Delayed Cell Cycle Progression Research Articles

FOXM1 Deubiquitination by USP21 Regulates Cell Cycle Progression and Paclitaxel Sensitivity in Basal-like Breast Cancer

SUMMARYThe transcription factor FOXM1 contributes to cell cycle progression and is significantly upregulated in basal-like breast cancer (BLBC). Despite its importance in normal and cancer cell cycles, we lack a complete understanding of mechanisms that regulate FOXM1. We identified USP21 in an RNAi-based screen for deubiquitinases that control FOXM1 abundance. USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship. Depleting USP21 downregulates the FOXM1 transcriptional network and causes a signifi-cant delay in cell cycle progression. Significantly, USP21 depletion sensitized BLBC cell lines and mouse xenograft tumors to paclitaxel, an anti-mitotic, frontline therapy in BLBC treatment. USP21 is the most frequently amplified deubiquitinase in BLBC patient tumors, and its amplification co-occurs with the upregulation of FOXM1 protein. Altogether, these data suggest a role for USP21 in the proliferation and potentially treatment of FOXM1-high, USP21-high BLBC.

The effects of rooibos (Aspalathus linearis) on 3T3-L1 preadipocytes after the induction of mitochondrial dysfunction

Abstract Rooibos (Aspalathus linearis) is a South African fynbos plant, well-known for its strong anti-oxidant capacity and use in cosmetic products. The anti-ageing properties of fermented and green (unfermented) rooibos were investigated using a cell culture model designed to evaluate the involvement of mitochondrial dysfunction in the age-related decline in preadipocyte function. Mitochondrial dysfunction was induced through long-term exposure of 3T3-L1 preadipocytes to ethidium bromide (EtBr). Mitochondrial DNA (mtDNA) depleted (ρ0) 3T3-L1 preadipocytes showed a significantly reduced rate of proliferation, delayed cell cycle progression (G0/G1 phase arrest), decreased mitochondrial membrane potential (ΔΨm), and increased glucose utilization and lactate production. Treatment with rooibos stimulated cell growth, attenuated G0/G1 phase arrest, improved ΔΨm, and increased glucose utilization and lactate production. Cellular ATP was significantly improved and AMPK activation was observed. The results obtained indicate that rooibos, particularly green rooibos, exhibit effects which preserve the functional capacity of preadipocytes exposed to ageing related insults.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

INDUCED POLYPLOIDY AND SORTING OF DAMAGED DNA BY MICRONUCLEATION IN RADIORESISTANT RAT LIVER EPITHELIAL STEM-LIKE CELLS EXPOSED TO X-RAYS.

Rat liver stem-like epithelial cells (WB-F344) that under certain conditions may differentiate into hepa- tocyte and biliary lineages were subjected to acute X-irradiation with the aim to examine cell cycle peculiarities dur- ing the course of survival. Suspensions of WB-F344 cells that grew as a monolayer and reached sub-confluence were irradiated with 1, 5, and 10 Gy of X-rays (2 Gy/min). As an intact control, sham-irradiated cells were used. After irra- diation, cells were plated into 25-cm2 tissue culture flasks to culture them for over several days without reaching contact inhibition. On days 1, 2, 3, and 5 post-irradiation, cells were harvested and examined for nuclear morpholo- gy and DNA ploidy by stoichiometric toluidine blue reaction and image cytometry. On days 7 and 9 post-irradiation, only heavily irradiated (10 Gy) cells were examined. Also, 10 Gy-irradiated cells were chosen for immunofluorescence staining to monitor persistence of DNA lesions (γ-H2AX), cell proliferation (Ki-67), and self-renewal factors charac- teristic for stem cells (OCT4 and NANOG). Radioresistance of WB-F344 cells was evidenced by the findings that they do not undergo rapid and mas- sive cell death that in fact was weakly manifested as apoptotic even in heavily irradiated cells. Instead, there was cell cycle progression delay accompanied by polyploidization (via Ki-67-positive mitotic slippage or via impaired cytokinesis) and micronucleation in a dose-dependent manner, although micronucleation to some extent went ahead of polyploidization. Polyploid cells amenable for recovering from DNA damage can mitotically depolyploidize. Many micronuclei contained γ-H2AX clusters, suggesting isolation of severely damaged DNA fragments. Both factors, OCT4 and NANOG, were expressed in the intact control, but became enhanced after irradiation. Although the fact of micronucleation is indicative of genotoxic effect, WB-F344 cells can probably escape cell death via sorting of damaged DNA by micronuclei. Induction of polyploidy in these cells can be adaptive to promote cell survival and tissue regeneration with possible involvement of self-renewal mechanism.

Loss of Nuclear Functions of HOXA10 Is Associated With Testicular Cancer Proliferation.

Background: HOXA10 is a key transcriptional factor that regulates testis development as reported from previous transgenic mouse models and human inherited diseases. However, whether it also plays important roles in promoting the development of testicular cancer is not well-understood.Objective: To study the expression of HOXA10 and its regulated signaling pathways in testicular cancers.Design, Setting, and Participants: A tissue microarray was constructed with benign and cancerous testis. TCam2, NT-2, and NCCIT cell models were applied in this study.Intervention: Immunohistochemistry and immunofluorescence were performed to measure the expression and cellular localization of HOXA10 in testicular cancer tissues and cell models. Cell proliferation and cell cycling rates were determined by BrdU incorporation and flow cytometry assays. HOXA10 transcriptomes were profiled with Ampliseq RNA-seq in testicular cancer cells. Immunoblotting assays were used to detect HOXA10-regulated signaling.Results: HOXA10 is a nuclear protein in benign spermatocytes. Reduced nuclear expression and increased cytoplasmic expression of HOXA10 are associated with testicular cancers. These changes are consistent in both seminoma and non-seminoma. Enhanced HOXA10 expression in testicular cancer cell models inhibits cell proliferation and delays cell cycle progression through G2/M phases. These functions of HOXA10 mainly affect the TP53, cKit, STAT3, AKT, and ERK signaling pathways.Conclusions: Loss of nuclear functions of HOXA10 enhances proliferation of testicular cancer cells, suggesting that downregulation of HOXA10 transcription activity may promote the development of testicular cancers.

Centromere protein U expression promotes non-small-cell lung cancer cell proliferation through FOXM1 and predicts poor survival.

PurposeCentromere protein U (CENPU) abnormally exhibits high expression in various types of human tumor tissues and participates in tumor progression; however, its expression pattern and biological function in lung cancer have not yet been elucidated. In the present study, we explored the clinical significance and biological function of CENPU in lung cancer.Materials and methodsThe Cancer Genome Atlas (TCGA) data analyses, quantitative real-time PCR (RT-PCR), and Western blotting were performed to quantify CENPU and FOXM1 expression in non-small-cell lung cancer (NSCLC) samples. Survival data were obtained from Kaplan–Meier plotter or PROGgene V2 prognostic database. The function of CENPU in lung cancer cell proliferation was determined using 5-ethynyl-2′-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and cell cycle assays, and the underlying mechanism was determined through bioinformatic analyses and validated by in vitro siRNA or plasmid transfection experiments.ResultsCENPU was abnormally overexpressed in NSCLC samples compared with matched paired normal tissues. Higher expression of CENPU predicted worse overall survival (OS) and relapse-free survival (RFS) in NSCLC patients. Knockdown of CENPU expression by siRNA significantly inhibited proliferation and delayed cell cycle progression of lung cancer cells. To figure out the mechanism, bioinformatic analyses were performed and the results showed that the transcription factor, FOXM1, positively correlated with CENPU. Further in vitro experiments indicated that FOXM1 was the possible downstream transcription factor of CENPU as the knockdown of CENPU led to lower expression of FOXM1 and the overexpression of FOXM1 significantly reversed the inhibition of proliferation caused by CENPU knockdown. Furthermore, FOXM1 was highly expressed in NSCLC. The knockdown of FOXM1 also attenuated proliferation and induced G1 arrest in lung cancer cells.ConclusionCENPU was highly expressed in NSCLC tissues, wherein it promoted lung cancer cell proliferation via the transcription factor, FOXM1, which could be a potential target for therapeutic strategies.

Restoration of miRNA-148a in pancreatic cancer reduces invasion and metastasis by inhibiting the Wnt/β-catenin signaling pathway via downregulating maternally expressed gene-3.

Various microRNAs (miRNA) have been recognized potential novel tumor markers and have a critical role in cancer development and progression. Recently, methylation of miRNA-148a was identified as a crucial biochemical process in the progression of cancer. However, its potential role and in pancreatic cancer as well as the underlying mechanisms have remained largely elusive. The present study investigated the potential antitumor effect of miR-148a as well as its impact on invasion and metastasis in pancreatic cancer. It was found that the expression of miRNA-148a and the potential predictive biomarker maternally expressed gene-3 (MEG-3) were obviously decreased in human pancreatic cancer tissues compared with those in adjacent non-tumorous tissues. Furthermore, miR-148a was found to be downregulated in pancreatic cancer cell lines compared with normal pancreatic cells through promoter methylation. An MTT assay and a clonogenic assay demonstrated that restoration of miRNA-148a inhibited the proliferation and colony formation of pancreatic cancer cells. In addition, miR-148a transduction led to the upregulation of MEG-3 expression and promoted apoptosis of pancreatic cancer cells. Western blot analysis revealed that transduction of miR-148a markedly decreased the expression levels of C-myc, cyclin D1 and β-catenin in pancreatic cancer cells. Methylation of miR-148a not only decreased the endogenous β-catenin levels but also inhibited the nuclear translocation of β-catenin to delay cell cycle progression. Furthermore, ectopic miR-148a methylation inhibited pancreatic cancer cell migration and invasion via causing an upregulation of MEG-3 expression. Most importantly, ectopic overexpression of miR-148a in pancreatic cancer cells inhibited tumor formation in an animal experiment. Taken together, miR-148a methylation is a crucial regulatory process to inhibit the proliferation and invasion of pancreatic cancer cells, and transduction of miR-148a suppressed the proliferation of pancreatic cancer cells through negative regulation of the Wnt/β-catenin signaling pathway. The findings of the present study suggested that miRNA-148a acts as a tumor suppressor in pancreatic cancer and may contribute to the development of novel treatments for pancreatic cancer.

A tight balance of Karyopherin \u03b21 expression is required in cervical cancer cells

BackgroundKaryopherin β1 (Kpnβ1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnβ1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnβ1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. Here, the biological effects associated with Kpnβ1 overexpression were investigated in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype.MethodsTo evaluate the effect of Kpnβ1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnβ1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis.ResultsOur data revealed that Kpnβ1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnβ1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnβ1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively.ConclusionsOverall, our results suggest that a tight balance of Kpnβ1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes.

RNF138 confers cisplatin resistance in gastric cancer cells via activating Chk1 signaling pathway

ABSTRACT Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.

A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression.

DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here, we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which gives rise to aneuploid progeny.

SiSTL2 Is Required for Cell Cycle, Leaf Organ Development, Chloroplast Biogenesis, and Has Effects on C4 Photosynthesis in Setaria italica (L.) P. Beauv.

Deoxycytidine monophosphate deaminase (DCD) is a key enzyme in the de novo dTTP biosynthesis pathway. Previous studies have indicated that DCD plays key roles in the maintenance of the balance of dNTP pools, cell cycle progression, and plant development. However, few studies have elucidated the functions of the DCD gene in Panicoideae plants. Setaria has been proposed as an ideal model of Panicoideae grasses, especially for C4 photosynthesis research. Here, a Setaria italica stripe leaf mutant (sistl2) was isolated from EMS-induced lines of “Yugu1,” the wild-type parent. The sistl2 mutant exhibited semi-dwarf, striped leaves, abnormal chloroplast ultrastructure, and delayed cell cycle progression compared with Yugu1. High-throughput sequencing and map-based cloning identified the causal gene SiSTL2, which encodes a DCD protein. The occurrence of a single-base G to A substitution in the fifth intron introduced alternative splicing, which led to the early termination of translation. Further physiological and transcriptomic investigation indicated that SiSTL2 plays an essential role in the regulation of chloroplast biogenesis, cell cycle, and DNA replication, which suggested that the gene has conserved functions in both foxtail millet and rice. Remarkably, in contrast to DCD mutants in C3 rice, sistl2 showed a significant reduction in leaf cell size and affected C4 photosynthetic capacity in foxtail millet. qPCR showed that SiSTL2 had a similar expression pattern to typical C4 genes in response to a low CO2 environment. Moreover, the loss of function of SiSTL2 resulted in a reduction of leaf 13C content and the enrichment of DEGs in photosynthetic carbon fixation. Our research provides in-depth knowledge of the role of DCD in the C4 photosynthesis model S. italica and proposed new directions for further study of the function of DCD.

Abstract 4129: RUNX2 as a regulator of high-risk T-ALL

Abstract For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is <10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p <0.0005) and those who failed induction therapy (p <0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p < 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p < 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p < 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p <0.001) and bone marrow (p <0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival. Citation Format: Nitesh Devinarayan Sharma, Christian K. Nickl, Stuart S. Winter, Huining Kang, Ksenia Matlawska Wasowska. RUNX2 as a regulator of high-risk T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4129.

Abstract 3548: EPAC-RAP1, the alternative cyclic AMP signaling pathway, regulates cell cycle progression in primary melanoma cells

Abstract Cyclic AMP (cAMP) is an important second messenger that acts as a key mediator in a wide range of cellular pathways. In melanocytes, the G-Protein Coupled Receptor (GPCR) Melanocortin-1-Receptor (MC1R), an important regulator of melanocyte proliferation and melanogenesis, activates cAMP signaling. Polymorphisms in MC1R lead to cAMP signaling impairments which are accompanied by higher risk for melanoma development. Despite the importance of cAMP signaling in melanocytes, very little is known about its role in melanoma. Exchange Protein directly Activated by cAMP (EPAC) constitutes an alternative pathway to cAMP signaling and is known to mediate the crosstalk between the cAMP and MAPK pathways. In this study, we show that pharmacological inhibition of EPAC, delays cell cycle progression and inhibits the growth of human primary melanoma cells. Flow cytometry and western blot analysis of cell cycle proteins showed that inhibition of EPAC arrests cells in G1-S and delays their mitotic progression. Paradoxically, inhibition of EPAC also caused an increase in AKT phosphorylation, suggesting a cell survival response elicited upon the cell cycle arrest caused by inhibition of EPAC allows continued survival of primary melanoma cells. Inhibition of EPAC in metastatic melanoma cells, on the other hand, stimulated their growth. These data show differential role of EPAC in primary and metastatic melanoma cells and provide a basis for targeting EPAC for prevention of cutaneous melanoma progression. Citation Format: Aishwarya Krishnan, Kirthana Prabhakar, Mary Ndiaye, Nihal Ahmad, Carlos Ivan Rodriguez, Vijayasaradhi Setaluri. EPAC-RAP1, the alternative cyclic AMP signaling pathway, regulates cell cycle progression in primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3548.

PGC-1α 在U251 细胞中协同Bcl-2 通过降低ROS 来调控细胞周期

B-cell lymphoma 2 (Bcl-2) has a dual function, acting as both an oncogene and an anti-tumor gene. It is well known that Bcl-2 exerts its tumor promoting function through the mitochondrial pathway. However, the mechanism by which it suppresses tumor formation is not well understood. We have previously shown that Bcl-2 inhibits cell cycle progression from the G0/G1 to the S phase after serum starvation, and that quiescent Bcl-2 expressing cells maintain a significantly lower level of mitochondrial reactive oxygen species (ROS) than control cells. Based on the fact that ROS mediate cell cycle progression and are controlled by peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α), a key molecule induced by prolonged starvation and involved in mitochondrial metabolism, we hypothesized that PGC-1α might be related to the cell cycle function of Bcl-2. In this paper, we show that PGC-1α is upregulated by Bcl-2 overexpression and downregulated following Bcl-2 knockdown or downregulation after serum starvation. However, Bcl-2 is negatively regulated by PGC-1α expression. Further, co-immunoprecipitation (co-IP) experiments showed that PGC-1α protein is co-precipitated with Bcl-2 at the G0/G1 phase. Taken together, our results suggest that PGC-1α interacts with Bcl-2 after serum depletion, and that Bcl-2 might recruit PGC-1α to reduce ROS, which in turn delays cell cycle progression in coordination with Bcl-2.

B-Myb Mediates Proliferation and Migration of Non-Small-Cell Lung Cancer via Suppressing IGFBP3

B-Myb has been shown to play an important oncogenic role in several types of human cancers, including non-small-cell lung cancer (NSCLC). We previously found that B-Myb is aberrantly upregulated in NSCLC, and overexpression of B-Myb can significantly promote NSCLC cell growth and motility. In the present study, we have further investigated the therapeutic potential of B-Myb in NSCLC. Kaplan–Meier and Cox proportional hazards analysis indicated that high expression of B-Myb is significantly associated with poor prognosis in NSCLC patients. A loss-of-function study demonstrated that depletion of B-Myb resulted in significant inhibition of cell growth and delayed cell cycle progression in NSCLC cells. Notably, B-Myb depletion also decreased NSCLC cell migration and invasion ability as well as colony-forming ability. Moreover, an in vivo study demonstrated that B-Myb depletion caused significant inhibition of tumor growth in a NSCLC xenograft nude mouse model. A molecular mechanistic study by RNA-seq analysis revealed that B-Myb depletion led to deregulation of various downstream genes, including insulin-like growth factor binding protein 3 (IGFBP3). Overexpression of IGFBP3 suppressed the B-Myb-induced proliferation and migration, whereas knockdown of IGFBP3 significantly rescued the inhibited cell proliferation and motility caused by B-Myb siRNA (small interfering RNA). Expression and luciferase reporter assays revealed that B-Myb could directly suppress the expression of IGFBP3. Taken together, our results suggest that B-Myb functions as a tumor-promoting gene via suppressing IGFBP3 and could serve as a novel therapeutic target in NSCLC.

Mitochondria Transcription Factor A: A Putative Target for the Effect of Melatonin on U87MG Malignant Glioma Cell Line.

The disruption of mitochondrial activity has been associated with cancer development because it contributes to regulating apoptosis and is the main source of reactive oxygen species (ROS) production. Mitochondrial transcription factor A (TFAM) is a protein that maintains mitochondrial DNA (mtDNA) integrity, and alterations in its expression are associated with mitochondrial damage and cancer development. In addition, studies have shown that mitochondria are a known target of melatonin, the pineal gland hormone that plays an important anti-tumorigenic role. Thus, we hypothesized that melatonin decreases the expression of TFAM (RNA and protein) in the human glioblastoma cell line U87MG, which disrupts mtDNA expression and results in cell death due to increased ROS production and mitochondrial damage. Our results confirm the hypothesis, and also show that melatonin reduced the expression of other mitochondrial transcription factors mRNA (TFB1M and TFB2M) and interfered with mtDNA transcription. Moreover, melatonin delayed cell cycle progression and potentiated the reduction of cell survival due to treatment with the chemotherapeutic agent temozolomide. In conclusion, elucidating the effect of melatonin on TFAM expression should help to understand the signaling pathways involved in glioblastoma progression, and melatonin could be potentially applied in the treatment of this type of brain tumor.

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

ABSTRACTPrecise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pHi, pHe) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na+/H+ exchanger NHE1 and Na+, HCO3− transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pHe to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pHi changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pHi regulation by the two transporters.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay.

Manipulation of a glycolytic regulator alters growth and carbon partitioning in the marine diatom Thalassiosira pseudonana

The regulation of carbon partitioning in diatoms is key to understanding mechanisms related to their high productivity, and the mechanisms controlling carbon flux towards different metabolic fates are currently not well understood. A unique variant of 6-phosphofructo-2-kinase/fructose 2,6 bisphosphatase (PFK2/F2BP), a regulator of glycolytic flux previously uncharacterized in single-cell photosynthetic eukaryotes, was identified in diatom genomes and overexpressed in Thalassiosira pseudonana. Overexpression resulted in increased activity of the glycolytic rate-determining enzyme phosphofructokinase (PFK) and altered carbon partitioning among intracellular carbohydrate, lipid, and protein levels. Higher PFK activity in transformant lines correlated with reduced growth rate and an extension of the G1 phase of the cell cycle, demonstrating a link between carbon metabolism and the control of cell cycle processes. While the relationship between growth and carbon flux under environmental stress (such as nutrient limitation) is well documented in diatoms, our data demonstrate that manipulation of carbon metabolism, independent of an environmental trigger (such as nutrient limitation), is sufficient to delay cell cycle progression. The inverse relationship between growth and PFK activity (as an indicator of glycolytic flux) differs from those described for other unicellular eukaryotes and supports an alternate organization and regulation of central carbon metabolism in diatoms, including a prominent regulatory role for PFK2/F2BP.

Metformin exerts multitarget antileukemia activity in JAK2V617F-positive myeloproliferative neoplasms

The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor ruxolitinib indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2V617F-positive cells. In JAK2V617F-expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.