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Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: Lung cancer is a leading cause of cancer-related mortality and remains one of the most poorly prognosed disease worldwide. Therefore, it is necessary to identify novel molecular markers with potential therapeutic effects. Recent findings have suggested that dual-specificity tyrosine-regulated kinase 2 (DYRK2) plays a tumor suppressive role in colorectal, breast, and hepatic cancers; however, its effect and mechanism in lung cancer remain poorly understood. Therefore, this study aimed to investigate the tumor-suppressive role and molecular mechanism of DYRK2 in lung adenocarcinoma (LUAD) by in vitro experiments and xenograft models., Materials and Methods: The evaluation of DYRK2 expression was carried out using lung cancer cell lines and normal bronchial epithelial cells. Overexpression of DYRK2 was induced by an adenovirus vector, and cell proliferation was assessed through MTS assay and Colony Formation Assay. Cell cycle analysis was performed using flow cytometry. Additionally, proliferative capacity was evaluated in a xenograft model by subcutaneously implanting A549 cells into SCID mice (C·B17/Icr-scidjcl-scid/scid)., Results: Immunoblotting assays showed that DYRK2 was downregulated in most LUAD cell lines. DYRK2 overexpression using adenovirus vectors significantly suppressed cell proliferation compared with that in the control group. Additionally, DYRK2 overexpression suppressed tumor growth in a murine subcutaneous xenograft model. Mechanistically, DYRK2 overexpression inhibited the proliferation of LUAD cells via p21-mediated G1 arrest, which was contingent on p53., Conclusion: Taken together, these findings suggest that DYRK2 may serve as potential prognostic biomarker and therapeutic target for LUAD., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study explores the function of FANCA gene, a pivotal member of the Fanconi anaemia (FA) pathway crucial for preserving genomic stability and preventing cancer, particularly in the context of gastric cancer (GC). Using immunohistochemistry, quantitative real-time PCR, and western blot analysis, we evaluate FANCA mRNA and protein expressions in GC cell lines. The relationship between FANCA expression and clinicopathological characteristics is also explored. Various assays, including CCK8, colony formation, wound healing, and Transwell assays, are used to assess functional changes in cells associated with FANCA. Flow cytometry is utilized to evaluate alterations in the cell cycle resulted from FANCA knockdown and overexpression. Our findings show elevated FANCA expression in GC cell lines, with levels correlated with pathologic stage and lymphatic metastasis. FANCA knockdown impedes cell proliferation, migration, and invasion and induces G1/S phase cell cycle arrest. Conversely, FANCA overexpression stimulates cell proliferation, migration, and invasion. In vivo xenograft experiments confirm the promotional role of FANCA in GC tumor progression. Moreover, FANCA overexpression is associated with the activation of cell cycle. Collectively, our results suggest that FANCA drives malignant cell behaviors in GC through the cell cycle pathway, highlighting its potential as a therapeutic target for the treatment of GC.

Oral cancer is one of the commonly reported malignancies of the human oral cavity and pharynx. It accounts for a significant level of cancer-based mortality across the globe. Long non-coding RNAs (lncRNAs) are emerging as important study targets in cancer therapy. The present study aimed to characterize the role of lncRNA GASL1 in regulating the growth, migration, and invasion of human oral cancer cells. The qRT-PCR showed significant (P<0.05) upregulation of GASL1 in oral cancer cells. Overexpression of GASL1 led to the loss of viability of HN6 oral cancer cells by inducing apoptosis which was associated with upregulation of Bax and downregulation of Bcl-2. The apoptotic cell percentage increased from 2. 81% in control to 25.89% upon GASL1 overexpression. Cell cycle analysis showed that overexpression of GASL1 increased the G1 cells from 35.19% in control to 84.52% upon GASL1 overexpression indicative of G0/G1 cell cycle arrest. Cell cycle arrest was also accompanied by inhibition of cyclin D1 and CDK4 protein expression. Wound healing and transwell assays showed that overexpression of GASL1 significantly (P<0.05) inhibited the migration and invasion of HN6 oral cancer cells. The invasion of the HN6 oral cancer cells was found to be decreased by more than 70%. Finally, the results of in vivo study revealed that GASL1 overexpression inhibits the xenografted tumor growth in vivo. Thus, the results are thus suggestive of the tumor-suppressive molecular role of GASL1 in oral cancer cells.

Protein kinase Cα (PKCα), belonging to ser/thr protein kinase, perform various biological functions. Overexpression of PKCα has been observed in multiple human malignancies including lymphoma. However, the molecular pathogenesis and involvement of PKCα in Non-Hodgkin lymphoma (NHL) are not clearly understood. Hence, deciphering the role of PKCα in NHL management may provide a better therapeutic option. In the present study, we used selective pharmacological inhibitors Gö6976 and Ro320432 that potentially inhibit PKCα-mediated signaling in DL cells, resulting in the inhibition of cell growth and mitochondrial-dependent apoptosis. PKCα inhibition by these inhibitors also displays cell cycle arrest at the G1 phase and causes growth retardation of DL cells. Our results extended the mechanism of PKCα in NHL, and provided potential implications for its therapy., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.

Aims: We aimed to investigate the role of G protein subunit alpha Z(GNAZ) in the progression and prognosis of patients with hepatocellular carcinoma (HCC)., Methods: Oncomine, GEO, TCGA, GEPIA2, Kaplan-Meier Plotter, TIMER2, Metascape, CCLE, LinkedOmics, and UALCAN databases were used to analyze the differential expression of GNAZ in HCC and normal liver tissues, relationship between GNAZ expression and prognosis of patients with HCC, and expression of GNAZ in common human HCC cell lines. Western blotting was performed to analyze GNAZ expression, while the Cell Counting Kit 8 assay was used to determine cell proliferation, and flow cytometry was used to evaluate the cell cycle and apoptosis. Wound healing and transwell invasion assays were used to investigate cell metastasis and invasion., Results: Using Oncomine, Gene Expression Omnibus (GEO), and GEPIA2 databases, GNAZ was found to be overexpressed in HCC tissues compared with that in adjacent normal liver tissues, and western blotting analysis showed GNAZ overexpression in seven patients with HCC who underwent surgical resection of HCC and para-cancerous tissues (p < 0.01). Survival analysis revealed that high GNAZ expression was negatively associated with overall survival (OS), recurrence-free survival, progression-free survival, and disease-specific survival in patients with HCC (p < 0.05). GNAZ overexpression was associated with worse 4- month, 6- month, 12- month, 24- month, 36- month, 48- month, and 60-month OS, as well as with different clinicopathological characteristics of patients with HCC, including hepatitis virus infection state; alcohol consumption state; male; female; Asian; microvascular invasion, Stage I-II, Stage II-III, and Stage III-IV; and grade II (Cox regression, p < 0.05). KEGG/GO biological process enrichment indicated that the genes similar to GNAZ in HCC were mainly enriched in the cell cycle, cell cycle phase transition, DNA replication checkpoint, and regulation of G0 to G1 transition. siRNA-GNAZ significantly reduced the viability of JHH-2 and SNU-761 cells from 12 to 96 h; increased the percentage of cells in the G0/G1 phase and decreased that of cells in the S and G2/M phases (p < 0.05); and markedly downregulated the expression of cyclin D, cyclin E, and CDK2 protein. siRNA-GNAZ also significantly increased the percentage of JHH-2 and SNU-761 cell apoptosis at late stages, while the number of surviving cells decreased (p < 0.05), and upregulated the expression of apoptosis-related proteins Bax and caspase 3 protein. Furthermore, siRNA-GNAZ remarkably reduced the healing of scratch wounds in JHH-2 and SNU-761 cells and the number of invasive cells compared with that in the control group (p < 0.001)., Conclusion: Our study demonstrated that GNAZ plays a pivotal role as a potential oncogene and predicts poor prognosis in patients with HCC. It promotes tumor proliferation via cell cycle arrest, apoptosis, migration, and invasion. Thus, GNAZ may be a potential candidate biomarker providing useful insight into hepatocarcinogenesis and aggressiveness., (Copyright © 2021 Elsevier B.V. All rights reserved.)

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cell division cycle must be strictly regulated during both development and adult maintenance, and efficient and well-controlled DNA replication is a key event in the cell cycle. DNA replication origins are prepared in G1 phase of the cell cycle in a process known as origin licensing which is essential for DNA replication initiation in the subsequent S phase. Appropriate origin licensing includes: (1) Licensing enough origins at adequate origin licensing speed to complete licensing before G1 phase ends; (2) Licensing origins such that they are well-distributed on all chromosomes. Both aspects of licensing are critical for replication efficiency and accuracy. In this minireview, we will discuss recent advances in defining how origin licensing speed and distribution are critical to ensure DNA replication completion and genome stability., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (© 2021. The Author(s).)

Polo-like kinase 4 (PLK4) has been reported to contribute to tumor growth, invasion, and metastasis. However, the role of PLK4 in human bladder cancer (BC) remains unclear. Here, we demonstrate the regulatory function of PLK4 in human BC progression. PLK4 is overexpressed in BC cell lines and tissues, and its overexpression correlated with poor prognosis. Our transcriptome analysis combined with subsequent functional assays indicated that PLK4 inhibition can suppress BC cell growth and induce cell cycle arrest at G1 phase via activation of the p38/p53/p21 pathway in vitro and in vivo. Overall, our data suggest that PLK4 is a critical regulator of BC cell proliferation, and thus, it may have potential as a novel molecular target for BC treatment., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Osteosarcomas are aggressive primary tumors of bone that are typically detected in locally advanced stages; however, which genetic mutations drive the cancer before its clinical detection remain unknown. To identify these events, we performed longitudinal genome-sequencing analysis of 12 patients with metastatic or refractory osteosarcoma. Phylogenetic and molecular clock analyses were carried out next to identify actionable mutations, and these were validated by integrating data from additional 153 osteosarcomas and pre-existing functional evidence from mouse PDX models. We found that the earliest and thus clinically most promising mutations affect the cell cycle G1 transition, which is guarded by cyclins D3, E1, and cyclin-dependent kinases 2, 4, and 6. Cell cycle G1 alterations originate no more than a year before the primary tumor is clinically detected and occur in >90% and 50% of patients of the discovery and validation cohorts, respectively. In comparison, other cancer driver mutations could be acquired at any evolutionary stage and often do not become pervasive. Consequently, our data support that the repertoire of actionable mutations present in every osteosarcoma cell is largely limited to cell cycle G1 mutations. Since they occur in mutually exclusive combinations favoring either CDK2 or CDK4/6 pathway activation, we propose a new genomically-based algorithm to direct patients to correct clinical trial options. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Momordin Ic (MI) is a natural pentacyclic triterpenoid enriched in various Chinese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. Studies have shown that MI presents antitumor properties in liver and prostate cancers. However, the activity and potential mechanisms of MI against colorectal cancer remain elusive. Here, we showed that MI inhibited cell proliferation with G0/1 phase cell cycle arrest in colon cancer cells. Moreover, it was observed that MI increased apoptosis compared to untreated cells. Further investigation showed that the SUMOylation of c-Myc was enhanced by MI and led to the down-regulated protein level of c-Myc, which is involved in regulating cell proliferation and apoptosis. SENP1 has been demonstrated to be critical for the SUMOylation of c-Myc. Meanwhile, knockdown of SENP1 by siRNA abolished the effects of MI on c-Myc level and cell viability in colon cancer cells. Together, these results revealed that MI exerted an anti-tumor activity in colon cancer cells via SENP1/c-Myc signaling pathway. These finding provide an insight into the potential of MI for colon cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Cell proliferation and cell death abnormalities are strongly linked to the development of cancer, including lung cancer. The purpose of this study was to investigate the effect of pterostilbene on cell proliferation and cell death via cell cycle arrest during the transition from G1 to S phase and the p53 pathway. A total of 24 female Balb/C mice were randomly categorized into four groups (n = 6): N-nitroso-tris-chloroethyl urea (NTCU) induced SCC of the lungs, vehicle control, low dose of 10 mg/kg PS + NTCU (PS10), and high dose of 50 mg/kg PS + NTCU (PS50). At week 26, all lungs were harvested for immunohistochemistry and Western blotting analysis. Ki-67 expression is significantly lower, while caspase-3 expression is significantly higher in PS10 and PS50 as compared to the NTCU (p < 0.05). There was a significant decrease in cyclin D1 and cyclin E2 protein expression in PS10 and PS50 when compared to the NTCU (p < 0.05). PS50 significantly increased p53, p21, and p27 protein expression when compared to NTCU (p < 0.05). Pterostilbene is a potential chemoprevention agent for lung SCC as it has the ability to upregulate the p53/p21 pathway, causing cell cycle arrest., (© 2021. The Author(s).)

Background: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined., Results: We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired., Conclusion: Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics., (© 2021. The Author(s).)

Purpose: This study aimed to confirm the association between developmentally regulated GTP-binding protein 2 (DRG2) expression and docetaxel-induced apoptosis and to determine whether prostate cancer responses to docetaxel treatment differ with DRG2 expression., Materials and Methods: PC3, DU145, and LNCaP prostate cancer cell lines were used. The MTT assay was used to determine cell viability. Western blotting analysis was performed using anti-DRG2 antibodies. Cells were transfected with 50 nmol DRG2 siRNA using an siRNA transfection reagent for DRG2 knockdown. The cell cycle was analyzed by using flow cytometry, and apoptosis was detected by using the Annexin V cell death assay., Results: DRG2 expression differed in each prostate cancer cell line. Docetaxel reduced DRG2 expression in a dose-dependent manner. Upon DRG2 knockdown in prostate cancer cells, an increase in the sub-G1 phase was observed without a change in the G1 or G2/M phases. When 4 nM docetaxel was administered to DRG2 knockdown prostate cancer cell lines, an increase in the sub-G1 phase was observed without increasing the G2/M phase, which was similar to that in DU145 cells before DRG2 knockdown. In PC3 and DU145 cell lines, DRG2 knockdown increased docetaxel-induced Annexin V (+) apoptosis by 8.7 and 2.7 times, respectively., Conclusions: In prostate cancer cells, DRG2 regulates G2/M arrest after docetaxel treatment. In prostate cancer cells with DRG2 knockdown, apoptosis increases without G2/M arrest in response to docetaxel treatment. These results show that inhibition of DRG2 expression can be useful to enhance docetaxel-induced apoptosis despite low-dose administration in castration-resistant prostate cancer., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2021.)

Cyclin D1 is an essential regulator of the G1-S cell-cycle transition and is overexpressed in many cancers. Expression of cyclin D1 is under tight cellular regulation that is controlled by many signaling pathways. Here we report that PARP14, a member of the poly(ADP-ribose) polymerase (PARP) family, is a regulator of cyclin D1 expression. Depletion of PARP14 leads to decreased cyclin D1 protein levels. In cells with a functional retinoblastoma (RB) protein pathway, this results in G1 cell-cycle arrest and reduced proliferation. Mechanistically, we found that PARP14 controls cyclin D1 mRNA levels. Using luciferase assays, we show that PARP14 specifically regulates cyclin D1 3'UTR mRNA stability. Finally, we also provide evidence that G1 arrest in PARP14-depleted cells is dependent on an intact p53-p21 pathway. Our work uncovers a new role for PARP14 in promoting cell-cycle progression through both cyclin D1 and the p53 pathway., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Long non-coding RNAs exert vital roles in several types of cancer. The objective of this study was to explore the role of LINC&#95;00355 in gastric cancer (GC) progression and its potential mechanism., Methods: The expression levels of LINC&#95;00355 in GC tissues and cells were detected by quantitative real-time PCR, followed by assessing the effects of LINC&#95;00355 knockdown or overexpression on cell properties. Dual-luciferase reporter assay was utilized to identify the relationship between LINC&#95;00355 and microRNA (miR)-15a-5p and miR-15a-5p and PHD finger protein 19 (PHF19), followed by the rescue experiments., Results: The results showed that LINC&#95;00355 was highly expressed in GC tissues and cells compared with the corresponding control. LINC&#95;00355 knockdown decreased the viability, migration, and invasion and increased the accumulation of GC cells in G1 phase and apoptosis. Meanwhile, LINC&#95;00355 downregulation markedly increased cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase protein levels, whereas decreased cyclin D1, cyclin E, matrix metalloproteinase (MMP) 9, MMP2, and N-cadherin protein levels in GC cells. However, LINC&#95;00355 overexpression had the opposite effects. It was verified that LINC&#95;00355 upregulated the expression of PHF19 through sponging miR-15a-5p. Furthermore, PHF19 overexpression reversed the effect of LINC&#95;00355 knockdown on GC cell properties, including cell viability, migration, invasion, and apoptosis., Conclusions: Collectively, these results suggest that LINC&#95;00355 promotes GC progression by up-regulating PHF19 through sponging miR-15a-5p. Our findings may provide an important clinical basis for reversing the malignant phenotype of GC.

Introduction: Pancreatic adenocarcinoma (PAAD) is among the deadliest forms of cancer globally. Carbonic anhydrase 12 (CA12) is known to play central roles in regulating many cancers, but its function in the context of PAAD is rarely discussed. This study was, therefore, designed to assess the expression of CA12 in PAAD and to explore its underlying mechanistic role in this cancer type., Methods: Immunohistochemical staining was used to measure CA12 expression in PAAD samples. The functionality of pancreatic cancer cells expressing varying levels of CA12 was assessed through wound healing, Transwell, and CCK-8 assays. In addition, flow cytometry was used to measure apoptosis and cell cycle progression in these same cells, while Western blotting was used to analyze the expression of proteins associated with the NF-κB signaling pathway., Results: PAAD tissue samples exhibited significant CA12 downregulation (P < 0.001), and lower CA12 expression was, in turn, associated with poorer overall survival (P < 0.001). CA12 overexpression significantly impaired the proliferation of PAAD cell lines, instead inducing their apoptotic death and G0/G1 phase cell cycle arrest (P < 0.05). We additionally found that CA12 may exert its tumor suppressive roles via modulating the NF-κB signaling pathway., Conclusion: These results indicate that CA12 functions as a tumor suppressor in PAAD and may thus be a novel therapeutic target that can be used to guide PAAD patient treatment.

Background: Melanoma is one of the deadliest skin cancers. The treatment of advanced melanoma has been dramatically improved by immune checkpoint inhibitors and targeted therapies. However, many patients still do not respond to these therapies., Objective: To investigate whether NAP1L4 can be a new therapeutic target for melanoma., Methods: Immunohistochemical analysis of human nevus and melanoma tissues was performed. Real-time RT-PCR and immunoblotting were performed using human samples and melanoma cell lines. Next, we examined the effect of NAP1L4 knockdown in melanoma cell lines using cell migration and invasion assays. To investigate the molecular mechanism related to these results, immunoblotting of p21 and Slug was examined. MMP-2 and MMP-9 activity assays were also performed. Further, pathway analysis between NAP1L4 and MMP-2 was performed. Finally, the effects of NAP1L4 knockdown on cell proliferation, apoptosis, and cell cycle were analyzed., Results: NAP1L4 was overexpressed in melanoma tissues compared to the nevus tissue. NAP1L4 knockdown reduced melanoma cell migration and invasion. NAP1L4 knockdown upregulated p21 and downregulated Slug expression in melanoma cells. NAP1L4 knockdown decreased the active levels of MMP-2 in the supernatant from melanoma cells. NAP1L4 knockdown inhibited apoptosis in camptothecin-induced DNA damage, induced cell cycle arrest at the G1/S phase, and inhibited cell proliferation., Conclusions: NAP1L4 may play a role in cell migration and invasion in melanoma cells through the regulation of Slug. We propose that NAP1L4 can be a new therapeutic target for proliferation and invasion of melanoma cells., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Defensins, a class of small cysteine-rich cationic polypeptides across cellular life, are identified as antimicrobial compounds that display direct antimicrobial and immune signaling activities that are involved in the host defense. In addition to their roles in the innate immune system, accumulating studies have reported that some members of defensins are expressed and involved in some cancer cells, such as colon cancer, colorectal cancer, lung cancer and renal cell carcinomas. However, the roles of α-Defensin 5 (DEFA5) in tumorigenesis and development remain unknown. In the present study, bioinformatics analysis and quantitative PCR results showed that the expression level of DEFA5 was dramatically downregulated in human gastric cancer. Overexpression of human DEFA5 in gastric cancer cell lines SGC7901 and BGC823 effectively diminished cell proliferation and reduced the colony forming ability. Moreover, DEFA5 overexpression induced cell cycle arrest by significantly increasing the number of G1-phase cells. Consistently, in vivo tumor formation experiments in nude mice showed the suppression of the tumor growth by DEFA5 overexpression, suggesting an inhibitory effect of DEFA5 in gastric cancer. Mechanistically, DEFA5 directly binds to BMI1, which subsequently decreased its binding at the CDKN2a locus and upregulated the expression of 2 cyclin-dependent kinase inhibitors, p16 and p19. Taken together, we concluded that DEFA5 showed an inhibitory effect in gastric cancer cell growth and may serve as a potential tumor suppressor in gastric cancer., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: ZNF674-AS1, a recently characterized long noncoding RNA, shows prognostic significance in hepatocellular carcinoma and glioma. However, the expression and function of ZNF674-AS1 in non-small cell lung cancer (NSCLC) are unclear., Methods: In this work, we investigated the expression of ZNF674-AS1 in 83 pairs of NSCLC specimens and adjacent noncancerous lung tissues. The clinical significance of ZNF674-AS1 in NSCLC was analyzed. The role of ZNF674-AS1 in NSCLC growth and cell cycle progression was explored., Results: Our data show that ZNF674-AS1 expression is decreased in NSCLC compared to normal tissues. ZNF674-AS1 downregulation is significantly correlated with advanced TNM stage and decreased overall survival of NSCLC patients. Overexpression of ZNF674-AS1 inhibits NSCLC cell proliferation, colony formation, and tumorigenesis, which is accompanied by a G0/G1 cell cycle arrest. Conversely, knockdown of ZNF674-AS1 enhances the proliferation and colony formation of NSCLC cells. Biochemically, ZNF674-AS1 overexpression increases the expression of p21 through downregulation of miR-423-3p. Knockdown of p21 or overexpression of miR-423-3p blocks ZNF674-AS1-mediated growth suppression and G0/G1 cell cycle arrest. In addition, ZNF674-AS1 expression is negatively correlated with miR-423-3p in NSCLC specimens., Conclusions: ZNF674-AS1 suppresses NSCLC growth by downregulating miR-423-3p and inducing p21. This work suggests the therapeutic potential of ZNF674-AS1 in the treatment of NSCLC.

Background: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized., Results: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover., Conclusion: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.

Cholangiocarcinoma is the most common biliary duct malignancy and the second most common primary liver cancer, accounting for 10‑20% of hepatic malignancies. With high mortality and poor prognosis, the 5‑year survival rate of cholangiocarcinoma is only 10%. A previous study demonstrated a significant association between aspirin use and a decreased risk of cholangiocarcinoma. However, the effect of aspirin on cholangiocarcinoma remains unknown. Therefore, the aim of the present study was to investigate the effects of aspirin on cholangiocarcinoma in vitro and in vivo. Three cholangiocarcinoma cell lines were used to analyze the effect of aspirin on cell proliferation, cell cycle progression, apoptosis, and the regulation of microRNAs. MicroRNAs are known to regulate the development and progression of various types of cancer. An HuCCT‑1 xenograft model was used for the in vivo study. It was determined that aspirin inhibited the proliferation of human cholangiocarcinoma cells (except TKKK cells). Aspirin induced cell cycle arrest in the G0/G1 phase and regulated cell‑cycle related proteins in cholangiocarcinoma cells (HuCCT‑1 cells) but did not induce apoptosis. The expression of miR‑340‑5p was significantly upregulated after treatment, and overexpression of miR‑340‑5p inhibited the proliferation of HuCCT‑1 cells and decreased the levels of cyclin D1. TKKK cells had low miR‑340‑5p expression, which may explain why aspirin had no effect on their proliferation. In vivo, aspirin reduced the growth of xenografted tumors. In conclusion, the present study indicated that aspirin partially inhibited cholangiocarcinoma cell proliferation and tumor growth by inducing G0/G1 phase cell cycle arrest, potentially through the miR‑340‑5p/cyclin D1 axis.

Ubiquitin-conjugating enzyme E2S (UBE2S) is involved in protein degradation and signal transduction, but its function in the development of melanoma is unclear. We focused on the role of UBE2S in melanoma development both in vitro and in vivo. UBE2S was overexpressed in malignant melanoma cells and tissues, and UBE2S expression was significantly different between tumor node metastasis staging T4 and T1/T2/T3. We designed UBE2S short hairpin RNA (shUBE2S) and transfected it into A375, SK-MEL-28, and MUM-2B cells using lentivirus. By whole-genome filtering, 247 genes and 265 genes were upregulated and downregulated, respectively, in shUBE2S-treated melanoma; these genes were mainly involved in immune reactions, apoptosis, DNA damage repair, and cell movement. The proliferation of melanoma cells was inhibited, apoptosis was increased, and cell cycle was arrested in G1/S in shUBE2S-treated melanoma. Expression of epithelial to mesenchymal transition-related proteins was significantly suppressed, and tumor growth was also suppressed in shUBE2S BALB/C nude mice. shUBE2S treatment may cause cell cycle arrest in G1/S phase, inhibit proliferation, induce apoptosis, and suppress tumor growth through DNA damage repair, epithelial to mesenchymal transition inhibition, protein kinase B-mTOR pathway, NF-κB signaling, and immune reactions, which provides a comprehensive understanding of the role of UBE2S in melanoma development and the need for advanced clinical research into UBE2S., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Long intergenic noncoding RNAs (lincRNAs) play a vital role in the occurrence and progression of cancer. The mechanism of lincRNAs in colorectal cancer (CRC) has not been fully elucidated. In this context, an integrated comparative long noncoding RNA (lncRNA) microarray technology was used to determine the expression profile of lncRNAs in CRC. The roles of LINC00908 are unclear. We found that LINC00908 was significantly upregulated in CRC. Inhibition of LINC00908 resulted in reduced cell proliferation and G1 cell cycle arrest, which was mediated by cyclin D1, cyclin-dependent kinase 4, and phosphorylated retinoblastoma. Moreover, inhibition of LINC00908-induced apoptosis through the intrinsic apoptosis signaling pathway, as shown by the activation of caspase-9 and caspase-3. Mechanistically, miR-143-3p directly bound to LINC00908. miR-143-3p expression was negatively correlated with LINC00908 expression in CRC tissue. Functional experiments revealed opposing roles for miR-143-3p and LINC00908, suggesting that LINC00908 negatively regulates miR-143-3p. Mechanistically, miR-143-3p directly targets LINC00908. The KLF5 inhibitor ML264 affected proliferation and apoptosis, indicating that LINC00908 may act as a competing endogenous RNA to facilitate the expression of the miR-143-3p target gene KLF5. Thus, LINC00908 has an important proliferative and antiapoptotic role in CRC by regulating the cell cycle and intrinsic apoptosis. LINC00908 could be a potential biomarker and a new therapeutic target for CRC., (© 2020 Wiley Periodicals LLC.)

Previous reports have shown that miR-30d-5p functions as a tumor suppressor in prostate cancer and gallbladder carcinoma, but its role in renal cell carcinoma (RCC) remains elusive. This study was designed to explore the functional role of miR-30d-5p in proliferation and autophagy of RCC. Our results show that miR-30d-5p is significantly down-regulated in RCC tissues compared with normal tissues. miR-30d-5p overexpression suppressed cell proliferation, cell-cycle G1/S transition and autophagy, but promoted apoptosis in RCC cell lines (786-O and ACHN). Intriguingly, autophagy-related gene 5 (ATG5) was directly targeted by miR-30d-5p, as shown using luciferase reporter assay and biotin-avidin pull-down assay. Moreover, overexpression of ATG5 attenuated the inhibitory effect of miR-30d-5p on proliferation and autophagy in 786-O cells. These results suggest that miR-30d-5p suppresses proliferation and autophagy in RCC cells by targeting ATG5, and this pathway may be a suitable basis for the design of novel cancer therapeutics., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The conserved acetyltransferase Gcn5 is a member of several complexes in eukaryotic cells, playing roles in regulating chromatin organization, gene expression, metabolism, and cell growth and differentiation via acetylation of both nuclear and cytoplasmic proteins. Distinct functions of Gcn5 have been revealed through a combination of biochemical and genetic approaches in many in vitro studies and model organisms. In this review, we focus on the unique insights that have been gleaned from suppressor studies of gcn5 phenotypes in the budding yeast Saccharomyces cerevisiae. Such studies were fundamental in the early understanding of the balance of counteracting chromatin activities in regulating transcription. Most recently, suppressor screens have revealed roles for Gcn5 in early cell cycle (G 1 to S) gene expression and regulation of chromosome segregation during mitosis. Much has been learned, but many questions remain which will be informed by focused analysis of additional genetic and physical interactions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pulmonary atresia with ventricular septal defect (PA/VSD) is a rare congenital heart disease (CHD) characterized by a lack of luminal continuity and blood flow from either the right ventricle or the pulmonary artery, together with VSDs. The prevalence of PA/VSD is about 0.2% of live births and approximately 2% of CHDs. PA/VSD is similar to tetralogy of Fallot (TOF) in terms of structural and pathological characteristics. The pathogenesis of these two CHDs remains incompletely understood. It was previously reported that N-myc downstream-regulated gene (NDRG)4 is required for myocyte proliferation during early cardiac development. In the present study, we enrolled 80 unrelated patients with PA/VSD or TOF and identified a probably damaging variant p.T256M of NDRG4. The p.T256M variant impaired the proliferation ability of human cardiac myocytes (hCM). Furthermore, the p.T256M variant resulted in G1 and G2 arrest of hCM, followed by an increase in p27 and caspase-9 expression. Our results provide evidence that the p.T256M variant in NDRG4 is a pathogenic variant associated with impaired hCM proliferation and cell-cycle arrest and likely contributes towards the pathogenesis of PA/VSD and TOF., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Rationale: SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB). Methods: We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468. Results: We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties. Conclusions: SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aims: Previous studies have shown that the forkhead transcription factor FoxO6 involved in memory consolidation and hepatic glucose homeostasis. Here we asked whether chicken FoxO6 may regulate preadipocyte proliferation, apoptosis and early adipogenesis., Main Methods: Overexpression and knockdown of FoxO6 were performed and evaluated through cell proliferation methods, Oil-Red-O staining, and specific marker expression. Chromatin immunoprecipitation (ChIP) assay was performed to confirm cyclin G2 (CCNG2) as a direct target gene of FoxO6., Key Findings: FoxO6 is ubiquitously expressed in different chicken tissues and highly expressed in liver, abdominal fat, and preadipocytes in cultured cell. FoxO6 overexpression decreased preadipocyte proliferation by causing G1-phase cell-cycle arrest, whereas inhibition of FoxO6 showed the opposite effects. Overexpression or knockdown of FoxO6 significantly altered the mRNA and protein levels of cell-cycle related markers, such as CCNG2, cyclin dependent kinase inhibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1A (CDKN1A) and cyclin D2 (CCND2). During preadipocyte proliferation, FoxO6 targets and induces expression of CCNG2, as confirmed by ChIP assay and qPCR. In addition, FoxO6 induces preadipocyte apoptosis through increasing the protein expression levels of cleaved caspase-3 and cleaved caspase-8. Moreover, FoxO6 at the early stage of adipogenesis suppressed mRNA and protein levels of the key early regulators of adipogenesis, such as PPARγ and C/EBPα., Significance: The results demonstrate that FoxO6 controls preadipocyte proliferation, apoptosis and early adipogenesis, and point to new approaches for further studies related to obesity., (Copyright © 2020. Published by Elsevier Inc.)

BACKGROUNDAlthough CDK4/6 inhibitors are an established treatment for hormone receptor-positive, HER2-negative metastatic breast cancers, their benefit in other malignancies remains limited.METHODSWe investigated factors associated with clinical outcomes from CDK4/6 inhibitor-based therapy among patients with G1/S phase cell-cycle alterations (CDK4/6 amplifications, CCND1/2/3 amplifications, or CDKN2A/B alterations).RESULTSOverall, 2457 patients with diverse solid tumors that underwent clinical-grade, next-generation sequencing (182-465 genes) and therapy outcome of (non-breast cancer) patients treated with matched CDK4/6 inhibitors were analyzed. G1/S phase cell-cycle alterations occurred in 20.6% (507 of 2457) of patients; 99% of those patients (n = 501) harbored ≥1 characterized co-alteration (median, 4; range, 0-24). In 40 patients with G1/S phase cell-cycle alterations given CDK4/6 inhibitors as part of their regimen, significantly longer median progression-free survival (PFS) was observed when CDK4/6 inhibitor-based therapies matched a larger proportion of tumor alterations, often because CDK4/6 inhibitors were administered together with other drugs that were matched to genomic co-alterations, hence achieving a high matching score (high vs. low [≥50% vs. <50%] matching score, PFS, 6.2 vs. 2.0 months, P < 0.001 [n = 40] [multivariate]) and higher rate of stable disease ≥6 months or an objective response (57% vs. 21%, P = 0.048).CONCLUSIONIn summary, in cell-cycle-altered cancers, matched CDK4/6 inhibitors, as part of an individualized regimen targeting a majority of genomic alterations, was independently associated with longer PFS.TRIAL REGISTRATIONClinicalTrials.gov NCT02478931.FUNDINGJoan and Irwin Jacobs Fund, National Cancer Institute (P30 CA023100, R01 CA226803), and the FDA (R01 FD006334).

Human umbilical cord mesenchymal stem cells can be obtained from different parts of the umbilical cord, including Wharton's jelly. Transplantation of Wharton's jelly umbilical cord stem cells (WJCMSCs) is a promising strategy for the treatment of various diseases. However, the molecular mechanisms underlying the proliferation of WJCMSCs are incompletely understood. Here, we report that overexpression of miR-196b-5p in WJCMSCs suppresses proliferation and arrests the cell cycle in G0/G1 phase, whereas knockdown of miR-196b-5p promotes WJCMSC proliferation and cell-cycle progression. Moreover, miR-196b-5p overexpression resulted in decreased levels of Cyclin A, Cyclin D, Cyclin E and cyclin-dependent kinases 2 and increased levels of p15 INK4b , whereas miR-196b-5p knockdown had the opposite effects. In conclusion, our data suggests that miR-196b-5p inhibits WJCMSC proliferation by enhancing G0/G1-phase arrest., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Downregulation of the myeloid master regulator Spi1 /PU.1 plays a pivotal role in leukemogenesis, and we previously showed that Spi1 /PU.1 directly represses metallothionein (MT)-1G through the epigenetic activity of PU.1. Furthermore, we recently demonstrated that overexpression of MT-1G inhibits retinoic acid-induced differentiation of acute promyelocytic leukemia NB4 cells. As PU.1 is a master regulator of growth and differentiation in myeloid cells, we examined its effects on cell proliferation of MT-1G -overexpressing NB4 (NB4MTOE) cells in the present study. Although there were no significant differences in total viable cell numbers between NB4MTOE cells and control cells during the time course examined, the proportion of S-phase cells was obviously increased in all NB4MTOE cells at 16-24 h after serum stimulation. Consistent with these findings, real-time PCR analyses revealed marked increases in the expression of cyclin E (G1/S-phase cyclin) and cyclin A (S-phase cyclin) in NB4MTOE cells during the same time period. Furthermore, NB4MTOE cells were significantly resistant to cytosine arabinoside (Ara-C), an S-phase-specific chemotherapeutic drug. Collectively, these findings suggest a role for MT-1G in G1/S transition during the growth phase of NB4 cells., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

A comprehensive molecular mechanistic role of lutein on adipogenesis is not well understood. The present study focused to evaluate the effect of lutein at the early and late phase of adipocyte differentiation in vitro using a 3T3-L1 cell model. The effect of purified carotenoid on the viability of normal and differentiated 3T3-L1 cells was analyzed by WST-1 assay. Oil Red O and Nile red staining were employed to observe lipid droplets in mature adipocytes. The effect of lutein on gene and protein expression of major transcription factors and adipogenic markers was analyzed by RT-PCR and western blotting, respectively. The role of lutein on mitotic clonal expansion was analyzed by flow cytometry. The results showed a significant reduction (p < 0.05) in the accumulation of lipid droplets in lutein-treated (5 μM) cells. Inhibition in lipid accumulation was associated with down-regulated expression of CEBP-α and PPAR-γ at gene and protein levels. Subsequently, lutein repressed gene expression of FAS, FABP4, and SCD1 in mature adipocytes. Interestingly, it blocks the protein expression of CEBP-α and PPAR-γ in the initial stages of adipocyte differentiation. This early-stage inhibition of adipocyte differentiation is linked with repressed phosphorylation AKT and ERK. Further, upregulated cyclin D and down-regulated CDK4 and CDK2 in lutein treated adipocytes enumerate its role in delaying the cell cycle progression at the G0/G1 phase. Our results emphasize that adipogenesis inhibitory efficacy of lutein is potentiated by halting early phase regulators of adipocyte differentiation, which strengthens the competency of lutein besides its inevitable presence in the human body., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background/aim: Forkhead box O4 (FOXO4) has been demonstrated to be a tumor suppressor and proposed as target for treatment of a variety of cancer types. However, the role of FOXO4 in cholangiocarcinoma (CCA), a dangerous cancer of bile-duct epithelium, has rarely been explored., Materials and Methods: The proliferative rate of CCA cell lines KKU-213B, KKU-055 and KKK-D068 was investigated using the sulforhodamine B (SRB) assay. Levels of FOXO4, cyclin E1 (CCNE1), CCNE2, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A) expression were measured using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The cell-cycle profile was explored using flow cytometry., Results: The SRB assay demonstrated that KKU-213B expressed low levels of FOXO4 but its proliferative rate was highest of all cell lines tested. Interestingly, ectopic expression of FOXO4 significantly suppressed proliferation of KKU-213B cells. Cell-cycle analysis revealed that the cell population in the G 0 /G 1 phase was significantly higher in FOXO4-transfected KKU-213B cells than in controls. RT-qPCR analysis demonstrated that the levels of expression of genes that play a role in the G 1 /S transition, namely CCNE1, CCNE2, CDK2 and CDC25A, were significantly lower in FOXO4-transfected KKU-213B cells compared to controls., Conclusion: FOXO4 suppressed CCA cell proliferation partly via down-regulating the expression of genes involved in the G 1 /S transition, leading to G 0 /G 1 arrest. Our findings suggest that induction of FOXO4 expression might be an alternative approach for the treatment of CCA., (Copyright © 2020 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Breast cancer (BC) is the most frequently diagnosed type of cancer, and the leading cause of cancer‑associated mortality in females worldwide. The aim of the present study was to investigate the prognostic and therapeutic potential of NUF2 in BC. The expression levels of NUF2 in BC tissues and cell lines were evaluated via bioinformatics, reverse transcription‑quantitative PCR, western blot analysis and immunohistochemistry (IHC). In addition, the effect of NUF2 knockdown on BC cell proliferation and apoptosis was investigated using small interfering RNA (siRNA) technology. Bioinformatics and IHC analysis showed that NUF2 was overexpressed in BC tissues. Furthermore, western blot and RT‑qPCR analyses demonstrated that NUF2 was upregulated in BC cells. In addition, BC cells transfected with NUF2 siRNA exhibited significantly decreased proliferation and colony formation, and increased apoptosis, compared with the control. Additionally, cell cycle analysis revealed that NUF2 induced G0/G1 cell cycle arrest by inhibiting cyclin B1 expression. Collectively, the present study suggested that NUF2 may represent a promising prognostic biomarker and a potential therapeutic target for BC.

Glioma is a primary, malignant, and aggressive brain tumor in adults. To develop new therapeutic strategies for glioma, we must determine its underlying mechanisms. In the present study, we aimed to investigate the potential role of miR-1272-ADAM9-CDCP1 signaling in the progression of glioma. We found that ectopic expression of miR-1272 produced significant inhibitory effects on cell proliferation and migration and was associated with cell cycle G0/G1 arrest in A172 and SHG44 glioma cells. Using the luciferase reporter assay, we identified ADAM9 as a target of miR-1272. The expression of ADAM9 was markedly decreased or increased after overexpression or inhibition, respectively, of miR-1272 in glioma cells. Moreover, overexpression of ADAM9 reversed the inhibitory effects of miR-1272 on glioma cell progression. Furthermore, CDCP1 served as a potential downstream molecule of miR-1272/ADAM9 signaling in glioma and promoted the proliferation and migration of glioma. Results derived from clinical samples and online databases confirmed correlations between the expression of ADAM9 and CDCP1 and both the severity and prognosis of glioma. In conclusion, these results suggest that miR-1272 and CDCP1 may act as novel regulators in glioma. The miR-1272/ADAM9/CDCP1 pathway may serve as a potential candidate pathway for the prevention of glioma.

Background: Dysregulation of cell cycle progression is a common feature of human cancer cells; however, its mechanism remains unclear. This study aims to clarify the role and the underlying mechanisms of Roquin1 in cell cycle arrest in breast cancer., Methods: Public cancer databases were analyzed to identify the expression pattern of Roquin1 in human breast cancers and its association with patient survival. Quantitative real-time PCR and Western blots were performed to detect the expression of Roquin1 in breast cancer samples and cell lines. Cell counting, MTT assays, flow cytometry, and in vivo analyses were conducted to investigate the effects of Roquin1 on cell proliferation, cell cycle progression and tumor progression. RNA sequencing was applied to identify the differentially expressed genes regulated by Roquin1. RNA immunoprecipitation assay, luciferase reporter assay, mRNA half-life detection, RNA affinity binding assay, and RIP-ChIP were used to explore the molecular mechanisms of Roquin1., Results: We showed that Roquin1 expression in breast cancer tissues and cell lines was inhibited, and the reduction in Roquin1 expression was associated with poor overall survival and relapse-free survival of patients with breast cancer. Roquin1 overexpression inhibited cell proliferation and induced G1/S cell cycle arrest without causing significant apoptosis. In contrast, knockdown of Roquin1 promoted cell growth and cycle progression. Moreover, in vivo induction of Roquin1 by adenovirus significantly suppressed breast tumor growth and metastasis. Mechanistically, Roquin1 selectively destabilizes cell cycle-promoting genes, including Cyclin D1, Cyclin E1, cyclin dependent kinase 6 (CDK6) and minichromosome maintenance 2 (MCM2), by targeting the stem-loop structure in the 3' untranslated region (3'UTR) of mRNAs via its ROQ domain, leading to the downregulation of cell cycle-promoting mRNAs., Conclusions: Our findings demonstrated that Roquin1 is a novel breast tumor suppressor and could induce G1/S cell cycle arrest by selectively downregulating the expression of cell cycle-promoting genes, which might be a potential molecular target for breast cancer treatment.

Background: A previous study revealed that matrin-3 is an essential component in maintaining fibroblast growth factor 2 (FGF2)-mediated undifferentiation of neural stem cells (NSCs) using a proteomics approach. Malignant melanoma (MM) arises from melanocytes that originate from neural crest stem cells during development. Additionally, it has been reported that the expression of FGF2 is positively correlated with the progression of MM., Objective: We expected that matrin-3, as a downstream component of FGF2, might be associated with the aggressiveness or differentiation of MM., Methods: Matrin-3 expression was measured in human melanoma patient tissues and human MM cell lines. We analyzed the effect of matrin-3 siRNA on the proliferation of human MM cell lines and focused on cell cycle progression and apoptosis. We carried out in vivo xenograft tumor experiments by implanting A375 cells transfected with matrin-3 shRNA., Results: Matrin-3 was highly expressed in MM, and matrin-3 knockdown inhibited the proliferation of melanoma cellsin vivo and in vitro. Furthermore, we found that matrin-3 knockdown led to an accumulation of cells in the G1 phase and an increase in apoptotic cell number., Conclusion: Our results suggest that matrin-3 could be a new therapeutic target for the treatment of MM., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Hypoxia in the microenvironment is related to chemotherapy resistance, tumor progression, and metastasis. Curcumin, as a phenolic compound extracted from the turmeric, has been used as an anti-cancer agent with low toxicity in recent years. Since curcumin has inhibitory activities against hypoxia-inducible factors (HIFs) in several cancers, this study was conducted to examine the effect of curcumin on MCF-7 cells and cancer stem-like cells (CS-LCs) under hypoxic and normoxic conditions. CS-LCs were isolated from MCF-7 cells using the magnet activated cell sorting (MACS) method based on CD 44 + / CD 24 - surface markers. The effects of curcumin on the viability of MCF-7 cells and CS-LCs were examined in hypoxic and normoxic conditions using the MTT test. The effects of curcumin on apoptosis and cell cycle of CS-LCs and MCF-7 cells were analyzed using flow cytometry. Moreover, the inhibitory effects of curcumin on the levels of HIF-1 and HIF-2α protein in CS-LCs were investigated using the western blot method. Early apoptosis occurred in CSC-LCs more than MCF-7 cells under hypoxic conditions. Flow cytometry assay showed that curcumin caused cell cycle arrest of CSC-LCs and MCF-7 at the G2/M phase under hypoxic conditions while under normoxic conditions, arrest occurred at the G0/G1 phase in MCF-7 cells and at S and G2/M phases in CS-LCs. Based on the results, the curcumin inhibited the expression of HIF-1 by degrading ARNT in CS-LCs.In conclusion, curcumin has inhibitory effects on MCF- 7 cells and CS- LCs and thus may be used as an antitumor agent., Competing Interests: The authors declare no potential conflicts of interest., (Thieme. All rights reserved.)

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Cell cycle dysregulation caused by aberrant cyclin D1 and CDK4 expression is a major determinant for proliferation of cancer cells in mantle cell lymphoma (MCL). Inhibition of CDK4/6 induces G1 arrest of MCL cells in patients, appearing to deepen and prolong the clinical response to partner agents. This article reviews aberrations of cell cycle genes in MCL cells and clinical trials of CDK4/6 inhibitors for MCL. Integrative longitudinal functional genomics is discussed as a strategy to discover genomic drivers for resistance in cancer cells and cancer-immune interactions that potentially contribute to the clinical response to palbociclib combination therapy in MCL., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The interaction between circRNAs and atherosclerosis has been extensively studied. However, more novel circRNAs need to be explored to help establish a perfect regulatory network. In the present research, hsa&#95;circ&#95;0000345 was demonstrated to regulate cellular development of oxygenized low-density lipoprotein (ox-LDL)-treated aortic smooth muscle cells (ASMCs), which was closely related to the occurrence and progress of atherosclerosis. Ox-LDL exposure remarkably decreased hsa&#95;circ&#95;0000345 expression in ASMCs. Transfection-induced hsa&#95;circ&#95;0000345 overexpression activated cell viability (detected by an MTT assay) and restrained cellular apoptosis (analysed by flow cytometry) in the atherosclerosis cellular model. While down-regulation of hsa&#95;circ&#95;0000345 reduced cell viability and promoted cell apoptosis. In addition, the data of the cell cycle distribution analysis and trans-well assay indicated that cell cycle progression was arrested at the G1 phase while cell invasion was enhanced in ASMCs following treatment of ox-LDL in the context of hsa&#95;circ&#95;0000345 OE plasmids. In addition, up-regulation of hsa&#95;circ&#95;0000345 supported HIF-1α at both the mRNA and protein level, and down-regulation of hsa&#95;circ&#95;0000345 reduced HIF-1α expression. Overall, the above findings revealed that hsa&#95;circ&#95;0000345 was a dramatic regulator of ASMCs proliferation, apoptosis and invasion in response to ox-LDL treatment. Hsa&#95;circ&#95;0000345 was identified as a protector of cell viability during ox-LDL induced cell development., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Genomic instability is a hallmark of cancer. The G1 checkpoint allows cells to repair damaged DNA that may lead to genomic instability. The high-risk human papillomavirus (HPV) E7 gene can abrogate the G1 checkpoint, yet the mechanism is still not fully understood. Our recent study showed that WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein 1) plays a role in regulating G1 checkpoint of E7 expressing cells. In this study, we explored the mechanism by which WDHD1 regulates G1 checkpoint in HPV E7 expressing cells., Methods: NIKS and RPE1 derived cell lines were used. Real-time PCR, Rescue experiment, FACS and BrdU labeling experiments were performed to examine role of GCN5 in G1 checkpoint abrogation in HPV-16 E7 expressing cells., Results: In this study, we observed that WDHD1 facilitates G1 checkpoint abrogation by modulating GCN5 in HPV E7 expressing cells. Notably, depletion of WDHD1 caused G1 arrest while overexpression of GCN5 rescued the inhibitory effects of WDHD1 knockdown on G1/S progression. Furthermore, siWDHD1 significantly decreased cell cycle proliferation and DNA synthesis that was correlated with Akt phosphorylation (p-Akt), which was reversed by GCN5 overexpression in HPV E7 expressing cells., Conclusions: In summary, our data identified a WDHD1/GCN5/Akt pathway leading to the abrogation of G1 checkpoint in the presence of damaged DNA, which may cause genomic instability and eventually HPV induced tumorigenesis.

Increasing evidence has indicated the roles of sirtuin 7 (SIRT7) in numerous human cancers. However, the effects and the clinical significance of SIRT7 in human lung cancer is largely unknown. The present research demonstrated that SIRT7 was increased in human lung cancer tumor tissues. SIRT7 upregulation was associated with clinicopathological characteristics of lung cancer malignancy including positive lymph node metastasis, high pathologic stage and large tumor size. SIRT7 was also upregulated in human non‑small cell lung cancer (NSCLC) cell lines. Furthermore SIRT7‑overexpressed A549 (A549‑SIRT7) and SIRT7‑knocked down H292 (H292‑shSIRT7) human NSCLC cell lines were established. Using these NSCLC cells and xenograft mouse models, it was revealed that SIRT7 overexpression markedly promoted growth and G1 to S cell cycle phase transition as well as migration, invasion and distant lung metastasis in A549 NSCLC cells, whereas SIRT7 knockdown suppressed these processes in H292 NSCLC cells. Mechanistically, in A549 NSCLC cells, SIRT7 overexpression significantly activated not only protein kinase B (AKT) signaling but also extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling. SIRT7 overexpression also significantly downregulated cyclin‑dependent kinase (CDK) inhibitors including p21 and p27 as well as upregulated cyclins including cyclin D1 and cyclin E1, and CDKs including CDK2 and CDK4. Notably, the epithelial‑mesenchymal transition (EMT) process of A549 NSCLC cells was facilitated by SIRT7 overexpression, as evidenced by E‑cadherin epithelial marker downregulation and mesenchymal markers (N‑cadherin, vimentin, Snail and Slug) upregulation. In addition, SIRT7 knockdown in H292 NSCLC cells exhibited the opposite regulatory effects. Moreover, inhibition of AKT signaling abated the promoting effects of SIRT7 in NSCLC cell proliferation and EMT progression. The present data indicated that SIRT7 accelerated human NSCLC cell growth and metastasis possibly by promotion of G1 to S‑phase transition and EMT through modulation of the expression of G1‑phase checkpoint molecules and EMT markers as well as activation of AKT and ERK1/2 signaling. SIRT7 could be an innovative potential target for human NSCLC therapy.