scholarly journals Restoration of miR-193a-5p and miR-146 a-5p Expression Induces G1 Arrest in Colorectal Cancer through Targeting of MDM2/p53

2019 ◽  
Vol 10 (1) ◽  
pp. 130-134 ◽  
Author(s):  
Saeed Noorolyai ◽  
Elham Baghbani ◽  
Leili Aghebati Maleki ◽  
Amir Baghbanzadeh Kojabad ◽  
Dariush Shanehbansdi ◽  
...  

Purpose: Colorectal cancer (CRC) remains a universal and lethal cancer owing to metastatic and relapsing disease. Currently, the role of microRNAs has been checked in tumorigeneses. Numerous studies have revealed that between the tumor suppressor miRNAs, the reduced expression of miR-146a-5p and -193a-5p in several cancers including CRC tissues are related with tumor progression and poor prognosis of patients. The purpose of this study is to examine the role of miR-146 a-5p and -193 a-5p in CRC cell cycle progression. Methods: The miR-193a-5p and -146 a-5p mimics were transfected into HT-29 CRC cells via jetPEI transfection reagent and their impact was assessed on p53, cyclin B, and NF-kB gene expression. The inhibitory effect of these miRNAs on cell cycle was assessed by flow cytometry. The consequence of miR-193a-5p and miR-146 a-5p on the protein expression level of Murine double minute 2 (MDM2) was assessed by western blotting. Results: miR193a-5p and -146a-5p regulated the expression of MDM2 protein and p53, cyclin B, and NF-kB gene expression in CRC cells. Treatment of HT-29 cells with miRNA-146a-5p and -193a-5p induced G1 cell cycle arrest. Conclusion: The findings of our study suggest that miR146a-5p and -193a-5p may act as a potential tumor suppressor by their influence on cell cycle progression in CRC cells. Thus, miRNA-146a-5p and -193a-5p restoration may be recommended as a potential therapeutic goal in the treatment of CRC patients.

2009 ◽  
Vol 185 (2) ◽  
pp. 193-202 ◽  
Author(s):  
Arne Lindqvist ◽  
Verónica Rodríguez-Bravo ◽  
René H. Medema

The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B–Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B–Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B–Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B–Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B–Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1655-1655
Author(s):  
Xiangao Huang ◽  
Maurizio Di Liberto ◽  
Jamieson Bretz ◽  
David Chiron ◽  
Peter Martin ◽  
...  

Abstract Abstract 1655 Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing. To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1. pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim. Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1537-1537 ◽  
Author(s):  
Zejin Sun ◽  
Donna Cerabona ◽  
Ying He ◽  
Grzegorz Nalepa

Abstract Cyclin dependent kinase inhibitor 3 (CDKN3) is a dual-specificity cell cycle regulatory phosphatase. In interphase, CDKN3 prevents premature G1/S transition by dephosphorylating interphase cyclin-dependent kinases (CDKs) to prevent premature inactivation of the RB pathway. During cell division, CDKN3 dephosphorylates the key mitotic kinase CDK1 at threonine-161 to extinguish CDK1 activity at the exit from mitosis. CDKN3 knockdown in cultured cells impairs the spindle assembly checkpoint (SAC), accelerates cell cycle progression and causes chromosomal instability, suggesting that it may function as a tumor suppressor. However, since CDKN3 has been reported as overexpressed in some malignancies and mutated or silenced in others, it is unclear whether it functions as an oncogene or a tumor suppressor. To understand the in vivo role of CDKN3 in carcinogenesis, we generated the first Cdkn3 conditional knockout mouse model. We found that Cdkn3-/- mice were viable, non-dysmorphic and born at expected Mendelian ratios, indicating that this gene is dispensable for normal embryonic development. In agreement with the postulated role of this phosphatase in cell cycle progression and regulation of CDKs, we found that Cdkn3-/- cells had increased CDK1, CDK2 and CDK4 activity; increased inhibitory phosphorylation of Rb; increased DNA replication and proliferation; and impaired SAC. Increased CDK activity and accelerated cell cycle progression caused genomic instability reflected by increased frequency of in vivo micronucleation during hematopoiesis as well as higher frequency of aneuploidy and multinucleation and accumulation of supernumerary centrosomes in Cdkn3-/- cells cultured ex vivo. Cdkn3-/- mice had increased myeloid colony-forming units in progenitor assays. Long-term observation of Cdkn3-/- mice revealed an increased risk of death from a variety of hematopoietic (leukemia and lymphoma) and non-hematopoietic (lung, prostate and ovarian) malignancies. Our findings establish Cdkn3 as an in vivo tumor suppressor in bone marrow and a variety of other tissues. In the long term, Cdkn3-/- mice will serve as a tool to dissect the function of this phosphatase in cell cycle control in more detail, and may prove useful in preclinical studies of chemotherapy of CDK-hyperactive, genomically unstable leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.


Open Medicine ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. 338-350
Author(s):  
Rui Xiang ◽  
Min Feng ◽  
Xin Zhou ◽  
Lihong Ma ◽  
Ningfei Dong

Abstract Background Colorectal cancer (CRC) is one of the deadliest cancers in the world. Increasing evidence suggests that circular RNAs (circRNAs) are implicated in CRC pathogenesis. This study aimed to determine the role of circAPLP2 and explore a potential mechanism of circAPLP2 action in CRC. Methods The expression of circAPLP2, miR-335-5p and helicase lymphoid-specific (HELLS) mRNA in CRC tissues and cells was measured by quantitative real-time polymerase chain reaction (qPCR). The functional effects of circAPLP2 on cell cycle progression/cell apoptosis, colony formation, cell migration, invasion and glycolysis metabolism were investigated by flow cytometry assay, colony formation assay, wound healing assay, transwell assay and glycolysis stress test. Glycolysis metabolism was also assessed by the levels of glucose uptake and lactate production. The protein levels of HELLS and HK2 were detected by western blot. The interaction between circAPLP2 and miR-335-5p, or miR-335-5p and HELLS was verified by dual-luciferase reporter assay. The role of circAPLP2 on solid tumor growth in nude mice was investigated. Results circAPLP2 and HELLS were overexpressed, but miR-335-5p was downregulated in CRC tissues and cells. Functional analyses showed that circAPLP2 knockdown suppressed CRC cell cycle progression, colony formation, migration, invasion and glycolysis metabolism, induced cell apoptosis and blocked solid tumor growth in nude mice. Moreover, miR-335-5p was a target of circAPLP2, and miR-335-5p could also bind to HELLS. Rescue experiments presented that miR-335-5p inhibition reversed the effects of circAPLP2 knockdown, and HELLS overexpression abolished the role of miR-335-5p restoration. Importantly, circAPLP2 could positively regulate HELLS expression by mediating miR-335-5p. Conclusion circAPLP2 triggered CRC malignant development by increasing HELLS expression via targeting miR-335-5p, which might be a novel strategy to understand and treat CRC.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 898-898
Author(s):  
Cassandra L Love ◽  
Dereje Jima ◽  
Zhen Sun ◽  
Rodney R. Miles ◽  
Cherie H. Dunphy ◽  
...  

Abstract Abstract 898 Burkitt Lymphoma (BL) is a highly proliferative form of non-Hodgkin lymphoma and is characterized by translocation of the C-MYC gene to the immunoglobulin gene loci resulting in deregulation. The role of collaborating gene mutations in BL is largely unknown. We performed whole exome sequencing and gene expression profiling of 57 Burkitt lymphoma and 94 DLBCL exomes. Mutational analysis revealed that ID3 is recurrently mutated in 38% of Burkitt lymphoma samples. ID3 mutations did not occur in any of the 94 DLBCL cases. ID3 gene expression was also found to be a distinguishing feature of Burkitt lymphomas (P<10−6), compared to DLBCL. We found a total of 27 distinct mutations in the ID3 genes among the 22 BL cases. These included five frameshift, four nonsense, and 18 missense mutations. We validated 16 of these events with Sanger sequencing with over 90% concordance. All of these mutations were located in the highly conserved helix-loop-helix region located on Exon 1. We explored the biological significance of ID3 mutations by initially comparing the gene expression profiles of BL cases that had mutated and wild-type ID3. Gene set enrichment analysis showed that those samples with mutated ID3 had higher expression of genes that were involved in cell cycle regulation, specifically those involved in the G1-S transition (P=0.01). In order to experimentally investigate the functional consequences of ID3 mutation, we generated mutant constructs corresponding to six different ID3 mutations observed in BLs. These mutant constructs were cloned into lentiviral vectors and overexpressed in BL cells that were wild type for ID3. We then performed cell cycle analysis for these wild type cells expressing GFP controls or the mutant constructs. We found that BL cells expressing each of the six mutant constructs demonstrated significant cell cycle progression from G1 to S phase compared to wild-type (P=0.01). Separately, we tested the effects of expressing mutant ID3 in cell proliferation assays and found that cells expressing mutant ID3 were considerably more proliferative than those expressing wild type (P=0.03). Conversely, we over-expressed the wild type form of ID3 in BL cells that had mutated ID3. These experiments completely rescued the observed phenotypes of the mutant ID3 constructs, with reduced cell cycle progression through increased G1 phase and decreased S-phase (P=0.04). We also noted decreased cell proliferation in these cells (P=0.03). These experiments support a role for ID3 as a novel tumor suppressor gene in Burkitt lymphoma. ID3 is a basic helix loop helix (bHLH) protein that binds to other E-proteins, blocking their ability to bind DNA. ID3 has been shown to be involved in a variety of biological processes including development and T and B cell differentiation. ID3 knockout mice have been shown to develop T cell as well as B cell lymphomas. Our data implicates this gene for the first time as a tumor suppressor in human cancer. Disclosures: No relevant conflicts of interest to declare.


2011 ◽  
Vol 2011 ◽  
pp. 1-8
Author(s):  
Jacek Z. Kubiak ◽  
Mohammed El Dika

Cyclin-Dependent Kinase 1 (CDK1) is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.


2019 ◽  
Vol 26 (11) ◽  
pp. 800-818
Author(s):  
Zujian Xiong ◽  
Xuejun Li ◽  
Qi Yang

Pituitary Tumor Transforming Gene (PTTG) of human is known as a checkpoint gene in the middle and late stages of mitosis, and is also a proto-oncogene that promotes cell cycle progression. In the nucleus, PTTG works as securin in controlling the mid-term segregation of sister chromatids. Overexpression of PTTG, entering the nucleus with the help of PBF in pituitary adenomas, participates in the regulation of cell cycle, interferes with DNA repair, induces genetic instability, transactivates FGF-2 and VEGF and promotes angiogenesis and tumor invasion. Simultaneously, overexpression of PTTG induces tumor cell senescence through the DNA damage pathway, making pituitary adenoma possessing the potential self-limiting ability. To elucidate the mechanism of PTTG in the regulation of pituitary adenomas, we focus on both the positive and negative function of PTTG and find out key factors interacted with PTTG in pituitary adenomas. Furthermore, we discuss other possible mechanisms correlate with PTTG in pituitary adenoma initiation and development and the potential value of PTTG in clinical treatment.


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