJECTIVE

The purpose of this study was to evaluate the effect of HCV core proteins of different genotypes on DNA methyltransferases (DNMTs) induction.

METHODS

We investigated DNMT1, DNMT3b and E-Cadherin (CDH1) mRNA and protein expression levels in an in vitro model of Huh-7 cells expressing the HCV core protein of different genotypes: 1b, 2a, 3a, 4h and 5a.

RESULTS

We found that both mRNA and protein expression levels of DNMT1 and 3b were upregulated in genotype 1b HCV core expressing cells as compared to control cells. DNMT3b mRNA levels did not change in genotypes 2a, 3a, 4h and 5a, but were upregulated at the protein level by genotype 1b, 2a, 3a. CDH1 mRNA expression was downregulated only in genotype 1b, whereas its protein expression resulted in downregulation by the HCV core of genotypes 1b, 2a and 3a. Conversely, no significant changes were observed for DNMTs and CDH1 investigated in Huh-7 cells expressing the genotypes 4h and 5a. Furthermore, we present evidence that HCV core 1b protein expression induces DNMTs overexpression through STAT3 protein as demonstrated by NSC74859 treatment. Moreover, SIRT1 inhibition affected DNMT1 and 3b expression only in HCV core protein genotype 1b expressing cells as demonstrated by treatment with its inhibitor sirtinol.

CONCLUSIONS

Our findings suggest that HCV core protein could play a role in HCC development at least in part by altering DNMTs expression.

Ubiquitin-mediated proteolysis has emerged as a paramount mechanism for regulating the cell division cycle. Changes in the activities of certain E3 ligases can promote the interconversion of cell cycle states or transitions. Recent studies have revealed how distinct E3 ligases control the activity of other E3 ligases and how the interplay between these degradation machines sets up the timing of cell cycle transitions. For example, during G1, the anaphase-promoting complex in conjunction with Cdh1 (APC(Cdh1)) catalyzes destruction of the S-phase activator Skp2, helping to define the G1 state. In response to poorly defined signals, APC(Cdh1) activity is reduced, allowing accumulation of Skp2 and therefore entry into S phase. In many cases, E3 ligases also function to ubiquitinate proteins that negatively regulate cell cycle transitions. Recent work indicates that cyclin-dependent kinase 2 and Polo kinase collaborate to phosphorylate Wee1, thereby promoting its ubiquitination by SCF(beta-TRCP). Thus, activation of the mitotic transition produces feedback signals that help to turn off the negative upstream pathway to further reenforce the transition.

Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of JARID2 was increased during TGF-ß-induced EMT of these cell lines and knockdown of JARID2 inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. JARID2 knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB family and microRNA-200 family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of CDH1 and microRNA-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.

OBJECTIVE

Epithelial ovarian cancer (EOC) remains the most lethal disease among gynecological malignancies. Prompt diagnosis is challenging because of the non-specific symptoms exhibited during the early stage of the disease. As a result, there is an urgent need for improved detection methods. In this study, we established a multiplex methylation-specific PCR (MSP) assay to improve the early detection of ovarian cancer, via identification of the methylation status of cell-free serum DNA.

METHODS

After screening, we chose seven candidate genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5 and OPCML) with a high frequency of methylation to construct the multiplex-MSP assay. When methylation of at least one of the seven genes was observed, the multiplex-MSP assay was considered positive. We performed retrospective and screening studies to verify the specificity and sensitivity of the assay in the detection of EOC.

RESULTS

The methylation status of cell-free serum DNA was examined in the preoperative serum of 202 patients, including 87 EOC patients (stage I, n=41; stage II-IV, n=46), 53 patients with benign ovarian tumors and 62 healthy controls. As expected, the multiplex MSP assay achieved a sensitivity of 85.3% and a specificity of 90.5% in stageI EOC, strikingly higher rates compared with a single CA125, which produced a sensitivity of 56.1% at 64.15% specificity [P=0.0036].

CONCLUSIONS

A multiplex MSP assay that analyzes the methylation status of cell-free serum DNA is a suitable and reliable approach to improve the early detection of ovarian cancer, potentially benefiting a broad range of applications in clinical oncology.

Recent advances in the field of brain energy metabolism strongly suggest that glutamate receptor-mediated neurotransmission is coupled with molecular signals that switch-on glucose utilization pathways to meet the high energetic requirements of neurons. Failure to adequately coordinate energy supply for neurotransmission ultimately results in a positive amplifying loop of receptor over-activation leading to neuronal death, a process known as excitotoxicity. In this review, we revisited current concepts in excitotoxic mechanisms, their involvement in energy substrate utilization, and the signaling pathways that coordinate both processes. In particular, we have focused on the novel role played by the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1, in cell metabolism. Our laboratory identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) -a key glycolytic-promoting enzyme- as an APC/C-Cdh1 substrate. Interestingly, APC/C-Cdh1 activity is inhibited by over-activation of glutamate receptors through a Ca(2+)-mediated mechanism. Furthermore, by inhibiting APC/C-Cdh1 activity, glutamate-receptors activation promotes PFKFB3 stabilization, leading to increased glycolysis and decreased pentose-phosphate pathway activity. This causes a loss in neuronal ability to regenerate glutathione, triggering oxidative stress and delayed excitotoxicity. Further investigation is critical to identify novel molecules responsible for the coupling of energy metabolism with glutamatergic neurotransmission and excitotoxicity, as well as to help developing new therapeutic strategies against neurodegeneration.

BACKGROUND

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors.

METHODS

MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets.

RESULTS

A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3'UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3'UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels.

CONCLUSIONS

Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis.

BACKGROUND

During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes.

METHODS

In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation.

RESULTS

Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration.

CONCLUSIONS

This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

E-cadherin has a determinant role in tumour progression, acting as an invasion and metastasis suppressor. Germline mutations of E-cadherin gene (CDH1) occur in 30% of families with Hereditary Diffuse Gastric Cancer (HDGC); of these 23% are missense mutations. The CDH1 missense mutations described to date span the entire gene and some lead to significant functional consequences. In this study, we explored the hypothesis that mutations affecting different E-cadherin protein domains have distinct effects on cell motility. To accomplish our objective we characterized the effect of eleven HDGC CDH1 germline missense mutations (T118R, L214P, G239R, A298T, T340A, P373L, R749W, E757K, E781D, P799R and V832M) on cell motility. Further, we studied their effect on the activation of signalling pathways known to be relevant for cell motility such as the EGFR, Src kinase and MAPKs. CDH1 mutations localized on the extracellular and juxtamembrane domains, both affecting the integrity of the extracellular domain, led to increased cell motility accompanied by increased EGFR activation. Moreover, we observed that cells expressing extracellular mutants exhibit increased activation of Src kinase and p38 MAPK. Our results allowed the identification of the E-cadherin domains pivotal for cell motility, further demonstrated a genotype-phenotype correlation, and defined a subset of HDGC cases which may benefit from EGFR inhibitors.

BACKGROUND

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome. Up to 30% of families with HDGC have mutations in the E-cadherin gene, CDH1. The role of prophylactic versus therapeutic gastrectomy for HDGC was studied prospectively.

METHODS

Eighteen consecutive patients with CDH1 mutations and positive family history were studied prospectively, including 13 without and 5 with symptoms. Proportions were compared by Fisher's exact test, and survival by the Breslow modification of the Wilcoxon rank-sum test.

RESULTS

Each patient underwent total gastrectomy (TG), and 17 (94%) were found to have signet ring cell adenocarcinoma. Twelve of 13 asymptomatic patients had T1, N0 cancer, and only 2/12 (16%) had it diagnosed preoperatively despite state-of-the-art screening methods. Each asymptomatic patient did well postoperatively, and no patient has recurred. For five symptomatic patients, each (100%) was found to have signet ring cell adenocarcinoma (P = 0.002 versus asymptomatic) by preoperative endoscopy; three (60%) had lymph node involvement and two (40%) had distant metastases at time of operation. Two-year survival was 100% for asymptomatic and 40% for symptomatic patients (P < 0.01).

CONCLUSIONS

The data show that asymptomatic patients with family history of HDGC and CDH1 mutation have high probability of having signet ring cell adenocarcinoma of the stomach that is not able to be diagnosed on endoscopy; when symptoms arise, the diagnosis can be made by endoscopy, but they have metastases and decreased survival. Surveillance endoscopy is of limited value, and prophylactic gastrectomy (PG) is recommended for patients with family history of HDGC and CDH1 mutations.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

We analyzed promoter methylation of RASSF1A, CTNNB1, CDH1, LAMB3, LAMC2, RUNX3, NORE1A, and CAV1 using methylation-specific PCR in 33 cases of small bowel carcinoid with both matched primary and metastatic tumors. The methylation status of RASSF1A and CTNNB1 were also determined in six primary appendiceal carcinoid tumors. Two neuroendocrine cell lines, NCI-H727 and HTB-119, were analyzed for promoter methylation. Immunohistochemical analyses for RASSF1A and beta-catenin were performed in 28 matched primary and metastatic tumors. Western blot analysis for RASSF1A and beta-catenin was also performed. Normal enterochromaffin cells were unmethylated in all eight genes examined. RASSF1A and CTNNB1 were unmethylated in appendiceal carcinoids. Methylation of RASSF1A and CTNNB1 promoters was more frequent in metastatic compared to primary tumors (p = 0.013 and 0.004, respectively). The NCI-H727 and HTB-119 cells lines were methylated in the RASSF1A promoter region, and after treatment with 5-aza-2'-deoxycytidine (5-AZA), RASSF1A mRNA was expressed in both cell lines. Western blot results for RASSF1A and beta-catenin supported the methylation-specific PCR findings. The other six genes did not show significant differences. These results suggest that increased methylation of RASSF1A and CTNNB1 may play important roles in progression and metastasis of small bowel carcinoid tumors.

Gastric cancer (GC), one of the most common human cancers, can be classified into gastric or intestinal phenotype according to mucin expression. TP53 mutation, allelic deletion of the APC gene and nuclear staining of β-catenin are frequently detected in the intestinal phenotype of GC, whereas <em>CDH1</em> gene mutation, microsatellite instability and DNA hypermethylation of MLH1 are common events in the gastric phenotype of GC. Our Serial Analysis of Gene Expression (SAGE) and Escherichia coli ampicillin secretion trap (CAST) analyses revealed that <em>CDH1</em>7, REG4, OLFM4, HOXA10, DSC2, TSPAN8 and TM9SF3 are upregulated in GC and that CLDN18 is downregulated in GC. Expression of <em>CDH1</em>7, REG4, HOXA10 and DSC2 and downregulation of CLDN18 are observed in the intestinal phenotype of GC. In contrast, OLFM4 is expressed in the gastric phenotype of GC. Expression of TSPAN8, TM9SF3 and HER2 are not associated with either gastric or intestinal phenotypes. Ectopic CDX2 expression plays a key function in the GC intestinal phenotype. MUC2, <em>CDH1</em>7, REG4, DSC2 and ABCB1 are direct targets of CDX2. Importantly, these genes encode transmembrane/secretory proteins, indicating that the microenvironment as well as cancer cells are also different between gastric and intestinal phenotypes of GC.

BACKGROUND

Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer; lobular breast cancer also occurs excessively in families with such condition.

METHODS

To determine if CDH1 is a susceptibility gene for lobular breast cancer in women without a family history of diffuse gastric cancer, germline DNA was analysed for the presence of CDH1 mutations in 318 women with lobular breast cancer who were diagnosed before the age of 45 years or had a family history of breast cancer and were not known, or known not, to be carriers of germline mutations in BRCA1 or BRCA2. Cases were ascertained through breast cancer registries and high-risk cancer genetic clinics (Breast Cancer Family Registry, the kConFab and a consortium of breast cancer genetics clinics in the United States and Spain). Additionally, Multiplex Ligation-dependent Probe Amplification was performed for 134 cases to detect large deletions.

RESULTS

No truncating mutations and no large deletions were detected. Six non-synonymous variants were found in seven families. Four (4/318 or 1.3%) are considered to be potentially pathogenic through in vitro and in silico analysis.

CONCLUSIONS

Potentially pathogenic germline CDH1 mutations in women with early-onset or familial lobular breast cancer are at most infrequent.

BACKGROUND

Present data are highly suggestive but do not unequivocally prove the cosegregation of germ-line CDH1 mutations with inherited invasive lobular breast cancer (ILBC).

METHODS

Two Caucasian families with 6 pathologically confirmed ILBC cases but no diffuse gastric cancer (DGC) were identified in our oncogenetics consultations. Screening for mutations of CDH1, BRCA1, and BRCA2 genes was performed on blood samples. When available, loss of heterozygosity (LOH) and immunohistochemistry (IHC) analyses were performed on tumor samples.

RESULTS

No BRCA1 or BRCA2 mutation was found. Deleterious CDH1 germ-line mutations c.283C>T and c.1582del were found in all the 4 living women with ILBC in family 1 and family 2, respectively. The mutation c.283C>T was also present in a healthy 71-year-old male and 2 obligate carriers in family 1. No DGC was observed in the 2 families. Loss of the wild-type CDH1 allele in 1 of the breast tumors was confirmed by LOH and IHC studies, in accordance with the "2-hit" model of tumor suppressor genes.

CONCLUSIONS

Germline CDH1 mutation can be cosegregated with ILBC in the absence of DGC. Present data do not support recommendation of prophylactic gastrectomy in CDH1 germline mutation carriers with ILBC. Cancer 2011. © 2011 American Cancer Society.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Hepatocellular Carcinoma (HCC) is one of the most common cancers in the world and it is often associated with poor prognosis. Liver transplantation and resection are two currently available curative therapies. However, most patients cannot be treated with such therapies due to late diagnosis. This underscores the urgent need to identify potential markers that ensure early diagnosis of HCC. As more evidences are suggesting that epigenetic changes contribute hepatocarcinogenesis, DNA methylation was poised as one promising biomarker. Indeed, genome wide profiling reveals that aberrant methylation is frequent event in HCC. Many studies showed that differentially methylated genes and CpG island methylator phenotype (CIMP) status in HCC were associated with clinicopathological data. Some commonly studied hypermethylated genes include p16, SOCS1, GSTP1 and CDH1. In addition, studies have also revealed that methylation markers could be detected in patient blood samples and associated with poor prognosis of the disease. Undeniably, increasing number of methylation markers are being discovered through high throughput genome wide data in recent years. Proper and systematic validation of these candidate markers in prospective cohort is required so that their actual prognostication and surveillance value could be accurately determined. It is hope that in near future, methylation marker could be translate into clinical use, where patients at risk could be diagnosed early and that the progression of disease could be more correctly assessed.

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces the expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

BACKGROUND

Besides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase.

RESULTS

We now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of ∼25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells.

CONCLUSIONS

Our results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation.

The anaphase-promoting complex/cyclosome (APC/C) controls the onset of anaphase by targeting securin for destruction. We report here the identification and characterization of a substrate of APC/C, RCS1, as a mitotic regulator that controls the metaphase-to-anaphase transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G(1). Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and degraded during mitotic exit in a Cdh1-dependent manner in vivo. APC/C recognizes a unique D-box at the N terminus of RCS1, as mutations of this D-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. RCS1 controls the timing of the anaphase onset, because the loss of RCS1 resulted in a faster progression from the metaphase to anaphase and accelerated degradation of securin and cyclin B. Biochemically, mitotic RCS1 associates with the NuRD chromatin-remodeling complex, and this RCS1 complex is likely involved in regulating gene expression or chromatin structure, which in turn may control anaphase onset. Our study uncovers a complex regulatory network for the metaphase-to-anaphase transition.

The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates cell proliferation by targeting multiple cell-cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability. By using a proteomic approach in Cdh1-null cells and mouse tissues, we have identified kinesin Eg5 and topoisomerase 2α as Cdh1 targets involved in the maintenance of genomic stability. These proteins are ubiquitinated and degraded through specific KEN and D boxes in a Cdh1-dependent manner. Whereas Cdh1-null cells display partial resistance to Eg5 inhibitors such as monastrol, lack of Cdh1 results in a dramatic sensitivity to Top2α poisons as a consequence of increased levels of trapped Top2α-DNA complexes. Chemical inhibition of the APC/C in cancer cells results in increased sensitivity to Top2α poisons. This work identifies in vivo targets of the mammalian APC/C-Cdh1 complex and reveals synthetic lethal interactions of relevance in anticancer treatments.

Loss of HOXA5 expression occurs frequently in breast cancer and correlates with higher pathological grade and poorer disease outcome. However, how HOX proteins drive differentiation in mammalian cells is poorly understood. In this paper, we investigated cellular and molecular consequences of loss of HOXA5 in breast cancer, and the role played by retinoic acid in HOXA5 function. Analysis of global gene expression data from HOXA5-depleted MCF10A breast epithelial cells, followed by validation, pointed to a role for HOXA5 in maintaining several molecular traits typical of the epithelial lineage such as cell-cell adhesion, tight junctions and markers of differentiation. Depleting HOXA5 in immortalized MCF10A or transformed MCF10A-Kras cells reduced their CD24+/CD44lo population, enhanced self-renewal capacity and reduced expression of E-cadherin (CDH1) and CD24. In the case of MCF10A-Kras, HOXA5 loss increased branching and protrusive morphology in Matrigel, all features suggestive of epithelial to basal transition. Further, orthotopically implanted xenografts of MCF10A-Kras-scr grew as well-differentiated pseudo-luminal carcinomas, while MCF10A-Kras-shHOXA5 cells formed aggressive, poorly differentiated carcinomas. Conversely, ectopic expression of HOXA5 in aggressive SUM149 or SUM159 breast cancer cells reversed the cellular and molecular alterations observed in the HOXA5-depleted cells. Retinoic acid is a known upstream regulator of HOXA5 expression. HOXA5 depletion in MCF10A cells engineered to express doxycycline-induced shHOXA5 slowed transition of cells from a less differentiated CD24-/CD44+ to the more differentiated CD24+/CD44+ state. This transition was promoted by retinal treatment, which upregulated endogenous HOXA5 expression and caused re-expression of occludin and claudin-7 (CLDN7). Expression of CDH1 and CD24 was transcriptionally upregulated by direct binding of HOXA5 to their promoter sequences as demonstrated by luciferase and ChIP analyses. Thus, loss of HOXA5 in mammary cells leads to loss of epithelial traits, an increase in stemness and cell plasticity, and the acquisition of more aggressive phenotypes.

Breast cancer, one of the leading causes of mortality and morbidity among females, is regulated in part by diverse classes of adhesion molecules one of which is known as cadherins. Located at adherens junctions, the members of this superfamily are responsible for upholding proper cell-cell adhesion. Cadherins possess diverse structures and functions and any alteration in their structures or functions causes impeding of normal mammary cells development and maintenance, thus leading to breast malignancy. E-, N-, P-, VE-, Proto-, desmosomal and FAT cadherins have been found to regulate breast cancer in positive as well as negative fashion, whereby both Ecadherin (CDH1) and N-cadherin (CDH2) contribute significantly towards transitioning from epithelial state to mesenchymal state (EMT) and enacting the abnormal cells to invade and metastasize nearby and distant tissues. Aberration in gene expression of cadherins can be either due to somatic or epigenetic silencing or via transcriptional factors. Besides other cadherins, E-cadherin which serves as hallmark of EMT is associated with several regulatory factors such as Snail, Slug, Twist, Zeb, KLF4, NFI, TBX2, SIX, b-Myb, COX-2, Arf6, FOXA2, GATA3 and SMAR1, which modulate E-cadherin gene transcription to promote or represses tumor invasion and colonization. Signaling molecules such as Notch, TGF-β, estrogen receptors, EGF and Wnt initiate numerous signaling cascades via these vital factors of cell programming, controlling expression of E-cadherin at transcriptional (mRNA) and protein level. Thus, interactions of cadherins with their roles in tumor suppression and oncogenic transformation can be beneficial in providing valuable insights for breast cancer diagnosis and therapeutics development.