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Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 μm diameter and 16 μm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 μm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and

Background: STAT3 is a pro-oncogenic transcription factor. Pyrimethamine (PYM) is a STAT3 inhibitor that suppresses the proliferation of some cancer cells through downregulation of STAT3 target proteins. Methodology & Results: We have used structure-based tools to design novel PYM-based compounds. Intracellular target validation studies revealed that representative compounds 11b–d and 15a downregulate STAT3 downstream proteins and inhibit STAT3 DNA binding domain (DBD). Relative to PYM, a cohort of these compounds are >100-fold more cytotoxic to cancer cells with constitutively active (high pSTAT3) and basal (low pSTAT3) STAT3 signaling, suggesting that STAT3 DBD inhibition is deleterious to the proliferation of cancer cells with low and high pSTAT3 levels. Conclusion: These are promising leads for further preclinical evaluation as therapeutic agents for STAT3-dependent cancers.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike β-protofibrils. At the close concentrations (∼7.5 μM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 μM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members., Human Microrchidia 4 (MORC4) ATPase has been implicated in acute and chronic pancreatitis, inflammatory disorders and cancer. Here the authors describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation.

Canine parvovirus (CPV) has been used in cancer control as a drug delivery vehicle or anti-tumor reagent due to its multiple natural advantages. However, potential host cell cycle arrest induced by virus infection may impose a big challenge to CPV associated cancer control as it could prevent host cancer cells from undergoing cell lysis and foster them regain viability once the virotherapy was ceased. To explore CPV-induced cell cycle arrest and the underlying mechanism toward improved virotherapeutic design, we focus on epidermal growth factor receptor (EGFR), a cellular receptor interacting with TfR that mediates CPV-host interactions, and alterations on its tyrosine phosphorylation sites in response to CPV infection. We found that CPV could trigger host G1/S cell cycle arrest via the EGFR (Y1086)/p27 and EGFR (Y1068)/STAT3/cyclin D1 axes, and EGFR inhibitor could not reverse this process. Our results contribute to our understandings on the mechanism of CPV-induced host cellular response and can be used in the onco-therapeutic design utilizing CPV by preventing host cancer cells from entering cell cycle arrest.

Long non‑coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)‑4429 were assessed in EC cell lines by reverse transcription‑quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV‑NC, miR‑4429 mimic and mimic‑NC, were transfected into RL95‑2 cells. Post‑transfection, Cell Counting Kit‑8, Transwell Matrigel and wound‑healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR‑4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual‑luciferase reporter system. Identified as a target of miR‑4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR‑4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR‑4429 markedly suppressed proliferation, migration and invasion of RL95‑2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR‑4429 mimic‑transfected RL95‑2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR‑4429 in EC. Therefore, targeting the SNHG12/miR‑4429 axis could serve as a potential future therapeutic target for treatment of EC.

Objectives Eurycoma longifolia Jack (Simaroubaceae) is commonly distributed in the Southeast Asia and Indo China, which has been shown to possess antianxiety, antibacterial, anticancer, antifungal, anti-inflammatory, antimalarial and antioxidant biological activities. 14,15β-dihydroxyklaineanone is a diterpene isolated from E. longifolia Jack, which is cytotoxic against human lung cancer and human breast cancer cell lines. However, the effects and underlying mechanisms of 14,15β-dihydroxyklaineanone on hepatocellular carcinoma remain unknown. Methods Cell viability assay and colony formation assay were used to measure HepG2 cell proliferation. Flow cytometry was used to analyse cell cycle and apoptosis. Wound-healing assay and transwell assay were used to observe cells migration. RNA sequencing and the enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to find and determine underlying pathways. Key findings We found that 14,15β-dihydroxyklaineanone inhibited the growth and migration of HepG2 cells but did not induce cell apoptosis. 14,15β-dihydroxyklaineanone induced S cell cycle arrest by downregulating the expression levels of cyclin A, p-CDK2, cyclin B1, p21, E2F-1 and PCNA. In addition, RNA sequencing showed that 14,15β-dihydroxyklaineanone regulated MAPK pathway by increasing the expression levels of phosphor-p38. Downregulating of p38 via both p38 inhibitor (SB203580) and p38-siRNA could antagonize the inhibition of cell proliferation and migration and reverse the changes in p-p38, E-cadherin, N-cadherin and PCNA expression induced by 14,15β-dihydroxyklaineanone treatment. Conclusions 14,15β-dihydroxyklaineanone inhibited cell proliferation and migration through regulating p38 MAPK pathway in HCC cells.

While proteasome inhibitors such as bortezomib showed satisfactory clinical benefits in the initial treatment of multiple myeloma (MM), drug resistance and relapse are unavoidable. Recent studies suggested inhibition of histone deacetylases (HDACs) restored sensitivity of bortezomib-resistant MM. Hence, we designed dual inhibitors targeting both HDACs and proteasomes to address the resistance of bortezomib. The most potent inhibitors, ZY-2 and ZY-13 showed excellent inhibition against proteasome and good selectivity against HDACs. In particular, ZY-2 not only exhibited good antiproliferative activities on the MM cell lines RPMI-8226, U266, and KM3 (IC50 values of 6.66, 4.31, and 10.1 nM, respectively) but also showed more potent antiproliferative activities against the bortezomib-resistant MM cell line KM3/BTZ compared with bortezomib (IC50 values of 8.98 vs. 226 nM, P < 0.01) and even better than the combination of the HDAC inhibitor MS-275 and bortezomib (1:1) (IC50 values of 8.98 vs. 98.0 nM, P < 0.01).

Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

The frequency of esophageal adenocarcinoma is rising despite widespread use of proton pump inhibitors (PPIs), which heal reflux esophagitis but do not prevent reflux of weakly acidic gastric juice and bile in Barrett’s esophagus patients. We aimed to determine if weakly acidic (pH 5.5) bile salt medium (WABM) causes DNA damage in Barrett’s cells. Because p53 is inactivated frequently in Barrett’s esophagus and p38 can assume p53 functions, we explored p38’s role in DNA damage response and repair. We exposed Barrett’s cells with or without p53 knockdown to WABM, and evaluated DNA damage, its response and repair, and whether these effects are p38 dependent. We also measured phospho-p38 in biopsies of Barrett’s metaplasia exposed to deoxycholic acid (DCA). WABM caused phospho-H2AX increases that were blocked by a reactive oxygen species (ROS) scavenger. WABM increased phospho-p38 and reduced bromodeoxyuridine incorporation (an index of S phase entry). Repair of WABM-induced DNA damage proceeded through p38-mediated base excision repair (BER) associated with reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease I (Ref-1/APE1). Cells treated with WABM supplemented with ursodeoxycholic acid (UDCA) exhibited enhanced p38-mediated responses to DNA damage. All of these effects were observed in p53-intact and p53-deficient Barrett’s cells. In patients, esophageal DCA perfusion significantly increased phospho-p38 in Barrett’s metaplasia. WABM exposure generates ROS, causing oxidative DNA damage in Barrett’s cells, a mechanism possibly underlying the rising frequency of esophageal adenocarcinoma despite PPI usage. p38 plays a central role in oxidative DNA damage response and Ref-1/APE1-associated BER, suggesting potential chemopreventive roles for agents like UDCA that increase p38 activity in Barrett’s esophagus. NEW & NOTEWORTHY We found that weakly acidic bile salt solutions, with compositions similar to the refluxed gastric juice of gastroesophageal reflux disease patients on proton pump inhibitors, cause oxidative DNA damage in Barrett’s metaplasia that could contribute to the development of esophageal adenocarcinoma. We also have elucidated a critical role for p38 in Barrett’s metaplasia in its response to and repair of oxidative DNA damage, suggesting a potential chemopreventive role for agents like ursodeoxycholic acid that increase p38 activity in Barrett’s esophagus.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions., Point mutations have been found in induced pluripotent stem cells (iPSCs) but when they arise is unclear. Here, the authors show that a G1/S cell cycle checkpoint deficiency transiently occurs early in genome reprogramming, suggesting a common developmental pathway between iPSC and tumorigenesis, and generate genetic burden-free human iPSCs.

Retrotransposons are genomic DNA sequences that copy them-selves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expres-sion. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

OBJECTIVES Doxorubicin is a DNA-damaging agent used to treat hematological cancers. Unfortunately, drug resistance can occur by defective DNA repair activity coupled with the ability of DNA polymerases to misreplicate unrepaired DNA lesions. This study demonstrates that the efficacy of doxorubicin can be improved by using an artificial nucleoside to efficiently and selectively inhibit this activity. METHODS In vitro studies using acute lymphoblastic leukemia cell lines define the mechanism of cell death caused by combining an artificial nucleoside with doxorubicin. RESULTS Flow cytometry experiments demonstrate that combining an artificial nucleoside with doxorubicin potentiates the cell killing effects of the drug by increasing apoptosis. The potentiation effect correlates with expression of TdT, a specialized DNA polymerase overexpressed in acute lymphoblastic leukemia. Cell cycle experiments demonstrate that this combination blocks cells at S-phase prior to inducing apoptosis. Finally, the unique chemical composition of the nucleoside analog was used to visualize the replication of damaged DNA in TdT-positive cells. This represents a potential diagnostic tool to easily identify doxorubicin-resistant cancer cells. CONCLUSION Studies demonstrate that a novel artificial nucleoside improves the therapeutic efficacy of doxorubicin, thereby reducing the risk of potential side effects caused by the DNA-damaging agent.

Translocase of outer mitochondrial membrane 20 (TOMM20) plays an essential role as a receptor for proteins targeted to mitochondria. TOMM20 was shown to be overexpressed in various cancers. However, the oncological function and therapeutic potential for TOMM20 in cancer remains largely unexplored. The purpose of this study was to elucidate the underlying molecular mechanism of TOMM20's contribution to tumorigenesis and to explore the possibility of its therapeutic potential using colorectal cancer as a model. The results show that TOMM20 overexpression resulted in an increase in cell proliferation, migration, and invasion of colorectal cancer (CRC) cells, while siRNA-mediated inhibition of TOMM20 resulted in significant decreases in cell proliferation, migration, and invasion. TOMM20 expression directly impacted the mitochondrial function including ATP production and maintenance of membrane potential, which contributed to tumorigenic cellular activities including regulation of S phase cell cycle and apoptosis. TOMM20 was overexpressed in CRC compared to the normal tissues and increased expression of TOMM20 to be associated with malignant characteristics including a higher number of lymph nodes and perineural invasion in CRC. Notably, knockdown of TOMM20 in the xenograft mouse model resulted in a significant reduction of tumor growth. This is the first report demonstrating a relationship between TOMM20 and tumorigenesis in colorectal cancer and providing promising evidence for the potential for TOMM20 to serve as a new therapeutic target of colorectal cancer. [BMB Reports 2019; 52(12): 712-717].

Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y., Vu, L., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7026–7030 and Nogi, Y., Yano, R., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3962–3966) developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a “genetics-like”approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tandem-winged helix), the heterodimerization, and the linker domain were essential. Analysis of the linker identified a putative helix–turn–helix (HTH) DNA-binding domain. This HTH constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.

The studies of iridium (III) complexes as potent anticancer reagents have attracted great attention. Here, a new iridium (III) complex [Ir(bzq)2(PYIP)](PF6) (Ir1, bzq = benzo[h]quinoline, PYIP = 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) was synthesized and its liposomes (Ir1Lipo) was prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cytotoxic activity of Ir1 and Ir1Lipo on HepG2, SGC-7901, BEL-7402, HeLa, B16, A549 and normal NIH3T3 cells. The complex Ir1 displays no obvious inhibitory effect on the growth of BEL-7402 cells, while the Ir1Lipo shows significant cytotoxic activity on BEL-7402 cells (IC50 = 2.6 ± 0.03 μM). In further studies, Ir1Lipo induced apoptosis by the mitochondrial pathways, such as increasing intracellular reactive oxygen species (ROS) content and intracellular Ca2+ level, decreasing the mitochondrial membrane potential (MMP). In addition, after incubation with Ir1Lipo, the colony formation of BEL-7402 cells was significantly inhibited. Moreover, flow cytometry was used to detect the impact of Ir1Lipo on cell cycle distribution, and western blot was used to detect the expression of caspases and Bcl-2 (B-cell lymphoma-2) family proteins. Furthermore, Ir1Lipo exhibited significant antitumor activity in vivo with an inhibitory rate of 65.8%. These results indicated that Ir1Lipo induces apoptosis in BEL-7402 cells through intrinsic mitochondrial pathway.

The facile modification of the ligands in organometallic Ru(II)-arene complexes offers more opportunities to optimize their pharmacological profiles. Herein, three Ru(II)-arene complexes containing a glutathione

Drug resistance and tumor heterogeneity limits the therapeutic efficacy in treating glioblastoma, an aggressive infiltrative type of brain tumor. GBM cells develops resistance against chemotherapeutic agent, temozolomide (TMZ), which leads to the failure in treatment strategies. This enduring challenge of GBM drug resistance could be rational by combinatorial targeted therapy. Here, we evaluated the combinatorial effect of phenolic compound (2-(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), GPR17 agonist 2-({5-[3-(Morpholine-4-sulfonyl)phenyl]-4-[4-(trifluoromethoxy)phenyl]-4H-1,2,4-triazol-3-yl}sulfanyl)-N-[4-(propan-2-yl)phenyl]acetamide (T0510.3657 or T0) with the frontline drug, TMZ, on the inhibition of GBM cells. Mesenchymal cell lines derived from patients' tumors, MMK1 and JK2 were treated with the combination of THTMP + T0, THTMP + TMZ and T0 + TMZ. Cellular migration, invasion and clonogenicity assays were performed to check the migratory behavior and the ability to form colony of GBM cells. Mitochondrial membrane permeability (MMP) assay and intracellular calcium, [Ca2+]i, assay was done to comprehend the mechanism of apoptosis. Role of apoptosis-related signaling molecules was analyzed in the induction of programmed cell death. In vivo validation in the xenograft models further validates the preclinical efficacy of the combinatorial drug. GBM cells exert better synergistic effect when exposed to the cytotoxic concentration of THTMP + T0, than other combinations. It also inhibited tumor cell proliferation, migration, invasion, colony-forming ability and cell cycle progression in S phase, better than the other combinations. Moreover, the combination of THTMP + T0 profoundly increased the [Ca2+]i, reactive oxygen species in a time-dependent manner, thus affecting MMP and leading to apoptosis. The activation of intrinsic apoptotic pathway was regulated by the expression of Bcl-2, cleaved caspases-3, cytochrome c, HSP27, cIAP-1, cIAP-2, p53, and XIAP. The combinatorial drug showed promising anti-tumor efficacy in GBM xenograft model by reducing the tumor volume, suggesting it as an alternative drug to TMZ. Our findings indicate the coordinated administration of THTMP + T0 as an efficient therapy for inhibiting GBM cell proliferation. publishedVersion

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes, including metastasis and therapy resistance. CB002 and structural-analogs restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2 checkpoint. Novel CB002-analogs induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogs target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression, and downregulation of essential proteins in DNA-synthesis and DNA-repair. CB002-analog #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs shows the activation of p53 pathway signaling in tumors with mutated p53, and targets an S-phase checkpoint.

This study investigated the roles of low-molecular-weight fucoidan (LMWF) in enhancing the anti-cancer effects of fluoropyrimidine-based chemotherapy. HCT116 and Caco-2 cells were treated with LMWF and 5-FU. Cell viability, cell cycle, apoptosis, and migration were analyzed in both cell types. Potential mechanisms underlying how LMWF enhances the anti-cancer effects of fluoropyrimidine-based chemotherapy were also explored. The cell viability of HCT116 and Caco-2 cells was significantly reduced after treatment with a LMWF-–5FU combination. In HCT116 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through the (1) induction of cell cycle arrest in the S phase and (2) late apoptosis mediated by the Jun-N-terminal kinase (JNK) signaling pathway. In Caco-2 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through both the c-mesenchymal–epithelial transition (MET)/Kirsten rat sarcoma virus (KRAS)/extracellular signal-regulated kinase (ERK) and the c-MET/phosphatidyl-inositol 3-kinases (PI3K)/protein kinase B (AKT) signaling pathways. Moreover, LMWF enhanced the suppressive effects of 5-FU on tumor cell migration through the c-MET/matrix metalloproteinase (MMP)-2 signaling pathway in both HCT116 and Caco-2 cells. Our results demonstrated that LMWF is a potential complementary therapy for enhancing the efficacies of fluoropyrimidine-based chemotherapy in colorectal cancers (CRCs) with the wild-type or mutated KRAS gene through different mechanisms. However, in vivo studies and in clinical trials are required in order to validate the results of the present study.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13–Stn1–Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine −/− mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing; (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Background Senataxin (SETX) is a DNA/RNA helicase critical for neuron survival. SETX mutations underlie two inherited neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and Amyotrophic Lateral Sclerosis type 4 (ALS4). Methods This review examines SETX key cellular processes and we hypothesize that SETX requires SUMO posttranslational modification to function properly. Results SETX is localized to distinct foci during S‐phase of the cell cycle, and these foci represent sites of DNA polymerase/RNA polymerase II (RNAP) collision, as they co‐localize with DNA damage markers 53BP1 and H2AX. At such sites, SETX directs incomplete RNA transcripts to the nuclear exosome for degradation via interaction with exosome component 9 (Exosc9), a key component of the nuclear exosome. These processes require SETX SUMOylation. SETX was also recently localized within stress granules (SGs), and found to regulate SG disassembly, a process that similarly requires SUMOylation. Conclusion SETX undergoes SUMO modification to function at S‐phase foci in cycling cells to facilitate RNA degradation. SETX may regulate similar processes in non‐dividing neurons at sites of RNAP II bidirectional self‐collision. Finally, SUMOylation of SETX appears to be required for SG disassembly. This SETX function may be crucial for neuron survival, as altered SG dynamics are linked to ALS disease pathogenesis. In addition, AOA2 point mutations have been shown to block SETX SUMOylation. Such mutations induce an ataxia phenotype indistinguishable from those with SETX null mutation, underscoring the importance of this modification., This mini‐review summarizes the most pertinent aspects of SETX biology that have recently been found to coalesce around its SUMO posttranslational modification. Senataxin (SETX) is a helicase that functions to maintain genomic stability, and SETX class‐specific mutations cause either a severe recessive ataxia (AOA2) or a dominant motor neuron disease (ALS4). RNA processing and binding proteins such as SETX remain of major focus in neurologic disease. Yet, the precise mechanism leading to pathology is elusive.

Background The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. Methods In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. Results Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. Conclusions FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-021-08836-y.

The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the βGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

Significance The intra-S phase checkpoint is essential to stabilize stalling/stalled replication forks. However, the mechanism underlying checkpoint regulation is poorly understood; two key questions remain to be answered: 1) why are stalling forks unstable when checkpoint is deficient, and 2) which protein(s) is the critical target of checkpoint regulation? Here, we demonstrate that CMG replicative helicase becomes uncoupled from stalled/blocked DNA polymerases in checkpoint-deficient cds1 Chk2 △ cells, likely reflecting physical separation of the helicase and polymerases. This uncoupling is prevented by the Cds1 Chk2 -mediated phosphorylation of Cdc45. We propose that replication elongation is directly regulated in response to replication fork stalling via phosphorylation of Cdc45, which significantly reduces CMG helicase processivity, in order to prevent helicase–polymerase uncoupling and promote replication fork stability.

Clioquinol is recently considered to be the most promising drug for treating cancer and neurodegenerative diseases. However, its mode of action varies from different disease models. In this study, we found that clioquinol inhibited cell growth in human neurotypic SHSY-5Y cells, which was attributed to both S-phase cell-cycle arrest and autophagic cell death. Clioquinol increased the intracellular contents of iron and zinc as well as calcium as measured by ICP-AES. Staining of Fluo-3 confirmed an increase in the level of calcium. Analysis of the metal-binding ability of clioquinol showed that it was not a chelating agent of calcium ions and the elevation of intracellular calcium content is not achieved by clioquinol as an ionophore. CaCl2 could simulate or even aggravate the cytotoxicity of clioquinol and it increased S-phase cell cycle arrest induced by clioquinol in a concentration dependent manner. Staining of acridine orange demonstrated that autophagy induced by clioquinol was not affected by addition of calcium ions. In contrast, the intracellular calcium ion chelator BAPTA-am abolished the clioquinol-induced S phase arrest and reduced the cell death caused by clioquinol. The WB assay of cell cycle-related proteins (CDK2, p21 and p27) further confirmed that S phase arrest is positively correlated with intracellular calcium elevation, which was due to the alterations of the mRNA and protein levels of calcium pumps (SERCA and SPCA). Taken together, these data indicate that clioquinol regulates the level of intracellular calcium ions to induce S-phase cell cycle arrest in human SH-SY5Y cells. Our results demonstrate for the first time that an increase of intracellular calcium content is one of the mechanisms of clioquinol in the inhibition of human neurotypic SHSY-5Y cells.

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.

Background DNA-crosslinking agents like cisplatin and mitomycin C (MMC) are indispensible for the treatment of many solid malignancies. These anticancer drugs generate DNA interstrand crosslinks (ICLs) that cause cell death by blocking replication forks. Many factors counteracting ICL-induced DNA replication stress, including the Fanconi anemia (FA) pathway, are regulated by ubiquitination and, therefore, ubiquitin ligases are potential targets for the sensitization of cancer cells to crosslinking agents. In this study, we investigated the function of the CRL4 ubiquitin ligase in modulating the response of cancer cells to ICL induction. Methods The two cullin paralogs CUL4A and CUL4B, which form the CRL4 ligase scaffold, were depleted in cancer cells by small interfering RNA followed by analysis of the cellular and biochemical responses to ICLs elicited upon cisplatin or MMC treatment. Results We report that the combined depletion of CUL4A and CUL4B weakens an FA pathway-dependent S phase checkpoint response. CRL4 positively stimulates the monoubiquitination of FANCD2 required for the recruitment of XPF-ERCC1, a structure-specific endonuclease that, in turn, contributes to the display of single-stranded DNA (ssDNA) at ICLs. After CRL4 down regulation, the missing ssDNA results in reduced recruitment of RPA, thereby dampening activation of ATR and CHK1 checkpoint kinases and allowing for S phase progression despite ICL induction. Conclusion Our findings indicate that CRL4 promotes cell survival by potentiating an FA pathway-dependent ssDNA-RPA signaling platform installed at ICLs. The anticancer efficacy of crosslinking agents may, therefore, be enhanced by down regulating CRL4 activity.

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

Glioblastoma is the most aggressive brain tumor. Although miR-141 has been demonstrated to primarily function as a tumor suppressor in numerous malignancies, including glioblastoma, the mechanisms involved remain poorly understood. Here, it is shown that miR-141 is downregulated in glioblastoma cell lines and tissues and may exert its biological function via directly targeting myelin transcription factor 1-like (MYT1L). Using two glioblastoma cell lines that differ from each other by the functionality of DNA-dependent protein kinase (DNAPK), a functional involvement of DNAPK in the miR-141 tumor suppression network was observed. In M059K cells with a normal function of DNAPK, the enforced expression of miR-141 attenuated MYT1L expression and suppressed cell proliferation. Conversely, the inhibition of miR-141 expression promoted cell proliferation; however, in M059J cells with a loss-of-function DNAPK, miR-141 constitutively inhibited cell proliferation upon ectopic overexpression or inhibition. An overexpression of miR-141 suppressed M059J cell migration, while it had no effect on M059K. Furthermore, the ectopic expression of miR-141 induced an S-phase arrest in both cell lines, whereas the inhibition of miR-141 caused a G1 arrest in M059J and accelerated the S phase in M059K. An overexpression and suppression of miR-141 resulted in an aberrant expression of cell-cycle proteins, including p21. Moreover, MYT1L may be a transcription factor of p21 in p53-mutant cells, whereas DNAPK may function as a repressor of MYT1L. The findings revealed the crucial role of DNAPK in miR-141-mediated suppression of gliomagenesis and demonstrated that it may be a target molecule in miR-141-associated therapeutic interventions for glioblastoma.