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Ross, Karen E., and Orna Cohen-Fix. "The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast." Genetics 165, no. 2 (October 1, 2003): 489–503. http://dx.doi.org/10.1093/genetics/165.2.489.
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Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.
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Alkebsi, Lobna A., Hiroshi Handa, Kenichi Tahara, Hiroaki Shimizu, Takuma Ishizaki, Kunio Yanagisawa, Makiko Takizawa, et al. "Chromosome 16q Located Genes CDH1, CDH13 and ADAMTS18 Are Correlated and Frequently Methylated But Not Associated With DNMTs levels In Human Lymphoma." Blood 122, no. 21 (November 15, 2013): 4289. http://dx.doi.org/10.1182/blood.v122.21.4289.4289.
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Abstract Hypermethylation of promoter contributes to the transcriptional repression of a number of cancer associated genes. In lymphoma, the promoter hypermethylation of many tumor suppressor genes (TSGs), such as p16, has been already known. Using methylation-specific PCR (MSP) and quantitative real-time PCR (qRT-PCR), we examined promoter methylation status and mRNA expression levels of E-cadherin (CDH1), H-cadherin (CDH13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS18) which are putative TSGs located on chromosome 16q, and also examined the mRNA expression levels of DNA methyltransferases (DNMT1, 3A and 3B) and studied the correlation between these different tested parameters in 36 of lymphomas [included 29 diffuse large B cell (DLBCL) and seven mantle cell lymphoma (MCL)] and 16 non-malignant lymphoid tissues after obtaining informed consent. The expression of DNMTs mRNA were significantly higher in lymphomas compared to non-malignant tissues (p<0.05). Promoter hypermethylation of CDH1, CDH13, and ADAMTS18 was detected in 31/36 (86%), 33/36 (91.6%) and 28/36 (77.7%) of lymphoma, respectively. The expression of CDH1 and ADAMTS18 was significantly reduced in the patients with hypermethylated promoter when compared to unmethylated (p<0.01 and p<0.05, respectively), while no significant difference was found in CDH13. CDH1 and ADAMTS18 expressions were significantly reduced in lymphomas compared to non-malignant tissues (p<0.01), while CDH13 showed no significant difference. Notably, there was significant positive correlation between the expression levels of CDH1 and CDH13 (r = 0.735, p<0.01) (Fig. 1A). Moreover, ADAMTS18 expression showed significant positive correlation with both CDH1 and CDH13 expression levels (r = 0.625, p<0.01; r = 0.720, p<0.01, respectively) (Fig. 1B, C). Also there was significant negative correlation between the expression levels of DNMT3A and 3B with ADAMTS18 (r = -0.396, p<0.01; r = -0.364, p<0.01, respectively), but not with CDH1 and CDH13 (Fig. 2A and B). We could not find any correlation between the levels of DNMTs mRNA and the methylation status of CDH1, CDH13 and ADAMTS18. We examined the effect of 5-Aza-2-deoxycytidine (5-aza-dC) treatment on CDH1, CDH13 and ADAMTS18 expression levels and their methylated promoters in 3 of lymphoma cell lines (Raji, CTB-1 and SLVL) and one patient primary DLBCL cell line. We found that 5-aza-dC treatment of CDH1, CDH13 and ADAMTS18-methylated cell lines led to restoration of their expression levels (Fig. 3A, B, and C). Our results showed that CDH1, CDH13 and ADAMTS18, tumor suppressor genes adjacently located at chromosome 16q, are remarkably correlated and frequently methylated in human lymphoma and their methylation could not be explained solely by the expression level of DNMTs mRNA. Disclosures: No relevant conflicts of interest to declare.
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Qiao, Xinxian, Liyong Zhang, Armin M. Gamper, Takeo Fujita, and Yong Wan. "APC/C-Cdh1." Cell Cycle 9, no. 19 (October 2010): 3904–12. http://dx.doi.org/10.4161/cc.9.19.13585.
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Höckner, Sebastian, Lea Neumann-Arnold, and Wolfgang Seufert. "Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1." Molecular Biology of the Cell 27, no. 14 (July 15, 2016): 2198–212. http://dx.doi.org/10.1091/mbc.e15-11-0787.
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The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.
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Listovsky, Tamar, and Julian E. Sale. "Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset." Journal of Cell Biology 203, no. 1 (October 7, 2013): 87–100. http://dx.doi.org/10.1083/jcb.201302060.
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The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.
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Arnold, Lea, Sebastian Höckner, and Wolfgang Seufert. "Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons." Molecular Biology of the Cell 26, no. 5 (March 2015): 843–58. http://dx.doi.org/10.1091/mbc.e14-09-1342.
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In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.
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Yanjun, Xu, Cao Wenming, Xu Qi, Guo Jianmin, Wang Xinbao, Cheng Xiangdong, and Ying Jieer. "Searching for CDH1 gene mutations in early-onset diffuse gastric cancer in Chinese patients." Journal of Clinical Oncology 32, no. 3_suppl (January 20, 2014): 23. http://dx.doi.org/10.1200/jco.2014.32.3_suppl.23.
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23 Background: CDH1 germline mutations are found to be associated with the development of hereditary diffuse gastric cancer (HDGC) and the early-onset diffuse gastric cancer (EODGC). But the impact of CDH1 gene mutations and large deletions on HDGC and EODGC has not been fully determined in Asians. Although the incidence of gastric cancer is relatively high in China, the detection rate of CDH1 germline mutations in Chinese patients with EODGC is rare compared to that in European patients. Methods: We investigated the mutation status of the CDH1 gene in 57 Chinese EODGC patients younger than 40 years old who met the clinical criteria for HDGC. Polymerase chain reaction-direct sequencing was performed, and multiplex ligation-dependent probe amplification (MLPA) was used to evaluate the patients with negative sequencing results. Associations between mutation, clinicopathologic, and overall survival data were analyzed by SPSS 19. Results: The germline mutations of CDH1gene were identified in 51 (89.5%) of the 57 EODGC patients. The nonsense mutation in exon 13 (c.2200T>C, p.Ala692*) occurred in fourty-six EODGC patients. The missense mutations were detected in twenty patients (Eighteen in exon 5: c.778G>C, p.Glu218Asp; Two in exon 12: c.2012C>G, p.Leu630Val). No deletion or duplication in any patient. Most of the patients carrying the CDH1 mutation in exon 13 had lymph node metastasis when compared with patients lacking CDH1 mutation (87.2% vs 60.0%) ( P < 0.05 ). EODGC patients, lacking germline CDH1 alterations, showed a longer median overall survival (mOS) than patients carrying CDH1 mutation in exon 13 ( P < 0.05 ). Moreover, the presence of CDH1 mutation in exon 13 was associated with the incidence of neural invasion ( P < 0.05 ). Conclusions: This study reveals novel CDH1 mutations in Chinese EODGC patients which had been poorly investigated. The presence of CDH1 mutation in EODGC patients may result in lymph node metastasis and poor prognosis. More research is needed to determine additional genetic targets that trigger EODGC.
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Martinez, Juan S., Dah-Eun Jeong, Eunyoung Choi, Brian M. Billings, and Mark C. Hall. "Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast." Molecular and Cellular Biology 26, no. 24 (October 9, 2006): 9162–76. http://dx.doi.org/10.1128/mcb.00603-06.
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ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/C Cdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.
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Krohs, Julika, Dominik Schnerch, Marie Follo, Julia Felthaus, Monika Engelhardt, and Ralph M. Waesch. "The Tumor Suppressor APC/CCdh1 and Its Role In Replication Stress and The Origin Of Genomic Instability." Blood 122, no. 21 (November 15, 2013): 2489. http://dx.doi.org/10.1182/blood.v122.21.2489.2489.
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Abstract Introduction We have previously proposed that Cdh1 is a tumor suppressor by maintaining genomic stability. We also found Cdh1 downregulated in several tumor cell lines including AML (Oncogene 2008; 27:907-17). Heterozygous Cdh1 knockout mice develop epithelial tumors, myelodysplasia and plasma cell dyscrasias (Nat. Cell Biol. 2008;10:802-11). By analyzing primary AML samples from bone marrow (BM) or peripheral blood (PB) we detected downregulation of Cdh1 in the vast majority of samples when compared to normal CD34+ HSCs. Progression through the cell cycle is tightly regulated by different cyclin-dependent kinases (Cdks) and their activating cyclin subunits. Stage-specific proteolysis of cyclins and other cell cycle regulators is important for transition to the next cell cycle phase. The anaphase-promoting complex/cyclosome (APC/C) is an E3-ubiquitin ligase that controls mitosis and G1 through degradation of these proteins. Through its activating subunits Cdh1 and Cdc20 the APC/C ensures substrate-specifity. While Cdc20 regulates progression through mitosis, Cdh1 is activated in late mitosis to coordinate accurate entry into S-phase. Thereby, the APC/C is crucial for maintaining genomic stability during the cell cycle. Suppression of APC/C-Cdh1 can lead to unscheduled cyclin expression and Cdk activity, which can cause cell cycle defects leading to the accumulation of DNA alterations and further to malignant transformations. However, the exact nature of the origin of genomic instability upon downregulation of Cdh1 is unclear. Methods To investigate stability of cyclins in Cdh1-knockdown (kd) cells, origin loading and start of replication, cells were released from a mitotic block and samples were taken every 2 h until S-phase entry for FACS and immunoblotting. For live-cell imaging cells were seeded 24 h before imaging in chambered coverslips, after which progression through the cell cycle was analyzed by automated microscopy. Results Characterization of a Cdh1-kd revealed strong stabilization of the substrates cyclin A/B leading to diminished loading of mini-chromosome maintenance (MCM) proteins on replication origins in G1. Stabilization of cyclin A/B and unscheduled Cdk1/2 activity may cause the observed premature entry into S-phase, while the reduced loading of MCMs in G1 could be responsible for the prolonged replication in S-phase seen in Cdh1-kd cells. Accordingly, treatment with the Cdk1 inhibitor RO-3306 restored reduced MCM loading. Polo-like kinase 1 (Plk1) was stabilized in Cdh1-kd cells, which may cause bypass of the Cdc14B-Cdh1-Plk1 dependent DNA damage checkpoint. Indeed, potential replication stress in Cdh1-kd cells did not lead to G2/M arrest, but was enforced by inhibition of the Cdh1 substrate Plk1. Underreplicated DNA and replication intermediates in mitosis may be the reason for increased genomic instability, namely lagging chromosomes, anaphase bridges and micronuclei in Cdh1-kd cells detected by live-cell imaging. In addition, aberrant cytokinesis and the development of polyploid cells generated by misseparation of chromosomes during mitosis were enhanced in Cdh1-kd cells. Finally, monitoring of 53BP1, a DNA-repair marker, in living cells showed amplified DNA-damage through increased double-strand breaks in Cdh1-kd cells. Conclusions Downregulation of the tumor suppressor APC/C-Cdh1 leads to deregulation of DNA-replication by stabilizing cyclin A and B in G1 and reduced loading of replication origins with MCM proteins resulting in the accumulation of enhanced genomic instability and DNA damage. Disclosures: No relevant conflicts of interest to declare.
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Schmidts, Andrea, Dagmar Wider, Julia Felthaus, Manuel Hein, Dominik Schnerch, Monika Engelhardt, and Ralph M. Waesch. "APC/C-Cdh1 as a Novel Regulator of Hematopoietic Stem Cell Differentiation." Blood 116, no. 21 (November 19, 2010): 1563. http://dx.doi.org/10.1182/blood.v116.21.1563.1563.
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Abstract Abstract 1563 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by targeting various cell cycle regulators for proteasomal destruction. APC/C in conjunction with its adaptor protein Cdh1, both stabilizes G1-Phase (a pre-condition for an accurate cell cycle progression) and is involved in the induction of cell cycle arrest and differentiation. Further evidence suggests that Cdh1 is involved in the differentiation of a variety of cells such as neurons, myocytes, hepatocytes and lens epithelial cells. During differentiation Cdh1 interacts with the TGFb signaling pathway, targets Id2 for destruction and indirectly leads to accumulation of p27 by Skp2 degradation. We have been able to demonstrate that the expression of Cdh1 is decreased in both, AML cell lines and in primary blast samples carrying the translocation t(8;21), which consequently leads to an AML1/Eto fusion protein and is one of the most common chromosomal rearrangements in AML. Furthermore, we have data suggesting that APC/C-Cdh1 significantly influences the differentiation of malignant myeloid cells. Here, we have analyzed the expression of Cdh1, its target proteins and relevant cell cycle regulators during normal myeloid differentiation. Methods: A cytokine cocktail consisting of SCF (50ng/ml), IL-3 (5 ng/ml) and G-CSF (100 ng/ml) was used to induce differentiation of CD34+ cells into CD11b+ macrophage-like cells over seven days. Daily protein isolation, CD11b-FACS and FACS analysis of propidium iodide staining were performed to analyze Cdh1 status, differentiation kinetics and cell cycle distribution. In addition, we have established a Cdh1 knockdown in CD34+ cells by lentiviral vector mediated RNA interference. By means of GFP-cell-sorting the initially achieved transduction efficiency of 30% in CD34+ cells was increased to 70–80%. Results: The differentiation experiments carried out with normal CD34+ cells showed that after 7 days of stimulation the predominant majority of them had lost the CD34 marker and about 30% expressed CD11b on their surface confirming previous results. During the differentiation process an initial rise in Cdh1 levels, followed by a continuously high expression, was observed. Furthermore, we detected a downregulation of the Cdh1 target proteins Id2 and Skp2 and stable protein levels of p27. The cell cycle profile indicated an initial proliferation with an incremental G2/M-peak and at day 6 increasing apoptosis with a high Sub-G1-peak. In the transduced CD34+ cells we have been able to confirm by Western blotting analysis and RT-PCR that the CD34 positive cells harboring a Cdh1 shRNA had significantly decreased protein and RNA levels of Cdh1 compared to CD34 positive cells harboring a control shRNA against GFP. Analysis of the influence of Cdh1 knockdown on differentiation of CD34+ cells is ongoing and will be presented at the meeting. Conclusion: These results are consistent with the important role of Cdh1 in initiating differentiation and also show its sustained function in post mitotic myeloid cells. Studying the differentiation characteristics of CD34+ cells with a Cdh1 knockdown is likely to help to further determine its function. Disclosures: No relevant conflicts of interest to declare.
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Ewerth, Daniel, Stefanie Kreutmair, Andrea Schmidts, Marie Follo, Dagmar Wider, Julia B. Schueler, Julia Felthaus, et al. "The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs." Blood 128, no. 22 (December 2, 2016): 2650. http://dx.doi.org/10.1182/blood.v128.22.2650.2650.
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Abstract Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.
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Bhattacharjee, Debanjan, Sreeram Kaveti, and Nishant Jain. "APC/C CDH1 ubiquitinates STAT3 in mitosis." International Journal of Biochemistry & Cell Biology 154 (January 2023): 106333. http://dx.doi.org/10.1016/j.biocel.2022.106333.
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Nagai, Masayoshi, Atsuko Shibata, and Takashi Ushimaru. "Cdh1 degradation is mediated by APC/C–Cdh1 and SCF–Cdc4 in budding yeast." Biochemical and Biophysical Research Communications 506, no. 4 (December 2018): 932–38. http://dx.doi.org/10.1016/j.bbrc.2018.10.179.
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Park, Hyun Jung, Robert H. Costa, Lester F. Lau, Angela L. Tyner, and Pradip Raychaudhuri. "Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Proteolysis of the Forkhead Box M1 Transcription Factor Is Critical for Regulated Entry into S Phase." Molecular and Cellular Biology 28, no. 17 (June 23, 2008): 5162–71. http://dx.doi.org/10.1128/mcb.00387-08.
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ABSTRACT The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G1 phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G1 phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G1 phase and that its proteolysis is important for regulated entry into S phase.
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Fanyi, Meng, Qiang Wang, Hongsheng Zhou, Mo Yang, and Liu Xiaoli. "Imatinib and Bortezomib Induce the Expression and Distribution of Anaphase-Promoting Complex Cdh1 in Blast Crisis of Chronic Myelogenous Leukemia." Blood 118, no. 21 (November 18, 2011): 4428. http://dx.doi.org/10.1182/blood.v118.21.4428.4428.
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Abstract Abstract 4428 Introduction: CML is well-known as the best understood human malignancy and a paradigm for cancer research from bench to bedside. However, treatment options for imatinib-resistant patients are still limited. Imatinib (IM) has already established as the standard first-line therapy for patients with chronic-phase CML. Bortezomib (BOR) not only prolonged life span for relapsed multiple myeloma(MM) patients, but also induced cell apoptosis in CML. However, the efficacy of TKIs and bortezomib on imatinib-resistant CML remains obscure. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. Cdh1, the adaptor protein of APC/C, in conjunction with APC/C in G1-phase, is required for preventing unscheduled proliferation and protecting primary mammalian cells from genomic instability. Cdh1-deficient cells showed a large variety of chromosomal aberrations. On the contrary, Cdc20, the substrate of Cdh1, could contribute to genetic aberration. However, it’s marginally addressed about the relationship between interaction of Cdh1-Cdc20 with IM resistance and genetic aberrations. Methods: We treated K562-WT and IM-resistant K562 cells with IM/nilotinib(AMN) and BOR, verified the effect of TKIs and BOR by FACS, and explored the change of the expression and the subcellular localization of Cdh1 by immunoblot and immunofluorescence microscopic analysis. Then, we enrolled ten patients with newly diagnosed CML-BC and dissected Cdh1-Skp2-p27 cascade in primary CML cells by immunoblot analysis. We explored the subcellular localization and interaction in space between Cdh1 and Skp2 by immunofluorescence in IM-sensitive or resistant primary cells and cell line. Moreover, the single siRNAs of Cdh1 and Skp2 were designed and were transiently transfected with HiPerFect Transfection Reagent. The expressions of Cdh1-Skp2-p27 pathway proteins were detected by immunoblot, the change of cell cycle and apoptosis were detected by flow cytometry. Result: We found that IM and BOR induced cell cycle quiescence and arrest, and changed the expression and nuclear relocation of Cdh1 in CML-BC cells. Moreover, AMN and BOR resulted in up-regulation of Cdh1 in IM-resistant cells. Our study revealed Cdh1 and Skp2 were co-localized more abundantly in the cytoplasm of IM-sensitive cells, but co-distributed to nuclears in IM-resistant cells. Furthermore, Cdh1 was lower level in IM-resistant CML-blast crisis (BC) than that of IM-sensitive patients. On the other side, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells. Conclusion: Expression and dynamic distribution of Cdh1, induced by IM and BOR, provide a novel interpretation of underlying mechanism to inhibit BCR-ABL downstream cascade via Cdh1-Skp2-p27 axis. Furthermore, we demonstrated BOR still exerted the regulation effect on expression and relocation of Cdh1 even in IM-resistant CML-BC cells, which provided evidences for synergy combination of TKI and proteasome inhibitor for overcoming IM-resistance. Besides, our results revealed Cdh1 tends to be related to genomic stability in CML-BC. Disclosures: No relevant conflicts of interest to declare. Footnotes:* Asterisk with author names denotes non-ASH members.
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Su, Hua, Qiang Wan, Xiu-Juan Tian, Fang-Fang He, Pan Gao, Hui Tang, Chen Ye, et al. "MAD2B contributes to podocyte injury of diabetic nephropathy via inducing cyclin B1 and Skp2 accumulation." American Journal of Physiology-Renal Physiology 308, no. 7 (April 1, 2015): F728—F736. http://dx.doi.org/10.1152/ajprenal.00409.2014.
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It is well documented that mitotic arrest deficiency (MAD)2B can inhibit the anaphase-promoting complex/cyclosome (APC/C) via cadherin (Cdh)1 and, consequently, can destroy the effective mitotic spindle checkpoint control. Podocytes have been observed to rapidly detach and die when being forced to bypass cell cycle checkpoints. However, the role of MAD2B, a cell cycle regulator, in podocyte impairment of diabetic nephropathy (DN) is unclear. In the present study, we investigated the significance of MAD2B in the pathogenesis of DN in patients, an animal model, and in vitro podocyte cultures. By Western blot and immunohistochemistry analyses, we found that MAD2B was evidently upregulated under high glucose milieu in vivo and in vitro, whereas Cdh1 was inhibited with high glucose exposure. Overexpression of MAD2B in podocytes by plasmid DNA transfection suppressed expression of Cdh1 and triggered the accumulation of cyclin B1 and S phase kinase-associated protein (Skp)2, two key molecules involving in cell cycle regulation, and the subsequent podocyte insult. In contrast, MAD2B deletion alleviated the high glucose-induced reduction of Cdh1 as well as the elevation of cyclin B1 and Skp2, which rescued the podocyte from damage. Taken together, our data demonstrate that MAD2B may play an important role in high glucose-mediated podocyte injury of DN via modulation of Cdh1, cyclin B1, and Skp2 expression.
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Ewerth, Daniel, Stefanie Kreutmair, Birgit Kügelgen, Dagmar Wider, Julia Felthaus, Julia Schüler, Andrea Schmidts, et al. "Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1." Blood 126, no. 23 (December 3, 2015): 2370. http://dx.doi.org/10.1182/blood.v126.23.2370.2370.
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Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.
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Edgerton-Morgan, Heather, and Berl R. Oakley. "γ-Tubulin plays a key role in inactivating APC/CCdh1 at the G1–S boundary." Journal of Cell Biology 198, no. 5 (August 27, 2012): 785–91. http://dx.doi.org/10.1083/jcb.201203115.
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A γ-tubulin mutation in Aspergillus nidulans, mipA-D159, causes failure of inactivation of the anaphase-promoting complex/cyclosome (APC/C) in interphase, resulting in failure of cyclin B (CB) accumulation and removal of nuclei from the cell cycle. We have investigated the role of CdhA, the A. nidulans homologue of the APC/C activator protein Cdh1, in γ-tubulin–dependent inactivation of the APC/C. CdhA was not essential, but it targeted CB for destruction in G1, and APC/CCdhA had to be inactivated for the G1–S transition. mipA-D159 altered the localization pattern of CdhA, and deletion of the gene encoding CdhA allowed CB to accumulate in all nuclei in strains carrying mipA-D159. These data indicate that mipA-D159 causes a failure of inactivation of APC/CCdhA at G1–S, perhaps by altering its localization to the spindle pole body, and, thus, that γ-tubulin plays an important role in inactivating APC/CCdhA at this point in the cell cycle.
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Tyagi, Apoorvi, Neha Sarodaya, Kamini Kaushal, Arun Pandian Chandrasekaran, Ainsley Mike Antao, Bharathi Suresh, Byung Ho Rhie, Kye Seong Kim, and Suresh Ramakrishna. "E3 Ubiquitin Ligase APC/CCdh1 Regulation of Phenylalanine Hydroxylase Stability and Function." International Journal of Molecular Sciences 21, no. 23 (November 28, 2020): 9076. http://dx.doi.org/10.3390/ijms21239076.
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Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.
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Caggiari, Laura, Mara Fornasarig, Mariangela De Zorzi, Renato Cannizzaro, Agostino Steffan, and Valli De Re. "Family’s History Based on the CDH1 Germline Variant (c.360delG) and a Suspected Hereditary Gastric Cancer Form." International Journal of Molecular Sciences 21, no. 14 (July 11, 2020): 4904. http://dx.doi.org/10.3390/ijms21144904.
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Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by germline pathogenic variant in CDH1, the gene encoding E-cadherin. The germline loss-of-function variants are the only proven cause of the cancer syndrome HDGC, occurring in approximately 10–18% of cases and representing a helpful tool in genetic counseling. The current case reports the family history based on a CDH1 gene variant, c.360delG, p.His121Thr in a suspected family for hereditary gastric cancer form. This frameshift deletion generates a premature stop codon at the amino acid 214, which leads to a truncated E-cadherin protein detecting it as a deleterious variant. The present study expands the mutational spectra of the family with the CDH1 variant. Our results highlight the clinical impact of the reported CDH1 variant running in gastric cancer families.
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Ondracka, Andrej, Jonathan A. Robbins, and Frederick R. Cross. "An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition." PLOS ONE 11, no. 7 (July 13, 2016): e0159166. http://dx.doi.org/10.1371/journal.pone.0159166.
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Lin, Bichen, Yang Liu, Lanxin Su, Hangbo Liu, Hailan Feng, Miao Yu, and Haochen Liu. "A Novel CDH1 Variant Identified in a Chinese Family with Blepharocheilodontic Syndrome." Diagnostics 12, no. 12 (November 24, 2022): 2936. http://dx.doi.org/10.3390/diagnostics12122936.
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The goal of the current study was to identify the pathogenic gene variant in a Chinese family with Blepharocheilodontic (BCD) syndrome. Whole-exome sequencing (WES) and Sanger sequencing were used to identify the pathogenic gene variant. The harmfulness of the variant was predicted by bioinformatics. We identified a novel heterozygous missense variant c.1198G>A (p.Asp400Asn) in the CDH1 gene in the proband and his mother with BCD syndrome. The sequencing results of three healthy individuals in this family are wild type. This result is consistent with familial co-segregation. According to ReVe, REVEL, CADD, gnomAD, dbSNP, and the classification of pathogenic variants with the standards of the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG), c.1198G>A (p.Asp400Asn) is predicted to be a likely pathogenic. We observed that variant c.1198G>A (p.Asp400Asn) was located in the extracellular cadherin-type repeats in CDH1. Amino acid sequence alignment of the CDH1 protein among multiple species showed that Asp400 was highly evolutionarily conserved. The conformational analysis showed that this variant might cause structural damage to the CDH1 protein. Phenotypic analysis revealed unique dental phenotypes in patients with BCD syndrome, such as oligodontia, conical-shaped teeth, and notching of the incisal edges. Our results broaden the variation spectrum of BCD syndrome and phenotype spectrum of CDH1, which can help with the clinical diagnosis, treatment, and genetic counseling in relation to BCD syndrome.
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Fujita, T., G. Wu, R. D. Wood, and Y. Wan. "Clinical significance of Cdh1-Skp2 cascade in breast cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 10609. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.10609.
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10609 Background: Skp2 (S-phase kinase-associated protein 2) is substrate-recognizing subunit of an ubiquitin E3 ligase SCF (Skp1, Cullin and F-box) complex and has a major role in p27 regulation. Overexpression of Skp2 is frequently seen in human tumors, often correlating with poor prognosis. Anaphase-promoting complex/cyclosome (APC/C) is another most prominent ubiquitin E3 ligase in cell cycle control, and its function is tightly controlled by activating subunit Cdh1 (Fizzy-related). APC-Cdh1 complex is supposed to be involved in Skp2 proteolysis and have essential role for S phase entry, however clinical significance of Cdh1 have not evaluated yet. Herein we examined the significance of Cdh1 in breast cancer. Methods: Firstly, MCF7 human breast cancer cells and MCF10A normal breast epithelial cells were analyzed in vitro and in vivo. Thereafter, using tissue microarray, we evaluate the expression profile of Cdh1, Skp2 and p27 in both breast cancer and normal breast epithelial tissue. Moreover, clinicopathological significance (age, tumor size, lymph node metastasis, distant metastasis, histological grade, and stage) of Cdh1 was analyzed from 126 breast cancer patients. Chi square-test, Fisher's exact test were used for statistical analysis of immunostaining results and clinicopathological data. p<0.05 was considered statistically significant. Results: Overexpression of Cdh1 induced attenuation of Skp2 and increased p27 protein expression, resulted in growth suppression. Moreover, knockdown of Cdh1 promoted higher Skp2 expression and S-phase population, reduced p27 and consequently induced cell transformation and proliferation in vitro and in vivo. Tissue microarray results appeared that positive Cdh1 and p27 was more frequently seen in normal breast tissue and statistically significant. On the other hand, Skp2 was less in normal tissue. Furthermore, Cdh1 positive breast cancer was more frequently seen in low histological grade tumors and statistically significant (p=0.04). Conclusions: Skp2 protein expression is regulated by Cdh1 in breast cancer. Cdh1 expression could be possible novel biomarker in patient with breast cancer. No significant financial relationships to disclose.
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Ke, Po-Yuan, and Zee-Fen Chang. "Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Pathway." Molecular and Cellular Biology 24, no. 2 (January 15, 2004): 514–26. http://dx.doi.org/10.1128/mcb.24.2.514-526.2004.
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ABSTRACT The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G1 phase of the cell cycle in mammalian cells.
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Imtiaz, Hasnain, Sharmin Afroz, Md Amir Hossain, Sm Faysal Bellah, Md Mostafizur Rahman, Md Shahin Kadir, Razia Sultana, Md Abdul Mazid, and Md Mustafizur Rahman. "Genetic polymorphisms in CDH1 and Exo1 genes elevate the prostate cancer risk in Bangladeshi population." Tumor Biology 41, no. 3 (March 2019): 101042831983083. http://dx.doi.org/10.1177/1010428319830837.
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The polymorphisms of invasion suppressor gene CDH1 and DNA mismatch repair gene Exo1 have been reported to play critical role in the development, tumorigenesis, and progression of several kinds of cancers including prostate cancer. This study was designed to analyze the contribution of single-nucleotide polymorphisms of the CDH1 (-160C/A) and Exo1 (K589E) to prostate cancer susceptibility in Bangladeshi population. The study included 100 prostate cancer cases and age-matched 100 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to determine the genetic polymorphisms. A significant association was found between CDH1 -160C/A (rs16260) and Exo1 (rs1047840, K589E) polymorphisms and prostate cancer risk. In case of CDH1 -160C/A polymorphism, the frequencies of the three genotypes C/C,C/A, and A/A were 45%, 48%, and 7% in cases and 63%, 32%, and 5% in controls, respectively. The heterozygote C/A genotype and combined C/A + A/A genotypes showed 2.10-fold (odds ratio = 2.1000, 95% confidence interval = 1.2956–4.0905, p = 0.013) and 2.08-fold (odds ratio = 2.0811, 95% confidence interval = 1.1820–3.6641, p = 0.011) increased risk of prostate cancer, respectively, when compared with homozygous C/C genotypes. The variant A allele also was associated with increased risk of prostate cancer (odds ratio = 1.6901, 95% confidence interval = 1.0740–2.6597, p = 0.0233). In case of Exo1 (K589E) polymorphism, G/A heterozygote, A/A homozygote, and combined G/A + A/A genotypes were found to be associated with 2.30-, 4.85-, and 3.04-fold higher risk of prostate cancer, respectively (odds ratio = 2.3021, 95% confidence interval = 2.956–4.0905, p = 0.0031; odds ratio = 4.8462, 95% confidence interval = 1.0198–23.0284, p = 0.0291; OR = 3.0362, 95% confidence interval = 1.7054–5.4053, p = 0.0001, respectively). The “A” allele showed significant association with increased susceptibility (2.29-fold) to prostate cancer (odds ratio = 2.2955, 95% confidence interval = 1.4529–3.6270, p = 0.0004). Our results suggest that CDH1 -160C/A and Exo1 K589E polymorphisms are associated with increased susceptibility to prostate cancer in Bangladeshi population.
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Norero, Enrique, M. Alejandra Alarcon, Christopher Hakkaart, Tomas de Mayo, Cecilia Mellado, Marcelo Garrido, Gloria Aguayo, et al. "Identification of c.1531C>T Pathogenic Variant in the CDH1 Gene as a Novel Germline Mutation of Hereditary Diffuse Gastric Cancer." International Journal of Molecular Sciences 20, no. 20 (October 9, 2019): 4980. http://dx.doi.org/10.3390/ijms20204980.
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Germline pathogenic variants in the CDH1 gene are a well-established cause of hereditary diffuse gastric cancer (HDGC) syndrome. The aim of this study was to characterize CDH1 mutations associated with HDGC from Chile, a country with one of the highest incidence and mortality rates in the world for gastric cancer (GC). Here, we prospectively include probands with family history/early onset of diffuse-type of GC. The whole coding sequence of the CDH1 gene was sequenced from genomic DNA in all patients, and a multidisciplinary team managed each family member with a pathogenic sequence variant. Thirty-six cases were included (median age 44 years/male 50%). Twenty-seven (75%) patients had diffuse-type GC at ≤50 years of age and 19 (53%) had first or second-degree family members with a history of HDGC. Two cases (5.5%) carried a non-synonymous germline sequence variant in the CDH1 gene: (a) The c.88C>A missense variant was found in a family with three diffuse-type GC cases; and (b) c.1531C>T a nonsense pathogenic variant was identified in a 22-year-old proband with no previous family history of HDGC. Of note, six family members carry the same nonsense pathogenic variant. Prophylactic gastrectomy in the proband’s sister revealed stage I signet-ring cell carcinoma. The finding of 1531C>T pathogenic variant in the CDH1 in proband with no previous family history of HDGC warrants further study to uncover familial clustering of disease in CDH1 negative patients. This finding may be particularly relevant in high incidence countries, such as the case in this report.
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Li, Min, J. Philippe York, and Pumin Zhang. "Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos." Molecular and Cellular Biology 27, no. 9 (February 26, 2007): 3481–88. http://dx.doi.org/10.1128/mcb.02088-06.
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ABSTRACT The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.
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Schmidts, Andrea, Daniel Ewerth, Dagmar Wider, Birgit Kuegelgen, Monika Engelhardt, and Ralph Waesch. "APC/CCdh1 Regulates Self-Renewal and Differentiation of Hematopoietic Stem Cells." Blood 118, no. 21 (November 18, 2011): 2379. http://dx.doi.org/10.1182/blood.v118.21.2379.2379.
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Abstract Abstract 2379 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by ubiquitinylation of various cell cycle regulators to tag them for proteasomal degradation. APC/C in conjunction with its adaptor protein Cdh1 (APC/CCdh1), both stabilizes G1-phase and is involved in the induction of cell cycle arrest and differentiation. Here, we have analyzed the influence of APC/CCdh1 on self-renewal capacity and differentiation potential and kinetics of human hematopoietic stem cells (HSCs). Methods: In order to study the expression levels of Cdh1 among different hematopoietic lineages, we stained mononuclear cells from bone marrow of healthy donors with antibodies against the cell surface markers CD11b, Glycophorin A, CD41a, CD34, CD3 and CD19, and isolated these subsets via cell sorting. We extracted protein from these subsets and performed Western Blot analysis. We established a strong lentiviral Cdh1 knock down (kd) in CD34+ cells and performed colony forming cell (CFC) assays: 1×104 Cdh1-kd-CD34+ cells and CD34+ cells transduced with a control vector (ctrl-CD34+) were plated in MethoCult H4534 and MethoCult H4534 + EPO (1 IU/ml). At 14 days, CFU-GEMM, CFU-GM, CFU-G, CFU-M and BFU-E were scored. CD11b- and Glycophorin A-FACS, FACS analysis of propidium iodide staining and Pappenheim's staining were carried out on individually picked colonies in order to assess differentiation kinetics and cell cycle distribution. Self-renewal capacity of ctrl- and Cdh1-kd-CD34+ cells was examined by performing replating assays (secondary CFCs) with the obtained CFU-GEMM colonies as previously described (Katayama et al., BMT, 1999). Secondary colonies were analyzed after another 14 days of incubation. Results: We observed Cdh1 protein levels to significantly vary among hematopoietic cell subsets: The highest Cdh1 levels were detected in CD34+ cells, lower levels in cells of the lymphoid lineage (CD3+; CD19+) and only marginal expression levels in cells arising from myeloid progenitors (CD11b; Glycophorin A; CD41a). By correlating Cdh1 levels of the individual cell subsets with their cell cycle profiles, we were able to exclude the possibility that this was merely due to differences in cell cycle distribution. Analysis of the CFC assays performed with the lentiviraly infected CD34+ cells showed a considerable decrease of about 40% in the number of BFU-E and 35% in the number of CFU-G- and CFU-M-numbers, when Cdh1-kd-CD34+ cells were plated compared to ctrl-CD34+ cells. Furthermore, we observed an increase of CFU-GEMMs with Cdh1 depletion. The expression levels of the cell surface markers CD11b and Glycophorin A were 10–20% lower among the colonies arisen from Cdh1-kd-CD34+ cells vs. ctrl-CD34+ cells. Upon manual counting of Pappenheim stained preparations, we found the early stages of both erythroid and myeloid differentiation to be more prevalent in the Cdh1-kd colonies. When studying the replating capacity, we observed that Cdh1-depleted cells gave rise to almost twice as many secondary colonies as compared to ctrl-cells. There was no difference with regard to the relative proportions of the colony types. Interestingly, tracking of GFP, which had been used as a reporter-gene for the lentiviral transduction of the ctrl- and Cdh1-kd-cells, showed that it was enhanced in Cdh1-kd-secondary colonies compared to ctrl-secondary colonies. Conclusion: By analyzing human bone marrow subsets we observed that Cdh1 levels diminish from HSCs to mature lymphoid and, to an even greater extent, mature myeloid cells, suggesting that Cdh1 is important to induce differentiation but dispensable for maintaining the differentiated state. Our in vitro results are consistent with an important role of APC/CCdh1 in both myeloid and erythroid differentiation of HSCs. The data suggests that depletion of Cdh1 in HSC interferes with normal differentiation both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. HSCs deficient in Cdh1 seem to increasingly undergo self-renewal. The stronger the Cdh1-kd the more likely the generation of secondary colonies appeared to be. In vivo models may be particularly helpful to further elucidate these phenomena. Disclosures: No relevant conflicts of interest to declare.
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Dohrn, Maike F., and Juan P. Bolaños. "Does APC/C CDH1 control the human brain size?" Journal of Neurochemistry 151, no. 1 (August 23, 2019): 8–10. http://dx.doi.org/10.1111/jnc.14835.
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Corso, Giovanni, Anna Berardi, Daniele Marrelli, Corrado Pedrazzani, Lorenzo Garosi, Enrico Pinto, and Franco Roviello. "CDH1 C-160A promoter polymorphism and gastric cancer risk." European Journal of Cancer Prevention 18, no. 1 (February 2009): 46–49. http://dx.doi.org/10.1097/cej.0b013e32830c8d70.
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Reis-Filho, Jorge S., Fresia Pareja, Fatemeh Derakhshan, David N. Brown, Jillian Sue, Pier Selenica, Yi Kan Wang, et al. "Abstract PD11-01: An artificial intelligence-based predictor of CDH1 biallelic mutations and invasive lobular carcinoma." Cancer Research 82, no. 4_Supplement (February 15, 2022): PD11–01—PD11–01. http://dx.doi.org/10.1158/1538-7445.sabcs21-pd11-01.
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Abstract Introduction: Invasive lobular carcinoma (ILC) is the most frequent special histologic subtype of breast cancer (BC). ILC is identifiable by pathologic assessment given its distinctive discohesive growth pattern, largely caused by CDH1 inactivation. Compared to common forms of BC, ILCs display lower responses to chemotherapy and selective estrogen receptor modulators. The low interobserver agreement for the diagnosis of ILC, however, renders the inclusion of histologic subtyping in therapeutic decision-making challenging. Artificial intelligence (AI)-based algorithms hold promise for improving pathologic diagnosis; their performance, however, depends on the ground truth labeling used. Here, we seek to develop an AI-based methodology for detection of ILC using ‘CDH1 biallelic mutations’ (i.e., mutation + loss-of-heterozygosity of the wild-type allele or two pathogenic somatic mutations) as ground truth, reasoning that in BC, >95% of CDH1 bi-allelic inactivation is found in ILCs.Materials and methods: We developed a convolutional neural network system to detect CDH1 biallelic genetic inactivation (AI-CDH1) using whole slide images (WSI) of 1,100 primary BCs with available targeted sequencing data. The model was trained using a 10-fold cross-validation method to detect biallelic mutations. The mean number of positive and negative samples in the training set was 85.2 (SD=2.57) and 562.8 (SD=10.51) per fold, respectively. The evaluation set consisted of a mean of 14.2 (SD=2.04) positive and 93.8 (SD=9.13) negative samples. We evaluated the performance of the AI-CDH1 classifier to predict the lobular phenotype and CDH1 status using original and revised labels, following a histopathologic re-review of the histologic type and CDH1 status curation. The latter was conducted by incorporating information on biallelic CDH1 inactivation beyond CDH1 mutations (homozygous deletions, deleterious structural rearrangements, and loss-of-heterozygosity and gene promoter methylation).Results: The AI-CDH1 classifier predicted biallelic CDH1 mutations with an area under the curve (AUC)=0.944 (95 CI: 0.925-0.963), sensitivity=91.6% and specificity=85.9%, PPV=49.8%, NPV=98.5% and accuracy=86.7%, and the original ‘lobular phenotype’ with an AUC=0.941 (95 CI: 0.922-0.960), sensitivity=89%, specificity=86.7%, PPV=55.6%, NPV=97.7% and accuracy=87.1%. Review of the CDH1 gene status revealed that 7/957 BCs lacking CDH1 biallelic mutations harbored biallelic CDH1 inactivation by promoter methylation, homozygous deletions or structural rearrangements. The AI-CDH1 classifier detected all seven reclassified BCs and predicted the revised CDH1 biallelic inactivation with an AUC=0.948 (95 CI: 0.930-0.966), sensitivity=92%, specificity=86.5%, PPV=52.3%, NPV=98.5% and accuracy=87.2%. Upon histologic re-review, which resulted in reclassification of 36/927 non-lobular BCs as ‘lobular’ and 5/173 ‘lobular’ BCs as ‘non-lobular’, the AI-CDH1 classifier detected the ‘lobular phenotype’ with an AUC=0.953 (95 CI: 0.935-0.971), sensitivity=90.7%, specificity=89.7%, PPV=66.8%, NPV=97.7% and accuracy=89.9%. Using the revised histologic re-classification and CDH1 biallelic inactivation status labels, the AI-CDH1 classifier predicted the lobular phenotype irrespective of CDH1 status (P>0.05).Conclusions: By training a machine learning system to detect ‘CDH1 biallelic mutations’, as ground truth rather than histologic diagnosis of lobular carcinoma, which might be confounded by human subjectivity, we developed an AI-based system that can detect ILCs accurately, providing a new paradigm for the development of AI-based cancer classification systems. Citation Format: Jorge S Reis-Filho, Fresia Pareja, Fatemeh Derakhshan, David N Brown, Jillian Sue, Pier Selenica, Yi Kan Wang, Arnaud Da Cruz Paula, Monami Banerjee, Zahra Ebrahimzadeh, Manuel Isava, Matthew Lee, Ran Godrich, Adam Casson, Ruben Padron, George Shaikovski, Alexander van Eck, Antonio Marra, Higinio Dopeso, Hannah Y Wen, Edi Brogi, Matthew G Hanna, Chris Kanan, Jeremy D Kunz, Felipe C Geyer, Carla Leibowitz, David Klimstra, Leo Grady, Thomas J Fuchs. An artificial intelligence-based predictor of CDH1 biallelic mutations and invasive lobular carcinoma [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD11-01.
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Schmidts, Andrea, Daniel Ewerth, Dominik Schnerch, Arunas Kvainickas, Dagmar Wider, Birgit Kügelgen, Heike L. Pahl, Monika Engelhardt, and Ralph Wäsch. "APC/CCdh1 As a Mediator Of Hematopoietic Differentiation, Self-Renewal and Malignant Transformation." Blood 122, no. 21 (November 15, 2013): 3676. http://dx.doi.org/10.1182/blood.v122.21.3676.3676.
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Abstract Introduction Cdh1 is an important activator of the anaphase-promoting complex/cyclosome (APC/C) and may play a major role in both the stabilization of G1-phase and the induction of cell cycle arrest and differentiation. Our work focuses on the function of APC/CCdh1in hematopoietic stem cells (HSCs) with regard to their potential for differentiation, self-renewal and malignant transformation. Methods Physiological expression levels of Cdh1 were studied among different human hematopoietic lineages (defined by staining for the cell surface markers CD11b, CD41a, CD34, CD3 and CD19) obtained from bone marrow (BM) of healthy donors and mobilized peripheral blood (PB). Next we established a strong lentiviral Cdh1 knock down (kd) in human CD34+ cells and performed colony forming cell (CFC) assays and replating assays. We also analyzed Cdh1-protein levels in 30 samples of BM or PB of patients first diagnosed with acute myeloid leukemia (AML). Finally experiments to further look into possible mechanisms of Cdh1 regulation during leukemogenesis were carried out. On the transcriptional level we reanalyzed published microarray data from CD34+-AML blasts and normal CD34+ cells (Leukemia 2011;25:1825-1833). On the post-transcriptional level we tested the hypothesis of Cdh1 degradation mediated by the ubiquitin-ligase SCF by expressing a dominant negative mutant of the core SCF subunit Cullin-1 (delta-Cul1) in the AML cell lines Kasumi-1 and HL-60. Results Western blot analysis of physiological Cdh1-distribution among the variable human hematopoietic lineages showed significant differences in Cdh1 protein levels. We saw diminishing levels of Cdh1 from HSCs to mature lymphoid and myeloid cells, suggesting that Cdh1 may be important to induce differentiation but dispensable for maintaining the differentiated state. In the Cdh1-kd-CFC assays a significant decrease of total colony numbers, CFU-Gs, CFU-GMs and BFU-Es >50% was observed. At morphological examination and FACS analysis these colonies proved to be more immature than the control colonies. Thus, depletion of Cdh1 in HSC hinders normal differentiation into the myeloid and erythroid lineage both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. Upon replating, we noticed a significant increase in the number of secondary colonies, with a doubling of total colony numbers, when using Cdh1 deficient HSC. This result indicates an advantage for self-renewal over differentiation in these cells, which seems to correlate with the intensity of the Cdh1-kd. Examination of Cdh1 protein levels in AML blasts revealed that basically all AML samples showed a strong down-regulation of Cdh1 protein levels compared to normal CD34+ cells, which may be a contributing factor to the differentiation block in leukemogenesis. Indeed, if we performed knockdown of Cdh1 in the HL-60 leukemia cell line they were in a less differentiated state as judged by CD11b expression. The evaluation of microarray data, in order to further address the mechanism of Cdh1 down-regulation in AML blasts, showed that Cdh1 transcription levels were not significantly different in CD34+ AML cells compared to normal CD34+ cells. This would be consistent with a posttranscriptional cause of decreased Cdh1-protein levels in AML blasts. Our ongoing work indicates SCF-dependent degradation of Cdh1, since inhibition of the SCF function (by expression of a dominant-negative form of the SCF subunit Cullin-1 (delta-Cul1)) in AML cell lines leads to a strong upregulation of Cdh1. Conclusions Our data establish Cdh1 as an important cell cycle regulator in the regulation of differentiation and self-renewal in HSCs. Its posttranscriptional downregulation by the SCF ubiquitin ligase may contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.
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Davis, Andrew A., Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, et al. "Abstract PD14-01: Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity." Cancer Research 82, no. 4_Supplement (February 15, 2022): PD14–01—PD14–01. http://dx.doi.org/10.1158/1538-7445.sabcs21-pd14-01.
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Abstract Background: ILC is the second most common type of breast cancer and accounts for approximately 10% of all invasive breast cancers. A hallmark of ILC is the lack of E-cadherin (CDH1) expression, which is frequently used to discriminate between lesions with borderline ductal and lobular histologies. While the genomic landscape of primary ILCs has been well described, less is known about patients (pts) with metastatic-ILC (mILC). Better characterization of the genomic landscape associated with mILC is critical for identifying biomarkers that may provide new insight into ILC tumor biology and ultimately improve long-term outcomes in pts with mILC. Methods: We retrospectively analyzed de-identified next-generation sequencing (NGS) data from 150 advanced/metastatic pts with ILC and 51 with mixed lobular/ductal histology, defined using the histology of the sequenced biopsy. Diagnoses were abstracted from pathology reports submitted at the time of sequencing. We used the stage documented closest in time to biopsy collection, and samples were excluded if the staging date was unknown or exceeded 180 days after the biopsy date. Our dataset consisted of samples that were molecularly profiled using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, full-transcriptome RNA-seq). The mutations identified for this study include somatic single-nucleotide variants and insertions/deletions. Furthermore, we examined the co-mutational landscape of CDH1-mutant disease and investigated transcript-level expression variation. Results: Mutations in CDH1 occurred in 65.3% of all mILC samples (98/150). CDH1 expression was similar between CDH1-mutant and WT mILC samples (Wilcoxon rank sum test, p=0.8). The median tumor mutational burden (TMB) score was significantly higher in CDH1-mutant samples (Wilcoxon rank-sum test, p=0.010). CDH1-mutant samples were more likely to have a high TMB (≥10 mutations/MB) when compared with the wild-type CDH1 cohort (10% vs. 6.2%), but this difference was not statistically significant (Fisher’s Exact test, p=0.5). Additionally, we observed that the ER+ subtype was more frequent in CDH1-mutant samples, although this difference was not statistically significant (97% vs 88%; Fisher’s exact test, p=0.063). PIK3CA mutations were enriched in CDH1-mutant mILC (Table 1). TBX3 and NCOR1 mutations were also mildly enriched in CDH1-mutant mILC, but these results were not significant when correcting for multiple testing (Table 1). CDH1-mutant mixed histology pts had lower CDH1 expression than WT pts (p<0.001, Wilcoxon rank sum exact test). PIK3CA mutations were enriched in CDH1-mutant mixed histology pts, but this difference was not statistically significant (50% vs. 31%; p=0.3, Fisher’s exact test). Log10 CDH1 expression across all mILC pts was lower than in mixed histology pts (3.01 vs 3.53; p<0.001, Wilcoxon rank-sum test). Conclusions: Our real-world dataset illustrates that the molecular landscape of CDH1-mutant mILC pts is distinct from CDH1-WT mILC pts. Additionally, mILC differs from mixed histology at a transcriptional level, with lower CDH1 expression regardless of CDH1 mutational status. Our findings suggest a use for CDH1 RNA expression levels in reclassifying mixed histology samples as mILC. Additionally, therapies targeting PIK3CA may be further investigated for their actionability in CDH1-mutant mILC cases. Table 1.Frequency of co-mutations in CDH1-mutant vs. WT mILC cohortsGenesCDH1-mutant (n=98)CDH1 WT (n=52)p-value1q-value2n (%)n (%)PIK3CA53 (54%)6 (12%)<0.001<0.001TBX313 (13%)0 (0%)0.0040.13NCOR111 (11%)0 (0%)0.0090.21Pearson’s Chi-squared test; Fisher’sexact test. 2Falsediscovery rate correction for multiple testing Citation Format: Andrew A Davis, Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, Ami N Shah, Paolo D’Amico, Lisa Flaum, William J Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-01.
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Hein, Manuel, Dominik Schnerch, Andrea Schmidts, Julia Felthaus, Dagmar Wider, Gabriele Ihorst, Monika Engelhardt, and Ralph M. Waesch. "Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML." Blood 114, no. 22 (November 20, 2009): 5045. http://dx.doi.org/10.1182/blood.v114.22.5045.5045.
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Abstract Abstract 5045 Introduction The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and Results We have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. Conclusion This is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. Disclosures No relevant conflicts of interest to declare.
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Aulia, Selina, and Bor Luen Tang. "Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer’s disease pathology." Biochemical and Biophysical Research Communications 339, no. 1 (January 2006): 1–6. http://dx.doi.org/10.1016/j.bbrc.2005.10.059.
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Raff, Jordan W., Kim Jeffers, and Jun-yong Huang. "The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time." Journal of Cell Biology 157, no. 7 (June 24, 2002): 1139–49. http://dx.doi.org/10.1083/jcb.200203035.
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In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box–mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]–GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM–GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM–GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.
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Min, Mingwei, Tycho E. T. Mevissen, Maria De Luca, David Komander, and Catherine Lindon. "Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages." Molecular Biology of the Cell 26, no. 24 (December 2015): 4325–32. http://dx.doi.org/10.1091/mbc.e15-02-0102.
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The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.
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Kim, Wantae, Yong Suk Cho, Xiaohui Wang, Ogyi Park, Xueyan Ma, Hanjun Kim, Wenjian Gan, et al. "Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1." Proceedings of the National Academy of Sciences 116, no. 19 (April 18, 2019): 9423–32. http://dx.doi.org/10.1073/pnas.1821370116.
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The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.
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Tsunematsu, Takaaki, Rieko Arakaki, Hidehiko Kawai, Jan Ruppert, Koichi Tsuneyama, Naozumi Ishimaru, William C. Earnshaw, Michele Pagano, and Yasusei Kudo. "APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit." Journal of Cell Science 133, no. 18 (September 15, 2020): jcs251314. http://dx.doi.org/10.1242/jcs.251314.
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ABSTRACTDuring mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.
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Barbosa-Matos, Rita, Rafaela Leal Silva, Luzia Garrido, Ana Cerqueira Aguiar, José Garcia-Pelaez, Ana André, Susana Seixas, et al. "The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing." Cancers 13, no. 17 (September 4, 2021): 4464. http://dx.doi.org/10.3390/cancers13174464.
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Hereditary diffuse gastric cancer (HDGC) caused by CDH1 variants predisposes to early-onset diffuse gastric (DGC) and lobular breast cancer (LBC). In Northern Portugal, the unusually high number of HDGC cases in unrelated families carrying the c.1901C>T variant (formerly known as p.A634V) suggested this as a CDH1-founder variant. We aimed to demonstrate that c.1901C>T is a bona fide truncating variant inducing cryptic splicing, to calculate the timing of a potential founder effect, and to characterize tumour spectrum and age of onset in carrying families. The impact in splicing was proven by using carriers’ RNA for PCR-cloning sequencing and allelic expression imbalance analysis with SNaPshot. Carriers and noncarriers were haplotyped for 12 polymorphic markers, and the decay of haplotype sharing (DHS) method was used to estimate the time to the most common ancestor of c.1901C>T. Clinical information from 58 carriers was collected and analysed. We validated the cryptic splice site within CDH1-exon 12, which was preferred over the canonical one in 100% of sequenced clones. Cryptic splicing induced an out-of-frame 37bp deletion in exon 12, premature truncation (p.Ala634ProfsTer7), and consequently RNA mediated decay. The haplotypes carrying the c.1901C>T variant were found to share a common ancestral estimated at 490 years (95% Confidence Interval 445–10,900). Among 58 carriers (27 males (M)–31 females (F); 13–83 years), DGC occurred in 11 (18.9%; 4M–7F; average age 33 ± 12) and LBC in 6 females (19.4%; average age 50 ± 8). Herein, we demonstrated that the c.1901C>T variant is a loss-of-function splice-site variant that underlies the first CDH1-founder effect in Portugal. Knowledge on this founder effect will drive genetic testing of this specific variant in HDGC families in this geographical region and allow intrafamilial penetrance analysis and better estimation of variant-associated tumour risks, disease age of onset, and spectrum.
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Kague, Erika, Cristiane Melissa Thomazini, Maria Inês de Campo Moura Pardini, Fabrício de Carvalho, Celso Vieira Leite, and Nídia Alice Pinheiro. "Methylation status of CDH1 gene in samples of gastric mucous from brazilian patients with chronic gastritis infected by Helicobacter pylori." Arquivos de Gastroenterologia 47, no. 1 (March 2010): 7–12. http://dx.doi.org/10.1590/s0004-28032010000100002.
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CONTEXT: Gastric cancer is one of the top list of cancer types that most leads to death in Brazil and worldwide. Helicobacter pylori(H. pylori) is a class I carcinogen and infect almost 90% of chronic gastritis patients. Some genotypes confer different virulent potential to H. pylori and can increase the risk of gastritis development. Methylation of CpG islands can inactivate tumor suppressor genes and therefore, it can be involved in the tumorigenic process. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is important in maintaining cell-cell contacts. The inactivation of this gene can increase the chance of metastasis. Promoter methylation of CDH1 at early steps of gastric carcinogenesis is not yet completely understood. OBJECTIVE: In this study, we investigated the methylation status of CDH1 in chronic gastritis samples and correlated it with the presence of H. pylori. METHODS: Sixty gastric mucosal biopsies were used in this study. The detection of H. pylori was performed with the PCR primers specific to urease C gene. H. pylori genotyping was performed by PCR to cagA and vacA (s and m region). The methylation status of these gene CDH1 was analyzed using methylation-specific polymerase chain reaction and direct sequencing of the PCR products was performed using primers methylated and unmethylated in both forward and reverse directions. RESULTS: H. pylori was detected in 90% of chronic gastritis samples; among these 33% were cagA positive and 100% vacA s1. The genotype vacA s2/m1 was not detected in any sample analyzed. Methylation of CDH1 was detected in 63.3% of chronic gastritis samples and 95% of them were also H. pylori-positive. CONCLUSION: This work suggests that CDH1 gene methylation and H. pylori infection are frequent events in samples from Brazilian patients with chronic gastritis and reinforces the correlation between H. pylori infection and CDH1 inactivation in early steps of gastric tumorigenesis.
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Ewerth, Daniel, Andrea Schmidts, Birgit Kuegelgen, Dagmar Wider, Julia Schüler, Monika Engelhardt, and Ralph M. Waesch. "Dissecting Stem Cell Proliferation and Differentiation In Association With The Central Cell Cycle Regulator APC/CCdh1In Vitro and In Vivo." Blood 122, no. 21 (November 15, 2013): 2446. http://dx.doi.org/10.1182/blood.v122.21.2446.2446.
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Abstract Introduction Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation. The stem cell fate is tightly connected with the cell cycle, where the major regulator anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks cell cycle regulatory proteins, such as cyclin A and B, for proteasomal degradation and thus controls their activity. Known targets of Cdh1 are involved in regulation of self-renewal and granulopoiesis. This raises the hypothesis that Cdh1 may be a critical mediator of HSC proliferation, self-renewal and differentiation. Methods CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokines. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with CD34 detection. The knockdown (kd) of Cdh1 was achieved by lentiviral delivery of specific shRNA into target cells. Results In vitro cultivation of CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed impressive downregulation of Cdh1 during culture. A high Cdh1 expression in CD34+ cells and lower expression in myeloid cells (CD41a+, CD15+, Glycophorin A+) reflects the situation we found in vivo in bone marrow (BM) subsets. Western blotting also revealed inactivation of Cdh1 by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the Cdk inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. When cultured under self-renewal conditions, CD34+ cells showed diminished proliferation with cells residing in lower generations, whereas during granulopoiesis, cells accumulated within higher generations. These experiments also revealed a more rapid decrease of CD34+ cells in granulopoiesis after three cell divisions in contrast to a moderate decline under self-renewal conditions. We also found a decreased colony-forming ability in cells divided more than twice during granulopoiesis, which correlates with their lower CD34 expression. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual commitment during granulopoiesis. Our current experiments extent these analyses to immunofluorescence of Numb distribution in individual cells to elucidate the impact of Cdh1 on symmetric/asymmetric cell division. We could already show that Cdh1-kd led to expansion of CD34+ HSCs in vitro. To further validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain-/- (NSG) xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of engrafted human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Further analyses of the contributing subsets to the pool of CD45+ human cells are ongoing. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Conclusions APC/CCdh1 mediates cell cycle entry and proliferation during self-renewal and differentiation in CD34+ HSCs in vitro and improves engraftment capacity in vivo. Disclosures: No relevant conflicts of interest to declare.
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Lambhate, Surbhi, Debanjan Bhattacharjee, and Nishant Jain. "APC/C CDH1 ubiquitinates IDH2 contributing to ROS increase in mitosis." Cellular Signalling 86 (October 2021): 110087. http://dx.doi.org/10.1016/j.cellsig.2021.110087.
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Toda, Kazuhiro, Kayoko Naito, Satoru Mase, Masaru Ueno, Masahiro Uritani, Ayumu Yamamoto, and Takashi Ushimaru. "APC/C-Cdh1-dependent anaphase and telophase progression during mitotic slippage." Cell Division 7, no. 1 (2012): 4. http://dx.doi.org/10.1186/1747-1028-7-4.
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de Boer, H. Rudolf, S. Guerrero Llobet, and Marcel A. T. M. van Vugt. "Controlling the response to DNA damage by the APC/C-Cdh1." Cellular and Molecular Life Sciences 73, no. 5 (December 9, 2015): 949–60. http://dx.doi.org/10.1007/s00018-015-2096-7.
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Almeida, Angeles. "Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival." Molecular Neurobiology 46, no. 3 (July 27, 2012): 547–54. http://dx.doi.org/10.1007/s12035-012-8309-2.
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García-Higuera, Irene, Eusebio Manchado, Pierre Dubus, Marta Cañamero, Juan Méndez, Sergio Moreno, and Marcos Malumbres. "Genomic stability and tumour suppression by the APC/C cofactor Cdh1." Nature Cell Biology 10, no. 7 (June 15, 2008): 802–11. http://dx.doi.org/10.1038/ncb1742.
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Sideridou, Maria, Roubini Zakopoulou, Konstantinos Evangelou, Michalis Liontos, Athanassios Kotsinas, Emmanouil Rampakakis, Sarantis Gagos, et al. "Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins." Journal of Cell Biology 195, no. 7 (December 26, 2011): 1123–40. http://dx.doi.org/10.1083/jcb.201108121.
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E-cadherin (CDH1) loss occurs frequently in carcinogenesis, contributing to invasion and metastasis. We observed that mouse and human epithelial cell lines overexpressing the replication licensing factor Cdc6 underwent phenotypic changes with mesenchymal features and loss of E-cadherin. Analysis in various types of human cancer revealed a strong correlation between increased Cdc6 expression and reduced E-cadherin levels. Prompted by these findings, we discovered that Cdc6 repressed CDH1 transcription by binding to the E-boxes of its promoter, leading to dissociation of the chromosomal insulator CTCF, displacement of the histone variant H2A.Z, and promoter heterochromatinization. Mutational analysis identified the Walker B motif and C-terminal region of Cdc6 as essential for CDH1 transcriptional suppression. Strikingly, CTCF displacement resulted in activation of adjacent origins of replication. These data demonstrate that Cdc6 acts as a molecular switch at the E-cadherin locus, linking transcriptional repression to activation of replication, and provide a telling example of how replication licensing factors could usurp alternative programs to fulfill distinct cellular functions.
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Ben Aissa-Haj, Jihenne, Maria Kabbage, Houcemeddine Othmen, Patrick Saulnier, Haifa Tounsi Kettiti, Amira Jaballah-Gabteni, Azer Ferah, et al. "CDH1 Germline Variants in a Tunisian Cohort with Hereditary Diffuse Gastric Carcinoma." Genes 13, no. 3 (February 23, 2022): 400. http://dx.doi.org/10.3390/genes13030400.
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Mutational screening of the CDH1 gene is a standard treatment for patients who fulfill Hereditary Diffuse Gastric Cancer (HDGC) testing criteria. In this framework, the classification of variants found in this gene is a crucial step for the clinical management of patients at high risk for HDGC. The aim of our study was to identify CDH1 as well as CTNNA1 mutational profiles predisposing to HDGC in Tunisia. Thirty-four cases were included for this purpose. We performed Sanger sequencing for the entire coding region of both genes and MLPA (Multiplex Ligation Probe Amplification) assays to investigate large rearrangements of the CDH1 gene. As a result, three cases, all with the HDGC inclusion criteria (8.82% of the entire cohort), carried pathogenic and likely pathogenic variants of the CDH1 gene. These variants involve a novel splicing alteration, a missense c.2281G > A detected by Sanger sequencing, and a large rearrangement detected by MLPA. No pathogenic CTNNA1 variants were found. The large rearrangement is clearly pathogenic, implicating a large deletion of two exons. The novel splicing variant creates a cryptic site. The missense variant is a VUS (Variant with Uncertain Significance). With ACMG (American College of Medical Genetics and Genomics) classification and the evidence available, we thus suggest a revision of its status to likely pathogenic. Further functional studies or cosegregation analysis should be performed to confirm its pathogenicity. In addition, molecular exploration will be needed to understand the etiology of the other CDH1- and CTNNA1-negative cases fulfilling the HDGC inclusion criteria.
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Ewerth, Daniel, Andrea Schmidts, Birgit Kuegelgen, Dagmar Wider, Julia Schüler, Monika Engelhardt, and Ralph Wäsch. "Dissecting Stem Cell Proliferation and Differentiation in Association with the Central Cell Cycle Regulator APC/CCdh1." Blood 120, no. 21 (November 16, 2012): 1236. http://dx.doi.org/10.1182/blood.v120.21.1236.1236.
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Abstract Abstract 1236 Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation. The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis. To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing. Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures: No relevant conflicts of interest to declare.