Cyclin-dependent Kinase Inhibitor P21 Research Articles

P2252Hyperglycemia drives myeloid CD34+ stem cells differentiation towards pro-inflammatory and senescent monocyte subpopulations

Abstract Background/Introduction Diabetes is characterized by a chronic low-grade inflammatory status mediated by cellular senescence and alterations of the circulating cytokine profile and of innate immune system cell components. An abnormal expansion of intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations with highly inflammatory and senescent-associated secretory phenotype (SASP) have been observed in T2DM patients with cardiovascular (CV) complications. It is unknown whether CD34+ hematopoietic stem cells (HSCs), players of CV prognosis, are involved in this process. Purpose To assess whether hyperglycemia might induce a pro-inflammatory priming of hematopoietic CD34+ HSCs that through senescence and acquisition of SASP skew their myeloid differentiation into more aggressive monocyte populations. Methods CD34+ cells were purified from cord blood of healthy donors and expanded in normal-glucose (NG; with 30 mM mannitol for osmotic control) or high-glucose (HG; 30 mM) serum-free medium plus cytokines. The cell were counted after 10, 20 and 30 days. The expression of p27, p21 RELA/p65, IL6, TNFα genes and telomere length was assessed by qPCR and secreted cytokines by ELISA. Apoptosis, ROS and monocyte subpopulations were evaluated by flow cytometry after Annexin V, CellRox and CD14/CD16 staining respectively. Results CD34+ HSCs cultured in HG (HG-CD34+) displayed a significant proliferation impairment when compared to their osmotic control (NG-CD34+). This loss of glucose tolerance was associated with a significant increase in mitochondrial ROS production (n=6; p≤0.01) without induction of apoptosis as showed by flow cytometry analysis for Annexin V. Moreover, qPCR assay revealed a significant telomere shortening (n=4; p≤0.05) and up-regulation of cyclin-dependent kinase inhibitors p27 and p21 in HG-CD34+cells (n=13; p≤0.05) along with an enhanced expression and secretion of TNFα (n=9; p≤0.05) and IL6 (n=10; p≤0.05) in HG-CD34+ when compared with NG-CD34+. SASP phenotype in HG-CD34+ was associated to a significant up-regulation of RELA/p65 gene in HG-CD34+ when compared with NG-CD34+ (n=8; p≤0.05). Furthermore, we found that in vitro HG-CD34+ differentiation into myeloid lineage generated higher levels of pro-inflammatory intermediate (n=3; p≤0.05) and non-classical (n=3; p≤0.01) monocyte subsets when compared with the normoglycemic counterpart. Conclusion(s) These data suggest that HG exposure primes HSCs myeloid differentiation towards inflammatory and senescent monocyte subpopulations. Acknowledgement/Funding This work was supported by Ricerca Finalizzata, Ministero della Salute [PE-2011-02348537]

Usnic Acid Inhibits Cell Proliferation Via Downregulation Of PCNA Expression In Gastric Carcinoma AGS Cells

Usnic acid is a secondary metabolite obtained from various species of lichen. Previous studies have shown various biological activities of usnic acid, such as anti-oxidant, anti-microbial, anti-viral, anti-protozoal, anti-inflammatory, and anti-proliferative activities in different models. Its anti-proliferative activities in gastric cancer cells are still unexplored. Herein, we have investigated the effects of usnic acid on cell proliferation and viability and associated molecular alterations in human gastric carcinoma AGS cells. The treatment of usnic acid (2.5-25μM) dose-dependently reduced cell proliferation. The mRNA expression of tumor suppressor gene phosphatase tensin homolog (PTEN)in the usnic acid-treated AGS cells was increased, which may play a role in the inhibition of cell proliferation and induction of cell death. We also observed a decrease in the expression of PCNA that regulates cell proliferation by playing an important role in DNA replication. The expression of cyclin-dependent kinase inhibitor p21, which may play a role in cell cycle and proliferation inhibition was found uninfluenced with usnic acid treatment. Thus, collectively these results revealed that usnic acid inhibits the cell proliferation of AGS cells through downregulating the expression of PCNA and can be further evaluated in vivo models for its therapeutic potentials.

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells.

Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 μg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 μg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 μM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 μg/mL induced a delay in G1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 μg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 μg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1–10 μg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis

Renal fibrosis is accompanied by the progression of chronic kidney disease. Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis using a mouse model of unilateral ureter obstruction. We found that in the initial stage of damage, where extracellular matrix was not yet deposited, proximal tubular cells arrested at G2 of the cell cycle. Further analyses indicated that the cyclin-dependent kinase inhibitor p21 is partially involved in the G2 arrest after the damage. A newly produced monoclonal antibody against p21 revealed that levels of p21 were sharply upregulated in response to the damage during the initial stage but dropped toward the later stage. To investigate the requirement of p21 for the progression of renal fibrosis, we constructed the novel p21 deficient mice by i-GONAD method. Compared with wild-type mice, p21 deficient mice showed exacerbation of the fibrosis. Thus we propose that during the initial stage of the renal damage, tubular cells arrest in G2 partially depending on p21, thereby safeguarding kidney functions.

3D Microenvironment Stiffness Regulates Tumor Spheroid Growth and Mechanics via p21 and ROCK.

The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments.

MicroRNA-139 is a predictor of prostate cancer recurrence and inhibits growth and migration of prostate cancer cells through cell cycle arrest and targeting IGF1R and AXL.

We previously identified a panel of five microRNAs (miRNAs) associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using next-generation sequencing-based whole miRNome sequencing and quantitative polymerase chain reaction-based validation analysis. In this study, we examined the mechanism of action of miR-139-5p, one of the downregulated miRNAs identified in the panel. Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan-Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 to confirm its targets as well as examine pathways through which miR-139 may function using cell-based assays. Low miR-139 expression was significantly associated with a variety of prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 expression was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (P = .0004 and .038, respectively). Validation in the TCGA data set showed a significant association between dichotomized miR-139 expression and biochemical recurrence (odds ratio, 0.52; 95% confidence interval, 0.33-0.82). Overexpression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared with control cells, with cells arrested in G2 of cell cycle. IGF1R and AXL were identified as potential targets of miR-139 based on multiple miRNA-binding sites in 3'-untranslated regions of both the genes and their association with prostate cancer growth pathways. Luciferase assays verified AXL and IGF1R as direct targets of miR-139. Furthermore, immunoblotting of prostate cancer cells demonstrated IGF1R and AXL protein expression were inhibited by miR-139 treatment, which was reversed by the addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated AKT and cyclin D1, with upregulation of the CDK inhibitor p21. miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through downregulation of IGF1R and/or AXL and associated signaling pathway components.

MicroRNA-223 is essential for maintaining functional β-cell mass during diabetes through inhibiting both FOXO1 and SOX6 pathways

The initiation and development of diabetes are mainly ascribed to the loss of functional β-cells. Therapies designed to regenerate β-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 β-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses β-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several β-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in β-cells could promote β-cell proliferation and improve β-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional β-cell mass and adaptation during metabolic stress.

Abstract 3023: Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia

Abstract Chemoresistance is a major burden for the treatment of many cancers, including acute myeloid leukemia (AML). AML is a clonal disease and chemoresistant cells may either evolved from the expansion of a subclone of the primary clone or from the clone gained additional mutations during therapy. We have generated chemoresistant cell lines from AML patient sample derived cell lines, OCI-AML2 and MV4-11, to study the chemoresistant mechanism. Interestingly, we found that both established resistant lines have TP53 mutations (Y220C or R248W) but not in the parental cell lines by Sanger sequencing. However, high resolution sequencing result shows that residual TP53 mutation is present in both parental cell lines. These small population of cells with TP53 mutation was expanded under the selection by chemotherapy and eventually became a dominant clone in chemoresistant lines. TCGA AML patient sample sequencing data also shows, there’re around 3% of patients have residual TP53 mutation reads. However, since the reads are low, these patients are considered TP53 wild type conventionally. Importantly, these patients have worse outcome of survival as compared to the TP53 wild type patients, indicating the residual TP53 mutation may play an important role on chemoresistance in patients. Consistent with TP53 mutation in chemoresistant cells, Gene Set Enrichment Analysis (GSEA) from RNA-seq data shows p53 target genes are repressed in resistant cells. The cyclin-dependent kinase inhibitor p21 is one of p53 target genes and is down regulated in chemoresistant cells. Knocking down of p21 in parental cells increased cell chemoresistance, indicating p21 gene repression contributes to chemoresistance. Data from TCGA also shows patients with lower p21 expression have worse outcome of survival. In chemoresistant cell line, there is reduced p53 recruitment at p21 promoter, leading to failure of p21 activation. The epigenetic drug Romidepsin can elevate histone acetylation at p21 promoter region, reactivates p21 gene expression and induces cell death. Furthermore, GSEA from RNA-seq data shows Romidepsin can reactivate genes that repressed by p53 mutation. Our data indicate that TP53 mutation is directly linked to chemoresistance. Residual TP53 mutation in patients was neglected previously, however, it can be an important driver for clonal evolution and chemoresistance in AML. In addition, epigenetic drug Romidepsin can be a potential therapeutic drug to prevent chemoresistance development especially for patients with TP53 mutation or residual TP53 mutation. Citation Format: Bowen Yan, Yi Qiu. Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3023.

Abstract 3038: Implication of DYRK1B Kinase in ovarian cancers and utilization of DYRK1B Inhibitors as a novel therapeutic strategy for ovarian cancer

Abstract Majority of tumors consist of two populations of cancer cells: actively proliferating and those in the reversible dormant or quiescent state, which contribute to the resistance of human tumors to chemotherapies. Conventional chemotherapeutic agents have limited efficacy in ovarian adenocarcinoma patients: while the appropriate chemotherapeutic agent kills proliferating cancer cells, dormant cancer cells survive the treatments and result in recurrent disease. The presence of dormant cancer cells is not revealed by standard H&E staining and morphological tumor analysis. CDK inhibitor p27 is known to be a marker of the dormant state. DYRK1B kinase (serine/threonine-protein kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B) is associated with survival of many types of cancers and was shown to play a key role in maintaining the cancer cells in quiescent state by stabilizing p27 and by inducing the breakdown of cyclin D isoforms. In this study, we investigated the functional and therapeutic relevance of DYRK1B in ovarian carcinomas. We have conducted an immunohistochemical study of 70 human ovarian tumors (35 primary/treatment-naive and 35 recurrent/standard chemotherapy resistant ones) and 20+ cases of normal and benign ovarian tissues for expression of DYRK1B, p27 and Ki-67. All FFPE tissue samples have been obtained from the consented patients and under the IRB-approved protocols clinical network, groups have been normalized for age and the disease type/stage. High levels of DYRK1B and p27 proteins expression in malignant treatment-naïve tumors and their significant correlation indicates the presence of dormant cancer cells mixed up with the proliferating ones within patient’s tumors. This effect was detected regardless of the tumor histological subtype (endometrial, serous, mucinous, etc.) and/or stage. While all advanced stage tumors (Stage IIIC and IV) overexpress both markers of dormancy; the early stage tumors (Stage I-II) also presented a higher number of cases with greatly positive scores. Tissue microarray and immunohistochemistry analysis in the literature showed that higher expression levels of DYRK1B correlated with a worse prognosis. We suggest that specific immunostaining of DYRK1B in tumor and endothelial cells can be explored as a predictive risk factor of time to progression/death in patients with primary ovarian tumors. Inhibition of DYRK1B kinase activity with FX9847, specific and selective DYRK1B inhibitor, inhibited ovarian cancer cell growth and induced apoptosis. Moreover, combination of FX9847 with chemotherapeutic agents such as taxanes or vinca alkaloids demonstrated an increased anti-cancer effect on ovarian cancer cells. Together, these findings suggest that DYRK1B is critically involved in the survival of ovarian carcinoma providing a new rationale for their treatment with the kinase inhibitors. Citation Format: Alexandra Kuznetsova, Arianna Damiani, Lita De Leon, Michael Frid, Menelik Duey, Jason Law, Olga Potapova, Maria Vilenchik. Implication of DYRK1B Kinase in ovarian cancers and utilization of DYRK1B Inhibitors as a novel therapeutic strategy for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3038.

Abstract 1156: Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation

Abstract Adult stem cells have been used as regenerative medicine for treating human diseases through gene therapy approach. These stem cells secrete cytokines, growth factors and extracellular vesicles including exosomes, which may induce antitumor activities. We have demonstrated that muscle progenitor cells (MPCs) from adult (murine and human) skeletal muscle have a high regenerative potential to repair bone, skeletal muscle, and cartilage. However, to date, the potential therapeutic use of MPCs in cancers has not yet been investigated. The objectives of this study were to identify and examine the roles of secreted factors including exosomes from MPCs on the proliferation/survival, motility/migration and invasive potential of prostate cancer (PCa) cells. Here we showed that co-culture of PCa cell lines, PC3 and C42B, with MPCs in trans-well inserts strongly suppressed PCa cell proliferation and survival by activating CDK inhibitors (p16 and p27) and apoptotic BBC3 and NOXA genes. Mass spectrometry analysis of factors secreted by MPCs identified several proteins with known anti-proliferative functions, including IGFBP6/7, pigment epithelium-derived factor (PEDF), HPCAL1, NO, and SPARC. Conditioned media (CM) from MPCs significantly increased docetaxel-induced cytotoxicity of PCa cells by suppressing the expression of key genes including AR, MLL, FOXA1, TWF1, and TGIF. Next, we isolated exosomes from cultured mouse and human MPCs through step-wise ultracentrifugation methods and tested their effect in the proliferation of cancer cells. We observed that treatment of PCa cells with MPC-derived exosomes inhibited cell proliferation as assayed with MTT and down-regulated the expression of AR and its target NKX3.1 genes. We also observed that the migration ability of ovarian cancer cells (OVAC5) and pancreatic cancer (Panc1), was markedly reduced following MPC-derived exosome treatments compared to untreated cells. Taken together, our data suggest that MPCs can suppress the proliferation and survival of cancer cells through secreted factors including exosomes. Thus, adult stem cell-based therapy along with existing therapies may be a promising strategy in treating various solid cancers as well as regenerating cancer-induced bone and muscle tissue lesions. Citation Format: Krishna M. Sinha, Rozita Yarmand, Nermin Kahraman, Fahriye Duzagac, Gabriel Lopez, Bulent Ozpolat, Johnny Huard. Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1156.

Abstract 3554: miR-139 is associated with improved prognosis in patients with localized prostate cancer

Abstract Background: We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy based on NGS-based whole miRNome discovery and qPCR-based validation analysis. In this analysis, we examine the effect of miR-139-5p, one of the down-regulated miRNAs identified in the panel, in greater detail. Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that over-expressed miR-139 or transiently transfected cells using miR-139 mimics for functional assays. Finally, we examined pathways through which miR-139 may function using prediction algorithms and confirmed targets by Western blotting and reporter assays. Results: MiR-139 down-regulation was significantly associated with a variety of accepted prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity and lymph node status. MiR-139 was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (p=0.0004 and 0.038, respectively). After adjusting for known prognostic factors, patients with high miR-139 expression had significantly lower risk of recurrence (HR 0.77, 95% 0.58-1.04). Validation in the TCGA dataset showed a significant association between dichotomized miR-139 expression and biochemical recurrence (OR 0.52, 95% CI 0.33-0.82). Over-expression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared to control cells, with cells arrested in G2 of cell cycle. IGF1R, RUNX1 and AXL were identified as potential gene targets of miR-139 based on their association with prostate cancer growth pathways and multiple miRNA binding site prediction tools. The reporter assays using luciferase gene constructs containing the predicted miRNA targeting sequence from IGFR1 and RUNX1 verified them as direct targets of miR-139. Furthermore, Western blotting of prostate cancer cells demonstrated RUNX1 and AXL expression were inhibited by miR-139 treatment, which was reversed by addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated Akt and cyclin D1, with upregulation of the CDK inhibitor p21. Conclusions: miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through inhibition of IGF1R, RUNX1 and/or AXL and their associated growth signaling pathways. Citation Format: Tania Benatar, Robert K. Nam, Elizabeth Kobylecky, Yutaka Amemiya, Arun K. Seth. miR-139 is associated with improved prognosis in patients with localized prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3554.

Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2

Neat1 is widely expressed in many tissues and cells and exerts pro-proliferation effects on many cancer cells. However, little is known about the function of Neat1 in myogenesis. Here we characterized the roles of Neat1 in muscle cell formation and muscle regeneration. Gain- or loss-of-function studies in C2C12 cells demonstrated that Neat1 accelerates myoblast proliferation but suppresses myoblast differentiation and fusion. Further, knockdown of Neat1 in vivo increased the cross-sectional area of muscle fibers but impaired muscle regeneration. Mechanically, Neat1 physically interacted with Ezh2 mainly through the core binding region (1001–1540 bp) and recruited Ezh2 to target gene promoters. Neat1 promoted myoblast proliferation mainly by decreasing the expression of the cyclin-dependent kinase inhibitor P21 gene but inhibited myoblast differentiation by suppressing the transcription of myogenic marker genes, such as Myog, Myh4, and Tnni2. Altogether, we uncover a previously unknown function of Neat1 in muscle development and the molecular mechanism by which Neat1 regulates myogenesis.

Knockdown of GC binding factor 2 by RNA interference inhibits invasion and migration of vascular smooth muscle cells.

GC binding factor 2 (GCF2) is a transcriptional repressor that inhibits the transcription of GC-rich promoters, thereby regulating biological processes, including proliferation. However, the role of GCF2 in vascular smooth muscle cells (VSMCs) remains unclear. The level of α-smooth muscle (α-SM) actin was determined by immunofluorescence. Cell viability, migration and invasion were analyzed using Cell Counting Kit-8, wound healing and Transwell assays, respectively. Apoptosis and cell cycle progression were determined using flow cytometry. The expressions of Bcl-2, Bax, cleaved caspase-3, cyclin E, CDK2 and the CDK inhibitor p21 were determined by reverse transcription-quantitative (RT-q)PCR and western blot analysis. RT-qPCR was performed to analyze the levels of GCF2 and western blot analysis was conducted to determine the phosphorylation levels of PI3K and AKT. α-SM actin was found to be expressed in VSMCs. Cell viability, migration and invasion were inhibited by small interfering (si)RNA targeting GCF2. Changes in the expression levels of Bcl-2, Bax and cleaved caspase-3 showed that the pro-apoptotic capacity of the cells was increased by siGCF2. Cell cycle arrest in the G0/G1 phase was induced by siGCF2, which was accompanied by changes in the levels of cyclin E, CDK2 and p21. Furthermore, phosphorylation of PI3K and AKT was suppressed by siGCF2. However, the inhibitory effects of siGCF2 on cell viability, migration and invasion were increased by insulin-like growth factor 1, which is a specific agonist of AKT. The anti-proliferative activity of siGCF2 may be associated with the PI3K/AKT pathway in VSMCs.

Control of Hematopoietic Stem and Progenitor Cell Function through Epigenetic Regulation of Energy Metabolism and Genome Integrity.

SummaryIt remains largely unclear how stem cells regulate bioenergetics and genome integrity to ensure tissue homeostasis. Here, our integrative gene analyses suggest that metabolic and genotoxic stresses may underlie the common functional defects of both fetal and adult hematopoietic stem and progenitor cells (HSPCs) upon loss of DPY30, an epigenetic modulator that facilitates H3K4 methylation. DPY30 directly regulates expression of several key glycolytic genes, and its loss in HSPCs critically impaired energy metabolism, including both glycolytic and mitochondrial pathways. We also found significant increase in DNA breaks as a result of impaired DNA repair upon DPY30 loss, and inhibition of DNA damage response partially rescued clonogenicity of the DPY30-deficient HSPCs. Moreover, CDK inhibitor p21 was upregulated in DPY30-deficient HSPCs, and p21 deletion alleviated their functional defect. These results demonstrate that epigenetic mechanisms by H3K4 methylation play a crucial role in HSPC function through control of energy metabolism and protecting genome integrity.

Long non-coding RNA slincRAD functions in methylation regulation during the early stage of mouse adipogenesis

ABSTRACT Adipocyte differentiation is a coordinated cellular process, which involves a series of dynamic molecular events. Up-regulation of long noncoding RNA slincRAD expression was found to occur in the early differentiation stages of 3T3-L1 cell, prior to the regulation of major transcription factors. By interacting with DNMT1 in S phase, slincRAD guides this essentially epigenetic factor to mediate promoter methylation of a batch of cell cycle-related genes, including cyclin-dependent kinase inhibitor p21. The regulation promotes the growth-arrested cells to re-enter into cell cycle under hormone induction and thereby advances the process of differentiation to clonal expansion stage. The abolishment of the interaction between slincRAD and DNMT1 by slincRAD knockdown results in a defective epigenetic regulation and finally compromised adipogenesis. Collectively, our study characterizes the epigenetic regulation of lncRNA involved in the early stage of adipogenesis.

Implication of DYRK1B kinase in dormant glioblastoma cancers and utilization of DYRK1B inhibitors as a novel therapeutic strategy for glioblastoma.

e14670 Background: A major determinant of cancer resistance and recurrence is the presence of quiescent cancer cells. It has long been known that quiescent cancers are resistant to a wide variety of anti-cancer therapeutics relative to actively proliferating cells. However, the presence and relative size of the quiescent compartment is not revealed by standard H&E staining and morphological tumor analysis and, therefore, the data demonstrating its importance and prevalence by tumor type are lacking. DYRK1B kinase (serine/threonine-protein kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B) is associated with growth and survival of many types of cancer cells and was shown to play a key role in maintaining the cancer cells in reversible quiescent state by stabilizing the CDK inhibitor p27 and by inducing the breakdown of cyclin D isoforms. Methods: We conducted an immunohistochemical study of 80 human glioblastoma (GBM) tumors (primary / treatment-naive, Grade IV) and 10 cases of normal postmortem brain tissues for expression of DYRK1B, and quiescence and proliferation markers p27 and Ki-67, respectively. For subsection of these tissues, high levels of DYRK1B and p27 expression were observed. Results: In this study, we investigated the functional and therapeutic relevance of DYRK1B expression in glioblastomas, the most common brain cancer in adults. It is an aggressive, highly resistant to treatment tumor type with median survival time of less than 16 months following surgery and treatment. Conclusions: There was a statistically significant correlation between immunostaining of DYRK1B and p27, indicating the presence of dormant cancer cells mixed with the proliferating cancer cells within the patients' tumors. We suggest that DYRK1B inhibitors in combination with chemotherapeutics agents have a potential to improve patient treatment outcome and prolong survival.

P21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

ABSTRACTThe Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response and Cell Cycle Regulation in Individual Cells

To enable reliable cell fate decisions, mammalian cells need to adjust their responses to dynamically changing internal states by rewiring the corresponding signaling networks. Here, we combine time-lapse microscopy of endogenous fluorescent reporters with computational analysis to understand at the single-cell level how the p53-mediated DNA damage response is adjusted during cell cycle progression. Shape-based clustering revealed that the dynamics of the CDK inhibitor p21 diverges from the dynamics of its transcription factor p53 during S phase. Using mathematical modeling, we predict and experimentally validate that S phase-specific degradation of p21 by PCNA-CRL4cdt2 is sufficient to explain these heterogeneous responses. This highlights how signaling pathways and cell regulatory networks intertwine to adjust the cellular response to the individual needs of a given cell.

Milk Oligosaccharides From Different Cattle Breeds Influence Growth-Related Characteristics of Intestinal Cells.

Oligosaccharides are present in human milk (HMO) in large amounts and in a high variety: Among other functions they are considered to influence the gut microbiota and gut maturation in infants. Due to the large volume of milk available bovine milk oligosaccharides (BMO) may be an alternative source of functional ingredients to potentially mimic HMO functions. Thus, we investigated direct effects of bovine milk oligosaccharides (BMO) from different cattle breeds on proliferation, differentiation and apoptosis in transformed (HT-29 and Caco-2) and non-transformed human intestinal cells (HIE cells). We observed a profound growth-inhibition effect induced by all BMO isolates in HT-29, Caco-2, and HIE cells in a dose-dependent manner. The effects varied not only between cell lines, i.e., HT-29 and Caco-2 cells were more sensitive than HIE cells, but also between the cattle breeds. Regarding the induction of differentiation, BMO induced differentiation only in HIE cells without affecting apoptosis. Cell cycle analysis via flow cytometry showed that growth inhibition was associated with a G2/M arrest in all cell lines. Expression levels detected by quantitative real-time RT-PCR revealed that this G2/M arrest was associated with changes in mRNA expression levels of cyclin A and B. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 and the tumor suppressor p53 were only enhanced in HIE cells necessary for arresting cells in the G2/M phase and induction of differentiation. In HT-29 and Caco-2 cells, a loss of p53 expression failed to induce G2/M associated induction of differentiation. The HIE cell specific differentiation induced by BMO was a result of influencing the phosphorylation states of EGFR (epidermal growth factor receptor) and MAP kinase, i.e., ERK1/2 (extracellular signal-regulated kinase 1/2), p38-α, and Akt2 phosphorylation. These results suggest that BMO inhibited intestinal cell proliferation and altered cell cycle dynamics by affecting corresponding regulator genes and mitogen-activated protein kinase signaling. As the development and maturation of digestive and absorptive processes depends on gut differentiation processes, our in vitro experiments show that breed-specific BMO are natural substances influencing various parameter which may be important in vivo in gastrointestinal development. This, however, needs to be proven in future studies.