scholarly journals A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Qian Liu ◽  
Lijuan Guo ◽  
Hongyan Qi ◽  
Meng Lou ◽  
Rui Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Ectopic Expression ◽  
Building Blocks ◽  
Small Subunit ◽  
S Phase ◽  
Clinical Samples ◽  
Dependent Manner

AbstractRibonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

scholarly journals HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

2001 ◽  
Vol 21 (5) ◽  
pp. 1854-1865 ◽  
Author(s):  
Caitlin Hall ◽  
David M. Nelson ◽  
Xiaofen Ye ◽  
Kayla Baker ◽  
James A. DeCaprio ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ectopic Expression ◽  
Cyclin A ◽  
S Phase ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Histone Gene Expression ◽  
Phase Progression

ABSTRACT Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

A Novel Circular RNA circPLCE1 Facilitates the Malignant Progression of Colorectal Cancer by Repressing the SRSF2-dependent PLCE1 pre-RNA Splicing

2021 ◽  
Author(s):  
Zhi-Lei Chen ◽  
Hong-Yu Chen ◽  
Lei Yang ◽  
Xiang-Nan Li ◽  
Zhenjun Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Rna Splicing ◽  
Ectopic Expression ◽  
Circular Rna ◽  
Malignant Progression ◽  
Circular Rnas ◽  
Normal Tissues

Abstract Background: Studies have demonstrated that circular RNAs (circRNAs) play important roles in various types of cancer; however, the mechanisms of circRNAs located in the nucleus have rarely been explored. Here, we reported a novel circular RNA circPLCE1 facilitates the malignant progression of colorectal cancer (CRC) via repression of SRSF2-dependent PLCE1 pre-RNA splicing. Methods: qRT-PCR was used to determine the expression of circPLCE1 in CRC tissues and cells. CCK-8, transwell and flow cytometric assays were used to assess the role of circPLCE1 in CRC cell proliferation, migration and apoptosis, respectively. Animal study was carried to test the role of circPLCE1 in vivo. Further, catRAPID and RPISeq were applied to predict the possible binding protein of circPLCE1. RNA fractionation and RIP assays were used to confirm the RNA-protein interaction. Results: In this study, we found that circPLCE1 was significantly downregulated in CRC tissues compared with adjacent normal tissues. However, circPLCE1 knockdown suppresses CRC cell proliferation, migration, invasion and increased apoptosis. Nude mice experiments showed that ectopic expression of circPLCE1 dramatically increased tumor growth in vivo. Mechanically, circPLCE1 directly binding to SRSF2 protein, repressing SRSF2-dependent PLCE1 pre-RNA splicing, resulting in the progression of CRC. Individually mutating the binding sites of circPLCE1 derepressed the production of PLCE1 mRNA. Conclusions: Our studies revealed a novel molecular mechanism in the regulation of PLCE1 and implicated a new function of circular RNA, supporting the pursuit of circPLCE1 as a potential tool for future CRC treatment.

scholarly journals TIFA Promotes CRC Cell Proliferation via RSK- and PRAS40- Dependent Manner

2021 ◽  
Author(s):  
Wenzhi Shen ◽  
Wenfei Du ◽  
Yanping Li ◽  
Yongming Huang ◽  
Xinyu Jiang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
Tissue Microarrays ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Site Mutation ◽  
Fha Domain

Abstract Background: Previous studies have shown that TIFA (TNF receptor associated factor (TRAF)-interacting protein with a Forkhead-associated (FHA) domain) plays different roles in various tumor types. However, the function of TIFA in colorectal cancer (CRC) remains unclear. The goal of this study is to uncover the biological function and molecular mechanism of TIFA in CRC.Methods: Tissue microarrays were used to evaluate TIFA expression. Cancer cell proferation assays were performed in TIFA knockdown and overexpressing cells in vitro and in a xenograft model in vivo. Human phosphokinase array, immunoprecipitation assays were performed to explore the underlying mechanism.Results: We disclosed that the expression of TIFA was marked increased in CRC versus normal tissue, and positively correlated with CRC TNM stages. In agreement, we found that the CRC cell lines show increased TIFA expression levels versus normal control. The knockdown of TIFA inhibited cell proliferation but have no effects on cell apoptosis in vitro and in vivo. Moreover, the ectopic expression of TIFA enhanced cell proliferation ability in vitro and in vivo. In contrast, the expression of mutant TIFA (T9A, oligomerization site mutation; D6, TRAF6 binding site deletion) alternatively abolished TIFA mediated cell proliferation enhancement. Exploration of the underlying mechanism demonstrated that the protein synthesis associated kinase RSK and PRAS40 activation were all responsible for TIFA mediated CRC progression. Conclusions: The above results described a model of TIFA in mediating CRC progression. This may provide a promising target for CRC therapy.

scholarly journals Silencing or inhibition of H3K79 methyltransferase DOT1L induces cell cycle arrest by epigenetically modulating c-Myc expression in colorectal cancer

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Liqun Yang ◽  
Qian Lei ◽  
Lin Li ◽  
Jie Yang ◽  
Zhen Dong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
S Phase ◽  
Self Renewal ◽  
Cycle Arrest ◽  
H3k79 Methylation

Abstract Background Epigenetic regulations play pivotal roles in tumorigenesis and cancer development. Disruptor of telomeric silencing-1-like (DOT1L), also known as KMT4, is the only identified histone methyltransferase that catalyzes the mono-, di-, and tri-methylation of lysine 79 histone 3 (H3K79). However, little is known about the effect of H3K79 methylation on the modulation of colorectal cancer (CRC) development. Methods DOT1L expression profiles in different subgroups of CRC tissues and its clinical significances were analyzed from some online datasheets. DOT1L in CRC cell lines was silenced by either lentivirus-mediated knockdown or inhibited by its specific inhibitor, EPZ004777. Then cell proliferation was detected by MTT assay, BrdU assay, and soft agar assay; cell cycle was detected by cytometry; and tumorigenicity was detected by using nude mice xenograft models. Clinical co-expression was analyzed between DOT1L and c-Myc. Chromatin immunoprecipitation (ChIP) assay was used to determine whether the translation of c-Myc was epigenetically regulated by H3K79me2 induced by DOT1L. c-Myc overexpression was used to rescue the cell cycle arrest and tumor growth induced by DOT1L silencing or inhibition in CRC. Results We found that DOT1L was highly expressed in colorectal cancer and was negatively related to the prognosis of patients with CRC. Silencing or inhibition of DOT1L blocked cell proliferation, BrdU incorporation, self-renewal capability in vitro, and tumorigenicity in vivo. Besides, inhibition or silencing of DOT1L also induced cell cycle arrest at S phase, as well as decreased the expression of CDK2 and Cyclin A2. Furthermore, in the clinical databases of CRC, we found that the expression of DOT1L was positively correlated with that of c-Myc, a major regulator in the upstream of cell cycle–related factors. Besides, c-Myc expression was downregulated after DOT1L knockdown and c-Myc restoration rescued decrease of cell proliferation, BrdU corporation, self-renewal capability, cell cycle progression in vitro and tumorigenicity in vivo induced by DOT1L silencing. Then we found that H3K79 methylation was decreased after DOT1L knockdown. ChIP assay showed that H3K79me2 was enriched on the – 682~+ 284 region of c-Myc promoter, and the enrichment was decreased after DOT1L inhibition. Conclusions Our results show that DOT1L epigenetically promotes the transcription of c-Myc via H3K79me2. DOT1L silencing or inhibition induces cell cycle arrest at S phase. DOT1L is a potential marker for colorectal cancer and EPZ004777 may be a potential drug for the treatment of colorectal cancer.

Identification of a new GLDC gene alternative splicing variant and its protumorigenic roles in lung cancer

2019 ◽  
Vol 15 (36) ◽  
pp. 4127-4139 ◽  
Author(s):  
Yingli Yuan ◽  
Luguo Sun ◽  
Xu Wang ◽  
Jingxian Chen ◽  
Mingnan Jia ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Colony Formation ◽  
Lactate Production ◽  
Cellular Transformation ◽  
Lung Cancer Cell Line ◽  
Stem Cell Marker ◽  
Brdu Incorporation ◽  
Clinical Samples

Aim: To clarify the regulatory roles of GLDCV1, the first identified truncated glycine decarboxylase (GLDC), on cancer stem cells and tumorigenesis. Materials & methods: RT-PCR or RT-qPCR, immunoblotting and immunohistochemical staining were applied to assess gene expression. MTT, BrdU incorporation and colony formation assays were used to examine cell proliferation capacity. Soft agar colony formation and in vivo transplantation were applied to evaluate cellular transformation and tumorigenesis. Results & conclusion: Expression of GLDCV1 or GLDC was enhanced in non-small-cell lung cancer cell line and clinical samples. GLDCV1 overexpression induced MRC5 cell proliferation, transformation and tumorigenesis. Additionally, GLDCV1 increased lactate production and cancer stem cell marker expression and activated ERK and P38 pathways. Our study gained deeper insight into GLDC oncogene.

scholarly journals Pyrvinium pamoate inhibits cell proliferation through ROS-mediated AKT-dependent signaling pathway in colorectal cancer

2021 ◽  
Vol 38 (2) ◽  
Author(s):  
Wenqian Zheng ◽  
Jinhui Hu ◽  
Yiming Lv ◽  
Bingjun Bai ◽  
Lina Shan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Epithelial To Mesenchymal Transition ◽  
Flow Cytometric Analysis ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Dependent Signaling Pathway ◽  
Pyrvinium Pamoate

AbstractThe use of the anthelmintic drug pyrvinium pamoate (PP) in cancer therapy has been extensively investigated in the last decade. PP has been shown to have an inhibitory effect in colorectal cancer (CRC), but the underlying mechanism remains elusive. We aimed to investigate the antitumor activity and mechanisms of PP in CRC. In the present study, we used CCK-8 assays, colony formation assays, and western blotting to reveal that PP effectively suppressed CRC cell proliferation and the AKT-dependent signaling pathway in a concentration-dependent and time-dependent manner. Flow cytometric analysis and fluorescence microscopy demonstrated that PP increased intracellular reactive oxygen species (ROS) accumulation. We found that the inhibitory effect of PP on cell proliferation and AKT protein expression induced by PP could be partially reversed by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, the results also demonstrated that PP inhibited cell migration by modulating epithelial-to-mesenchymal transition (EMT)-related proteins, including E-cadherin and vimentin. In conclusion, our data suggested that PP effectively inhibited cell proliferation through the ROS-mediated AKT-dependent signaling pathway in CRC, further providing evidence for the use of PP as an antitumor agent.

scholarly journals Sevoflurane inhibits malignant progression of colorectal cancer via hsa_circ_0000231-mediated miR-622

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Jingpeng Wang ◽  
Shuyuan Li ◽  
Gaofeng Zhang ◽  
Huihua Han
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Volatile Anesthetic ◽  
Tumor Formation ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas

Abstract Background Sevoflurane (Sev), a commonly used volatile anesthetic, has been reported to inhibit the process of colorectal cancer (CRC). Circular RNAs (circRNAs) are revealed to participate in the pathogenesis of CRC. This study aims to reveal the mechanism of hsa_circ_0000231 in Sev-mediated CRC progression. Methods The expression of hsa_circ_0000231 and microRNA-622 (miR-622) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein level was determined by western blot analysis. Cell proliferation was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell colony formation and DNA content quantitation assays. Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate and propidium iodide double staining and caspase 3 activity assays. Cell migration and invasion were investigated by wound-healing and transwell invasion assays, respectively. The putative relationship between hsa_circ_0000231 and miR-622 was predicted by circular RNA Interactome online database, and identified by dual-luciferase reporter and RNA immunoprecipitation assays. The impacts of hsa_circ_0000231 on Sev-mediated tumor formation in vivo were presented by in vivo assay. Results Hsa_circ_0000231 expression was upregulated, while miR-622 was downregulated in CRC tissues and cells compared with control groups. Sev treatment decreased hsa_circ_0000231 expression, but increased miR-622 expression in CRC cells. Sev treatment suppressed cell proliferation, migration and invasion, and induced cell apoptosis. Hsa_circ_0000231 overexpression restored Sev-mediated CRC progression in vitro. Additionally, hsa_circ_0000231 acted as a sponge of miR-622, and miR-622 inhibitors reversed the impacts of hsa_circ_0000231 silencing on CRC process. Furthermore, Sev treatment inhibited tumor growth by regulating hsa_circ_0000231 in vivo. Conclusion Hsa_circ_0000231 attenuated Sev-aroused repression impacts on CRC development by sponging miR-622. This findings may provide an appropriate anesthetic protocol for CRC sufferers undergoing surgery.

scholarly journals DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation, cell apoptosis, and cell migration

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Yixin Tong ◽  
Yuan Huang ◽  
Yuchao Zhang ◽  
Xiangtai Zeng ◽  
Mei Yan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Cell Apoptosis ◽  
Downstream Target ◽  
Normal Tissues ◽  
Physiological Processes ◽  
Mutual Regulation

AbstractAt present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.

scholarly journals Inhibition of MCF-7 breast cancer cell proliferation by 5alpha-dihydrotestosterone; a role for p21(Cip1/Waf1)

2004 ◽  
Vol 32 (3) ◽  
pp. 793-810 ◽  
Author(s):  
MA Greeve ◽  
RK Allan ◽  
JM Harvey ◽  
JM Bentel
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
S Phase ◽  
Cyclin D3 ◽  
P27 Kip1 ◽  
Mcf 7

Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.

scholarly journals The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
Author(s):  
A E Parker ◽  
R K Clyne ◽  
A M Carr ◽  
T J Kelly
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

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